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Characterization of Novel Yeast Receptors Implicated in Metal and Lipid Metabolic Pathways

Permanent Link: http://ufdc.ufl.edu/UFE0021703/00001

Material Information

Title: Characterization of Novel Yeast Receptors Implicated in Metal and Lipid Metabolic Pathways
Physical Description: 1 online resource (171 p.)
Language: english
Creator: Villa, Nancy Yaneth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: iron, izh, lipids, metabolism, nystatin, rafts, regulation, sphingolipids, yeast, zinc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the yeast Saccharomyces cerevisiae, a family of genes was identified by DNA microarrays. These genes were called IZH1-4 (Implicated in Zinc Homeostasis) because their expression was dependent on the zinc concentration in the cell. The IZH genes encode membrane proteins with seven transmembrane spanning domains and three highly conserved motifs. Muliple sequence alignments revealed that the IZH gene products (Izhs) belong to a large and ubiquitous family of proteins that includes the progestin and adiponetin receptors (PAQRs). Eleven members of this protein family have been identified in vertebrates, and homologues can be found in species ranging from bacteria to humans. The human PAQRs have started to capture the attention of researchers due to their implication in the development of diseases like obesity and type 2 diabetes. Sequence alignment has also indicated that Izh proteins share distant similarity with the yeast alkaline ceramidases Ypc1p and Ydc1p, two enzymes involved in the sphingolipid metabolic pathway. A first approach to the characterization and elucidation of the role(s) for IZHs is presented. First of all, IZH2 is shown to be induced under zinc deficiency via the zinc sensor Zap1p. Furthermore, the IZH2 gene and its homolog IZH4 respond to excess of zinc, cobalt, and nickel, as well as to iron deficiency via the hypoxic transcription factor Mga2p. Interestingly, when expressed in media replete in iron and zinc, the Izh2 protein (Izh2p) seems to be ubiquitinated. However, in a medium deficient in iron or zinc Izh2p does not appear to be ubiquitinated. On the other hand, different lines of evidence are presented that indicate that IZHs are also implicated in lipid metabolism. In this regard, we initially show that IZH1, and IZH4, are slightly inducted by the fatty acid palmitate, whereas IZH3 is induced by oleate. Interestingly, IZH2 is highly induced by myristateat the transcriptional and post-translational levels. Other lines of evidence suggesting a potential involment of the IZHs in lipid metabolism come from the observation that mutation of the IZH3 gene produces a nystatin resistant phenotype. Nystatin is an antifungal that interacts with ergosterol in the yeast plasma membrane. Sterol analysis indicates that izh3? mutant presents alterations in the sterol composition. Specifically, we demonstrate that izh3? has lower levels of free ergosterol than a wild type strain, thus suggesting a role for IZH3 in the sterol metabolic pathway. Despite the distant similarity with alkaline ceramidases, we could not demonstrate that Izhs function as ceramidases per se; however when overexpressed, the Izhs induced the synthesis of free sphingoid bases. Furtheremore, we show preliminary data indicating that the overexpression of Izhs can result in the de novo sphingolipid biosynthesis. Finally, strong evidence is presented indicating that Izh2p and Izh3p are plasma membrane proteins that are associated with microdomains formed by sterols and sphingolipids called lipid rafts. This result not only supports our hypothesis that Izhs are membrane receptors, but also it opens new and exciting avenues regarding the role of these proteins in lipid metabolism.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nancy Yaneth Villa.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Lyons, Thomas J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021703:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021703/00001

Material Information

Title: Characterization of Novel Yeast Receptors Implicated in Metal and Lipid Metabolic Pathways
Physical Description: 1 online resource (171 p.)
Language: english
Creator: Villa, Nancy Yaneth
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: iron, izh, lipids, metabolism, nystatin, rafts, regulation, sphingolipids, yeast, zinc
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In the yeast Saccharomyces cerevisiae, a family of genes was identified by DNA microarrays. These genes were called IZH1-4 (Implicated in Zinc Homeostasis) because their expression was dependent on the zinc concentration in the cell. The IZH genes encode membrane proteins with seven transmembrane spanning domains and three highly conserved motifs. Muliple sequence alignments revealed that the IZH gene products (Izhs) belong to a large and ubiquitous family of proteins that includes the progestin and adiponetin receptors (PAQRs). Eleven members of this protein family have been identified in vertebrates, and homologues can be found in species ranging from bacteria to humans. The human PAQRs have started to capture the attention of researchers due to their implication in the development of diseases like obesity and type 2 diabetes. Sequence alignment has also indicated that Izh proteins share distant similarity with the yeast alkaline ceramidases Ypc1p and Ydc1p, two enzymes involved in the sphingolipid metabolic pathway. A first approach to the characterization and elucidation of the role(s) for IZHs is presented. First of all, IZH2 is shown to be induced under zinc deficiency via the zinc sensor Zap1p. Furthermore, the IZH2 gene and its homolog IZH4 respond to excess of zinc, cobalt, and nickel, as well as to iron deficiency via the hypoxic transcription factor Mga2p. Interestingly, when expressed in media replete in iron and zinc, the Izh2 protein (Izh2p) seems to be ubiquitinated. However, in a medium deficient in iron or zinc Izh2p does not appear to be ubiquitinated. On the other hand, different lines of evidence are presented that indicate that IZHs are also implicated in lipid metabolism. In this regard, we initially show that IZH1, and IZH4, are slightly inducted by the fatty acid palmitate, whereas IZH3 is induced by oleate. Interestingly, IZH2 is highly induced by myristateat the transcriptional and post-translational levels. Other lines of evidence suggesting a potential involment of the IZHs in lipid metabolism come from the observation that mutation of the IZH3 gene produces a nystatin resistant phenotype. Nystatin is an antifungal that interacts with ergosterol in the yeast plasma membrane. Sterol analysis indicates that izh3? mutant presents alterations in the sterol composition. Specifically, we demonstrate that izh3? has lower levels of free ergosterol than a wild type strain, thus suggesting a role for IZH3 in the sterol metabolic pathway. Despite the distant similarity with alkaline ceramidases, we could not demonstrate that Izhs function as ceramidases per se; however when overexpressed, the Izhs induced the synthesis of free sphingoid bases. Furtheremore, we show preliminary data indicating that the overexpression of Izhs can result in the de novo sphingolipid biosynthesis. Finally, strong evidence is presented indicating that Izh2p and Izh3p are plasma membrane proteins that are associated with microdomains formed by sterols and sphingolipids called lipid rafts. This result not only supports our hypothesis that Izhs are membrane receptors, but also it opens new and exciting avenues regarding the role of these proteins in lipid metabolism.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Nancy Yaneth Villa.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Lyons, Thomas J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021703:00001


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CHARACTERIZATION OF NOVEL YEAST RECEPTORS IMPLICATED IN METAL AND
LIPID METABOLIC PATHWAYS





















By

NANCY YANETH VILLA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































2007 Nancy Yaneth Villa



























For my mom and my grandmom Teresa, whose faith and strength have been my driving force to
go on, and to face different challenges in my life.









ACKNOWLEDGMENTS

I would like to thank Dr. Thomas Lyons for the opportunity to work in his research

projects, for the guidance and the support that gave me during my Ph.D. I also want to thank Drs

Yusuf Hannun and Ashley Cowart for their help with some of my research projects, and for their

incredible generosity. I am very grateful to the people in the mass spectrometry laboratory at the

University of Florida, and the people from the lipidomic core at the Medical University of South

Carolina (MUSC). I want to give special thanks to Charlene Alford, at MUSC, for her technical

support. Thanks also to my committee members, for their time, their suggestions and useful

comments.

I am forever grateful to my family and my friends for their unlimited love, and support. I

am deeply grateful to my lovely brother Carlos Alberto, who despite the distance was always

close to me, enjoying my triumphs and absorbing each of my tears. Without his help and support,

getting to this point would never have been possible.

Finally, thanks to my lovely God for helping me to grow during this great academic

opportunity.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F TA B L E S .......... .... .............. ................................................................... 9

LIST OF FIGURES .................................. .. .... ..... ................. 10

A B S T R A C T ................................ ............................................................ 13

CHAPTER

1 INTRODUCTION ............... ................. ........... ......................... .... 15

Z inc H om eostasis......................................................................................... 15
The Budding Yeast Saccharomyces cerevisiae as a Model System............................. ... 17
Identification ofIZHs on the Saccharomyces cerevisiae Genome .................................. 17
Regulatory Sequences Surrounding the IZH Genes .................................... ............... 18
The IZH Gene Family Encodes Membrane Proteins............ .. ........... 19
The PAQR Fam ily of Proteins ......................................... ........................ ...............20
Izh2 is an Osm otin Receptor Protein................................................................. ............... 21
The IZHs and their Connection with Lipid Metabolic Pathways .......................................22
S u m m atio n ................... ............................... ......................... ................ 2 4

2 METALLOREGULATION OF IZHS................................................................ ................ 31

Intro du action ................... ................... ...................................................... .. 3 1
M materials and M methods ...................................... .. .......... ....... ...... 32
Y east Strains and Plasm ids........................................... ................... ............... 32
Y east M edia ........................................................ ............... ................. 33
Y east Transform nations and A says ........................................ ........................... 35
P reparation of M icrosom es................................................................... .....................36
W western Blot Analysis of Protein Expression ...................................... ............... 36
Immunoprecipitation of Izh2p and Western Blot Analysis .................. ..................37
R results ............... ....... ... ........... ........................... 38
IZH 2 is a Zaplp Target G ene ................................................ .............................. 38
IZH2 and IZH4 Are Part of the Hypoxic Response.................................. ....................39
Regulation oflZH4 by Excess of Several Transitions Metals is only Mga2p-
D dependent ................ ............. .. ........ ...... ....................................... 40
Iron Deficiency Affects the Expression of IZH2 and IZH4 ................. .................41
Ubiquitination of Izh2p is Dependent on Nutritional Conditions ..............................42
D isc u ssio n ................... ............................................................ ............... 4 3









3 DUAL REGULATION OF THE IZHS BY METALS AND FATTY ACIDS: THE
FIRST LINE OF EVIDENCE IMPLICATING THE IZH FAMILY IN LIPID
M E TA B O L ISM .......................................... ................ ...................... ........ 55

Intro du action ................... .......................................................... ................ 5 5
M materials and M methods ................................... ... .. .......... ....... ...... 57
Y east Strains and Plasm ids........................................... ................... ............... 57
B iochem ical A ssay s............ .... .............................................................. .......... ....... 58
Results ......................... ......................... ......... ..........58
Fatty Acids Exert a Regulatory Effect on the Expression of IZHs..............................58
The Expression of the Izh Proteins is Regulated by Fatty Acids ...................... ........59
Transcriptional Regulation of ZH2 and IZH4 in Presence of Metals and Fatty
A cids O ccurs via M ga2p ......... ..................... .................................... ............... 60
D iscu ssion ......... ......................................... ...............................6 1

4 NYSTATIN-RESISTANCE OF IZH3A, IS ASSOCIATED TO ALTERATIONS IN
THE ERGOSTEROL CONTENT .............. ............................... .................. ............... 70

Introduction ................. ......................................... ............................70
M materials and M methods ..................................... ... .. .......... ....... ...... 72
Y east Strains and R agents ..................................................................... ..................72
Y east Transform ations........ ........ .. ................................... .... ...... 73
Y east Growth M edia and Conditions ........................................ ......................... 73
Phenotypic Studies ........................ .. ........................ .. .... .......... ........ 74
C om plem entation Studies........................................................................ ..................75
Sterol Extractions .............................................................75
Sterol Analysis by Ultraviolet Spectroscopy ................................. ...................76
Analysis of Sterols by Gas Chromatography-Mass Spectrometry (GC-MS) ..................77
Analysis of Total and Free Ergosterol by High Performance Liquid
Chromatography-Atmospheric Pressure Chemical Ionization- Mass Spectrometry
(H PLC-APCI-M S) .......................................................... .. ........ .... 78
R results ........................... ............ .. .. .......... ....................................................78
IZH Genes Affect the Tolerance to the Antifungal Nystatin...............................78
Nystatin Induces Alterations in the Total Sterol Composition of Wild Type and
iz h 3 ........................................ .. ............... ............................ .............................8 0
Gas Chromatography-Mass Spectrometric (GC-MS) Analysis Revealed not
Significant Differences in the Basal Levels of Total Sterols for Wild Type and
iz h 3 A ....................................................................... ................... 8 1
Alterations in the Free Ergosterol Content Were Observed for the Mutant izh3A..........81
Addition of Certain Sphingolipids Ameliorate the Aberrant Nystatin Effects on
W ild Type and izhA M utants ............................................... ............................ 83
D iscu ssion ......... ....... .................................. .......... ............................... 83

5 POTENTIAL IMPLICATION OF IZHS IN THE SPHINGOLIPID BIOSYNTHETIC
P A T H W A Y .........................................................................97

In tro d u ctio n ................... ...................9...................7..........









M materials and M ethods ..................... ................................................ .. ........ ............ 99
Strains Plasmids and Yeast Transformations ...................................... ............... 99
Y east G row th C conditions ........................................................................ .................. 99
In Vitro C eram idase A ssay ................................................... ................................... 100
Phenotypic Studies .............................................. .. .. .... .................. 102
T total L ipid Extraction ......................... ............................................. 102
Phospholipid D eterm nation ........... ...... ............ ......................................... ...........103
High-Performance Liquid Chromatography Analysis of O-Phthalaldehyde-
Sphingoid B ase D erivatives......................................................... .. ...................... 104
Analysis of Radiolabeled Sphingolipids by One-Dimensional Thin Layer
Chrom atography .................. ......... ... .. ... ....... ......... ............... 105
Analysis of Sphingolipids by Electrospray-Ionization Tandem Mass Spectrometry
(E S I-M S/M S ) .................................................... ................ 10 6
R e su lts ............ ... .......................... ........ ........................... .. ....... ... ............... 10 8
Overexpression of IZHs Produces Increase in the Levels of Free Sphingoid Bases.....108
In Vitro Ceramidase Assays Suggest that Izhs May not be Alkaline Ceramidases.......109
Thin Layer Chromatography of Radioactive Sphingolipids Reveals Similar
Sphingolipid Profiles for YPC1 and the IZHs.........................................................111
Fumonisin B1 Induces the Acummulation of Sphingoid Bases in YPC1, IZH2, and
IZH 3 ........... ........ ... ...... ............ ... ........ ....... .. ............. ....... 111
Myriocin Inhibits the Increase in the Levels of Sphingolipid Biosynthesis Mediated
by IZH 2 and IZH 3 ......... .. .................................. ...... .............. ........ .... 112
D iscu ssion ......... ............ ......................... ............................113

6 CELLULAR LOCALIZATION OF THE Izh2 AND Izh3 PROTEINS BY
M EM BRANE FRACTIONATION .......................................................... ............... 128

Introduction ................... ........................................................ ................. 128
M materials an d M eth od s .............................................................................. ..................... 12 9
Plasm ids and Y east transform ations.................................... .......................... .. ........ 129
G row th C conditions ........ ...... ......... ............................................ .... ... ....... 130
Isolation Purification and Characterization of Yeast Plasma Membranes ....................130
Isolation of Lipid Rafts from Yeast Plasma Membrane: Procedure 1...........................132
Isolation of Lipid Rafts from Total Membranes: Procedure 2 ........... .....................133
R esu lts ................... ................................................................................13 5
Izh2p and Izh3p Were Found Enriched in Plasma Membrane.................................... 135
Izh2p and Izh3p Are Associated with Lipid Rafts ................................................137
D isc u ssio n ................... ......................................................................... 1 3 8

APPENDIX

A YEAST GROWTH MEDIA AND PROCEDURE ................................... ............... 146

B Y EA ST TR AN SFO RM A TION ............................................................................ ......... 150

P ro to c o l ................... ........................................................................... 1 5 0
S o lu tio n s ................... .......................................................................... 1 5 1









Litium Acetate Tris Base Ethylenediamminetetraacetic Acid (LITE) Solution ..........151
Polyethylenglycol Litium Acetate Tris Base Ethylenediamminetetraacetic Acid
(PE G -L iT E ) Solution ................. .. .............................. .. ............. ... ........ .... ...15 1
Tris Base Ethylenediamminetetraacetic Acid (10X-TE) Solution..............................151
C carrier D N A ......... ..... ................................................... ............................ 15 1

C GAS-CHROMATOGRAMS AND MASS SPECTRA OF TOTAL STEROLS ...............152

D CHROMATOGRAMS AND MASS SPECTRA OF ERGOSTEROL ANALYZED BY
H PLC-A PCI-M S ........... ... .......... .... ........................ .. ........ ........ .. .. 55

L IST O F R E F E R E N C E S ......... .. ............... ................. ..........................................................159

BIOGRAPHICAL SKETCH .......... ................... ...............171









LIST OF TABLES

Table page

2-1 List of strains used in Chapter 2 .............................................. .............................. 47

2-2 G enes induced > 2-fold by zinc excess........................................ .......................... 48

3-1 List of strains used in Chapter 3 .............................................. .............................. 63

4-1 Sources and genotypes of the strains used in Chapter 4 .................................................87

5-1 Strains and genotypes in Chapter 5........................................................... ............... 16









LIST OF FIGURES


Figure page

1-1 Zaplp activation is zinc dependent. ............................................................................26

1-2 A common budding yeast Saccharomyces cerevisiae cell, and some of the most
important and well characterized zinc transporters. .................................. .................26

1-3 Phylogentic analysis of the Izhp family and its closer and more distant homologues
in y east ......................................................... ..................................27

1-4 Regulatory elements surrounding IZH1, IZH2 and IZH4 genes suggest dual role in
m etal and lipid m etabolism ........................................................................ ..................28

1-5 Predicted topology for the Izhp fam ily. ................................................... .....................28

1-6 Sequence alignment of important conserved regions in PAQRs, hemolysins and
ceram idases. ................................................................................29

1-7 Phylogentic showing the Izhp family, its closer and more distant homologues in
yeast, and other organism s ........................................................................ ...................29

1-8 The Sphingolipid biosynthetic pathway in the yeast Saccharomyces cerevisiae ............30

2-1 Z inc regulation of IZH 2 ............................................................................ ................... 49

2-2 Transcriptional regulation of IZH2 and IZH4 by different metals......................... 50

2-3 Transcriptional activation of ZH4-lacZ in cells exposed to excess of different metals
is dependent on the presence of MGA2 but independent on SPT23. ................................51

2-4 Iron regulation of IZH 2 and IZH 4......... .................................................. ............... 52

2-5 Post-translational effect of iron deficiency on the overexpression of Izh2p. ....................53

2-6 Zinc-dependence on the translational response for Izh2p............................................54

3-1 Zaplp-dependent regulation of IZH2-lacZ. The IZH2-lacZ reporter responds to both
zinc and exogenous m yristate (C 14:0)........................................ ........................... 64

3-2 Transcriptional regulation of ZHs by exogenous fatty acids. 65

3-3 Oaflp/Pip2p dependence of the IZH2-lacZ activity in presence and in absence of
myristate (C14:0) and oleate (C18:1), (panels A and B, respectively)............................66

3-4 Post-translational response of the Izhp family upon addition several fatty acids. and
D, the expression of Izh2p is highly induced upon addition of myristate (C14:0) ............67









3-5 Dual regulation of IZH2 and IZH4 by exogenous myristate (C14:0) and cobalt...............68

3-6 Regulation of ZH2 and IZH4 by exogenous oleate (C18:1) and cobalt ..........................69

4-1 Chemical structures of the sterols analyzed in this chapter. ..................... ................88

4-2 Model proposed for the nystatin action in the yeast plasma membrane..........................89

4-3 N ystatin-dependent phenotypes. ........................................ .........................................90

4-4 Effect of nystatin on izh3A and the wild type strain BY4742 at different stages of
grow th ........................................................................................... 9 1

4-5 Ultraviolet spectrophotometric characterization of total A5-7 sterols in wild type
strain an d izh 3 ......... ............................ .. .. ......... ......................................92

4-6 Semi-quantitation of total ergosterol and 24(28)-DHE content in wild type and izh3A
by U V -spectrophotom etry ....................................... ..... ............................... ...............92

4-7 Quantitation of basal levels of total ergosterol and lanosterol of WT and izh3A by
GC-M S......................................................... 93

4-8 Total and free ergosterol content on WT and izh3. ................................. ...............94

4-9 Sphingoid bases override the toxic effects of nystatin. 95

4-10 Effect of ceramides and stearylamine in cells exposed to nystatin...............................96

5-1 Chemical structures of the sphingoid bases, ceramides, and sterylamine a structural
sphingoid base homolog. ............................................. .............. ..... ........ 117

5-2 Synthesis and hydrolysis of the yeast ceramides ................................. .... .................118

5-3 Overview of de novo biosynthetic pathway in yeast. ...................................................... 119

5-4 Overexpression of IZH2 and IZH3 produces an increase in the basal levels of the
sphingoid bases Cls-PH S and Cls-DH S..................................... .......................... 120

5-5 Overexpression of YPC1 and IZHs induces the increase in the levels of C1i-PHS,
C20-PH S, and C s1 -D H S. ................................................ .... ........ ........ 121

5-6 The fluorescent ceramide substrates and the fatty acid product used during the in
vitro ceram idase assay s............. .... ............................................................ ........ ........... 122

5-7 TLC analysis of in vitro ceramidase activity at different pHs ............... ................. 123

5-8 TLC-autoradiograph of radiolabeled lipids shows increased levels of PHS and DHS
when IZHs and YPC1 are overexpressed .................... ..... .............. 124









5-9 Effect of fumonisin B1 on the production of sphingoid bases ............................... 125

5-10 Effect of myriocin on the growth and the sphingolipid biosynthesis of IZH2 and
IZH 3 .......................................................... ...................................126

5-11 Overexpression of ZH2 and IZH3 produces an increase in the levels of C26-PHC and
C 26-D H C ................................................................................127

6-1 Lipid composition of the yeast lipid rafts. ......... ... ......................... ................. 142

6-2 Localization of Izh2p and Izh3p in plasma membrane ............... ............. ...............143

6-3 Izh2p and Izh3p are dissociated from lipid rafts prepared from plasma membranes......144

6-4 Localization of Izh2p and Izh3p in lipid rafts prepared from total membranes. .............145

C-l Gas chromatography-electron ionization in tandem with mass spectrometric analysis
(G C -E I-M S). ..............................................................................152

C-2 The NIST El mass spectra for the TMS derivatives of cholesterol (top), ergosterol
(m iddle) and lanosterol (bottom ). ............................................ ............................ 153

C-3 Total ion chromatograms (TICs) and electron ionization chromatograms (EICs) of
sylilated sterols. A represents wild type and B, is izh3 .............................................. 154

D-1 High perfomace liquid chromatography-atmospheric pressure chemical ionization in
tandem, with mass spectrometric analysis (HPLC-APCI-MS) of standards.................155

D-2 Atmospheric pressure chemical ionization chromatogram (APCI)-mass spectra for
th e sterol stan dard s.................................................. ................. 156

D-3 Base peak chromatogram (BPC) and electron ionization chromatograms (EICs) for
w ild ty p e ................... ........................................................................ 15 7

D-4 Base peak chromatogram (BPC) and electron ionization chromatograms (EICs) for
izh 3 ......................................................... ...................................158









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION OF NOVEL YEAST RECEPTORS IMPLICATED IN METAL AND
LIPID METABOLIC PATHWAYS

By

Nancy Yaneth Villa

December 2007

Chair: Thomas Lyons
Major: Chemistry

In the yeast Saccharomyces cerevisiae, a family of genes was identified by DNA

microarrays. These genes were called IZH1-4 (Implicated in Zinc Homeostasis) because their

expression was dependent on the zinc concentration in the cell. The IZH genes encode membrane

proteins with seven transmembrane spanning domains and three highly conserved motifs.

Muliple sequence alignments revealed that the IZH gene products (Izhs) belong to a large and

ubiquitous family of proteins that includes the progestin and adiponetin receptors (PAQRs).

Eleven members of this protein family have been identified in vertebrates, and homologues can

be found in species ranging from bacteria to humans. The human PAQRs have started to capture

the attention of researchers due to their implication in the development of diseases like obesity

and type 2 diabetes. Sequence alignment has also indicated that Izh proteins share distant

similarity with the yeast alkaline ceramidases Ypclp and Ydclp, two enzymes involved in the

sphingolipid metabolic pathway.

A first approach to the characterization and elucidation of the role(s) for IZHs is presented.

First of all, IZH2 is shown to be induced under zinc deficiency via the zinc sensor Zaplp.

Furthermore, the IZH2 gene and its homolog IZH4 respond to excess of zinc, cobalt, and nickel,

as well as to iron deficiency via the hypoxic transcription factor Mga2p. Interestingly, when









expressed in media replete in iron and zinc, the Izh2 protein (Izh2p) seems to be ubiquitinated.

However, in a medium deficient in iron or zinc Izh2p does not appear to be ubiquitinated.

On the other hand, different lines of evidence are presented that indicate that IZHs are also

implicated in lipid metabolism. In this regard, we initially show that IZH1, and IZH4, are slightly

inducted by the fatty acid palmitate, whereas IZH3 is induced by oleate. Interestingly, IZH2 is

highly induced by myristateat the transcriptional and post-translational levels.

Other lines of evidence suggesting a potential involment of the IZHs in lipid metabolism

come from the observation that mutation of the IZH3 gene produces a nystatin resistant

phenotype. Nystatin is an antifungal that interacts with ergosterol in the yeast plasma membrane.

Sterol analysis indicates that izh3A mutant presents alterations in the sterol composition.

Specifically, we demonstrate that izh3A has lower levels of free ergosterol than a wild type

strain, thus suggesting a role for IZH3 in the sterol metabolic pathway.

Despite the distant similarity with alkaline ceramidases, we could not demonstrate that Izhs

function as ceramidases per se; however when overexpressed, the Izhs induced the synthesis of

free sphingoid bases. Furtheremore, we show preliminary data indicating that the overexpression

of Izhs can result in the de novo sphingolipid biosynthesis.

Finally, strong evidence is presented indicating that Izh2p and Izh3p are plasma membrane

proteins that are associated with microdomains formed by sterols and sphingolipids called lipid

rafts. This result not only supports our hypothesis that Izhs are membrane receptors, but also it

opens new and exciting avenues regarding the role of these proteins in lipid metabolism.









CHAPTER 1
INTRODUCTION

Zinc Homeostasis

Zinc is an essential metal for growth and metabolism of yeast and higher eukaryotes (Wu

et al., 2006). Zinc is also an important catalytic cofactor for more than 300 enzymes and a

structural component of many proteins (Taylor et al., 2003; Wu et al., 2006). Because the

importance of zinc for cells, a variety of disorders have been attributed to alterations in the zinc

content. For instance, zinc deficiency is associated with impaired cell division and

differentiation, retarded growth, dysfunction of the immune system, anemia, and defects in

appetite among others (MacDiarmid et al., 2000; Wu et al., 2006). Zinc deficiency is also

associated with increased levels of lipid and protein oxidation as well as oxidative DNA damage

(Keen et al., 1989; Oteiza et al., 1995). In this regard, zinc deficiency represents a high risk

factor for cancer, and other human diseases (Ho, 2004). Notwithstanding the deleterious effects

of zinc deficiency to cells, excess of zinc is also toxic (Koh et al., 1996). Therefore, organisms

have evolved with intricate mechanisms that regulate the appropriate acquisition,

compartmentalization, and storage of zinc by the cell (zinc homeostasis).

Most of the progress in the understanding of zinc homeostasis at the molecular level,

comes from pioneering studies performed by David Eide, and coworkers in the budding yeast

Saccharomyces cerevisiae. Their findings have started to uncover the mysteries of subcellular

distribution of zinc and have revealed that zinc homeostasis is remarkably more complex than

was originally described.

A central component of metal ion homeostasis systems is the regulation of the ion flow

across lipid membranes. In this regard, lipids serve as barriers to the diffusion of charged and

hydrophobic particles. Compounds and small peptides that bind tightly to zinc may partially









shield the charge and act to ferry the ion across membranes. In eukaryotes, metal ion transport is

mediated by different families. One of them is named the ZIP family (so called for being 'Zrt-,

Irt- like proteins) because their members play prominent roles in transporting zinc and iron from

outside the cell to the cytoplasm (Gaither and Eide, 2001). ZIP transporters have also been found

to mobilize stored zinc by transporting the metal from within an intracellular compartment into

the cytoplasm. A second group of transporters, the CDF family (Cation Diffusion Facilitator),

transports zinc in the direction opposite to that of the ZIP proteins, promoting zinc efflux or

compartmentalization, by pumping zinc from the cytoplasm out of the cells, or into the lumen of

an organelle. In yeast, several members of the ZIP family have been identified. For instance

Zrtlp and Zrt2p are encoded by ZRT1 (Zhao and Eide, 1996a), and ZRT2 genes (Zhao and Eide,

1996b) (Zinc Regulated Transporter 1 and 2 respectively) and constitute the high, and low

affinity zinc uptake systems, respectively. Other characterized members of this family Zipl-

Zip4, and Irt of the plant Arabidopsis thaliana, transport zinc (and in the case of Irtlp, iron and

manganese) across the membranes of plant cells (Eide et al., 1996).

In S. cerevisiae, zinc uptake is controlled at transcriptional level in response to intracellular

zinc levels. For example, transcription of ZRT]and ZRT2 is highly induced in zinc-limited cells.

Regulation of these genes in response to zinc is mediated by the transcription factor Zapip (Zhao

and Eide, 1997). Zap p (Zinc activator protein) not only regulates the transcription of ZRT] and

ZRT2 but also, its own transcription (Gaither and Eide, 2001).

How does Zaplp activate transcription? Studies conducted by Eide and co-workers have

indicated that under zinc deficiency, Zaplp binds to a consensus sequence of 11 bp

(ACCTTNAAGGT), called ZRE (Zinc Responsive Element), in the promoter region ofZaplp-









target genes (Zhao etal., 1998) (Figure 1-1). The transcriptional regulatory effect of Zap p is

repressed in zinc-replete cells (e.g. 10 [LM ZnCl2).

The Budding Yeast Saccharomyces cerevisiae as a Model System

Saccharomyces cerevisiae is a unicellular and non-pathogenic eukaryote whose genome

was completely sequenced in 1996. Because it is a close relative of pathogenic fungi such as

Candida albicans and Candida glabrata (Figure 1-3), and the fact that it shares many

homologous genes with higher eukaryotes, this microorganism is a powerful tool to study

biochemical events that can further be extrapolated to more sophisticated and evolved systems.

In this regard, yeast combines a well-described biochemistry and ease of genetic

experimentation. These, characteristics are very useful for mapping out complex signal

transduction pathways (Chung and Obeid, 1999). Furthermore, metabolic pathways that are

poorly understood in higher eukaryotes, including mammals, are more easily understood as a

result of studies performed in S. cerevisiae

Identification of IZHs on the Saccharomyces cerevisiae Genome

The fact that single and multiple deletions of the zinc transporter genes ZRT1, ZRT2, and

ZRT3 were not lethal for the cells, suggests, at first glance, that these genes are not essential for

cell viability, and suggests the presence of other genes with overlapping functions.

Because the above mentioned genes are Zaplp target genes, the ZAP] regulon was screened by

DNA microarrays in order to identify new Zaplp-target genes (Lyons et al., 2000). The results of

this study revealed 46 new genes as potential Zaplp targets. Two of these genes, YDR492w and

YOL002c contain a putative ZRE consensus sequence in their promoters and were highly

expressed under zinc-limitation via Zaplp (Lyons et al., 2000; Lyons et al., 2004). YOL002c,

and a third homologous gene YOL101c, were induced by high levels of zinc, (Lyons et al.,

2004). A fourth homolog YLR023c, discoved by sequence alignments, revealed phenotypic









effects on zinc tolerance (Lyons et al., 2004). Due to their transcriptional metalloregulation, zinc

related phenotypes, and highly conserved motifs containing potential metal-binding residues, this

novel family of genes was renamed like the IZH family (Implicated in Zinc Homeostasis), and its

members were named as follows. IZH1 (YDR492w), IZH2 (YOL002c), IZH3 (YLR023c), and

IZH4 (YOL101c) (Lyons et al., 2004).

Regulatory Sequences Surrounding the IZH Genes

In the promoter regions of some of the IZHs, a group of regulatory sequences have been

identified by using RSA-TOOLS (http://rsat.ulb.ac.be/rsat) (Hertz et al., 1999). First of all, the

promoter regions of IZH1 and IZH2 have a zinc responsive element sequence (ZRE) (Figure 1-4

A and B, respectively).

On the other hand, IZH2 and IZH4 have in their promoter regions a putative low oxygen

response element (LORE), (Figure 1-4 B and C, respectively). Under hypoxia (low oxygen) or

conditions that mimic hypoxia such as excess of cobalt and nickel, the transcription factor

Mga2p regulates the transcription of target genes via the LORE. It has been proposed that the

mentioned transition metals may disrupt the production of reactive oxygen species which may be

important signaling molecules in the oxygen response pathway (Huang et al., 1996). Lyons et al

2004, demonstrated that not only cobalt and nickel, but also excess of zinc can mimic hypoxia

and induce the transcription oflZH2 and IZH4. Therefore, these two genes have been proposed

to be part of the hypoxic response (Lyons et al., 2004).

Another speculative regulatory element known as Oleate Response Element (ORE) has

been detected in the promoter region of IZH1, IZH2 and IZH4 (Lyons et al., 2004) (Figure 1-4 A,

B, and C, respectively It has been found that in the presence of fatty acids like oleate, the

transcription factors Oaflp and Pip2p form a heterodimer that binds to the ORE consensus

sequence in the promoter of target genes (Einerhand et al., 1993).









The binding of Oaflp and Pip2p to ORE is the proposed mechanism by which both

transcription factors regulate the fatty-acid dependent gene activation (Karpichev et al., 1997;

Kapichev and Small, 1998). Interestingly, despite the presence of ORE in the promoter region of

IZH2, this gene is transcriptionally regulated by exogenous myristate (a saturated fatty acid of a

carbon chain of 14 carbons, C14:0) (Karpichev et al., 2002; Lyons et al., 2004). In this regard,

the results shown in this dissertation contribute to expand our knowledge with the finding that

IZH1, IZH3, and IZH4 are also regulated by fatty acids at transcriptional level. The presence of

different regulatory elements in the promoter regions of the IZH suggests, at first glance, a dual

role for these genes in metal ion homeostasis and lipid metabolic pathways.

The IZH Gene Family Encodes Membrane Proteins

The IZH gene family encodes membrane proteins with predicted seven transmembrane

spanning domains (7TMs), and highly conserved motifs (Figures 1-5 and 1-6), which are

summarized as follows: (i) a long motif N-terminal to TM1 that generally resembles

PxnGYRxnNEX2Nx2T/SH; (ii) an Sx2Hx2S motif at the C-terminus of TM2; (iii) a DX9GS

motif at the beginning of TM3; (iv) a Px2H motif in TM5 where the H residue is generally only

conserved in higher eukaryotes; and (v) the loop between TM6 and TM7 containing PER/KxnPG

and Hx2F/WH motifs with a conserved histidine in the middle of TM7 being most common.

Furthermore, IZHs encode proteins that belong to the ubiquitous and recently discovered family

of membrane protein receptors and named as PAQRs (Progestin and Adipo Q Receptors).

Multiple sequence alignments (Figure 1-6) (Lyons et al., 2004), as well as phylogentic analysis

(Figure 1-7) performed for the four yeast proteins, revealed a high similarity between these

proteins and the PAQRs. Like the Izhs, PAQR genes encode proteins with seven transmembrane

domain proteins.The topology model presented in Figure 1-5 has been confirmed for the yeast

Izh2p and Izh4p (Kim et al., 2003), as well as the human PAQR1 and PAQR2 (Zhu et al., 2003).









.Because Izhs are membrane proteins with seven transmembrane domains, it has been we

have proposed that the Izhs are membrane receptors. Interetingly, phylogenetic analysis has

revealed that some members of the Izhp family such as Izhlp, Izh2p, and Izh3p have

homologous proteins in other fungi, some of which are very pathogenic (Figure 1-3). Since S.

cerevisiae is a non-pathogenic fungus, investigating the role of these proteins can shed light into

the mode of action of pathogenic fungi.

The PAQR Family of Proteins

Even though PAQRs are widely dispersed throughout eubacteria and ubiquitous in

eukaryotes, very little is known about PAQR genes from any of these organisms. In fact, the first

published characterization of a member of the PAQR protein family was of a protein cloned

from the opportunistic pathogen Bacillus cereus. This protein, when expressed in E. coli,

conferred hemolytic activity onto the host strain, and therefore, it was named hemolysin III,

(Hly3) (Baida and Kuzmin, 1995). A subsequent study suggested that Hly3 was capable of

inducing large pores in erythrocyte membranes on the order of 30-35 A, in diameter (Baida and

Kuzmi, 1996). Rehli et al., 1995 reported that the gene PAQR11 was highly induced during

differentiation of human macrophages from monocytes. The gene product, PAQR11, was

postulated to function as a receptor based on the fact that, like G-protein coupled receptors, it has

seven predicted transmembrane domains. However, no sequence similarity with known G-

protein coupled receptors was found (Rehli et al., 1995). The next relevant published finding was

the discovery of a PAQR-like gene in the a-proteobacterium Azospirillum brasilense. In this

case, the PAQR encodes a chimeric protein that consists of an N-terminal Hly3-like domain

fused to a C-terminal domain that is identical to the CheA hisidine kinase involved in chemotaxis

(Hauwaerts et al., 2002).









In humans, the PAQR family of genes encodes membrane protein receptors, which

mediate hormonal signaling (Hsieh et al., 2005). For example, PAQR1 and PAQR2 (also called

AdipoR1, and AdipoR2, respectively) function as receptors for the insulin-sensitizing hormone

adiponectin (AdipoQ), (Yamuchi et al., 2003a). Adiponectin is an adipocyte-derived

proteinaceous hormone related to tumor necrosis factor (Shapiro and Scherer, 1998) that acts as

an anti-diabetic and ant-atherogenic adipokine by enhancing insulin sensitivity (Yamauchi et al.,

2003b). In fact low circulating levels of AdipoQ are associated with type 2 diabetes, obesity and

coronary artery disease (Shimada et al., 2004). In a separate study the human PAQR5 (mPRy),

PAQR7 (mPRa), and PAQR8 (mPR3) proteins were identified as membrane receptors for the

hormone progesterone (Zhu et al., 2003a). These receptors transmit rapid and non-genomic

steroid signals (Zhu et al., 2003b). Interstingly, adiponectin and steroid receptors are part of the

same family, which remains to be mystery since they recognize hormones that are highly

different structurally.

Izh2 is an Osmotin Receptor Protein

Osmotin is a secreted polypeptide found in the tobacco leaves, which is closely related to

the natural sweetener thaumatin (Veronese et al., 2003). Both proteins belong to the PR-5 family

of plant defensins. Unlike thaumatin, osmotin is a potent antifungal that induces apoptosis in

Saccharomyces cerevisiae by signaling suppression of cellular stress responses via RAS2/cAMP

(3',5'-cyclic Adenyl Mono Phosphate) (Narasimhan et al., 2001). The antifungal activity of

osmotin is dependent on the fungal cell wall composition (Veronesse et al., 2003). For example,

while glycoproteins repress the osmotin action, phosphomannans enhance its toxicity (Yun et al.,

1997). In addition to this, the integration between osmotin and specific plasma membranes

receptors, is required for the osmotin antifungal activity (Narasimhan et al., 2005). Recently, it









was reported that the overexpression of the yeast Izh2 protein mediated the apoptotic effects of

osmotin via a RAS2 signaling pathway (Narasimhan et al., 2005). A possible mechanism by

which Izh2p mediates cell death involves the interaction of osmotin with Izh2p. This interaction

activates RAS2/cAMP pathway with the concomitant suppression of stress responses and the

subsequent accumulation of reactive oxygen species and cell death (Narasimhan et al., 2005).

Therefore, Izh2p has been recognized as an osmotin receptor (OsmoR).

The IZHs and their Connection with Lipid Metabolic Pathways

The first insight about the role of the IZHs in a pathway different from metal homeostasis

was postulated by Karpichev et al., 2002. In that report, the YOL002c (IZH2) gene was shown to

be highly induced in cells grown in the presence of myristate (C14:0) as the sole carbon source.

In contrast, mutations of this gene produced defects in the growth of cells were exposed to

myristate. This observation suggested a regulatory role for IZH2 in lipid metabolism. During

those studies, it was also found that the mutation of IZH2 (izh2A) produced a resistant phenotype

against the antibiotic nystatin. Because nystatin preferentially binds to ergosterol in the yeast

plasma membrane (Lees et al., 1995), it was proposed that the izh2A mutant has alterations in

sterol composition. Furthermore, Cherry et al., 1998 reported that the IZH3 transcription is

induced and the IZH4 transcription is repressed by defects in the ergosterol biosynthetic

pathway. These observations, combined with the fact that some Izhp vertebrate orthologs (e.g.

membrane progestin receptors) function as a receptors for structurally related steroids, identify

ergosterol metabolism as a likely biochemical pathway in which to place the IZH genes and

consequently the Izh proteins.

Besides sterols, the yeast membranes contain sphingolipids. In the yeast plasma

membrane, sterols like ergosterol and sphingolipids are the main components of the detergent-









resistant microdomains termed lipid rafts (Pike, 2003; Schnabl et al., 2004). Rafts are conceived

as platforms that mediate the sorting of proteins, and are implicated in membrane trafficking

pathways like endocytosis and exocytosis (Lawrens and Andre, 2006). Interestingly, the

sphingolipid pathway is also directly connected with other metabolic pathways, including fatty

acid synthesis, elongation and sterol metabolism (Sims et al., 2004). Furthermore, as mentioned

before, multiple sequence alignments and phylogenetic analysis have also revealed that Izhs (or

yeast PAQRs) have distant similarity to a family of yeast membrane proteins known as alkaline

ceramidases (Figures 1-6 and 1-7).

Ceramidases are enzymes that catalyze the deacylation of ceramides to generate sphingoid

bases and fatty acids (Figure 1-8). Ceramide and its metabolites act as signaling molecules for

apoptosis and proliferation. Therefore, the ceramide/sphingoid base ratio is considered to be a

'rheostat' that governs these events. Three types of ceramidases have been identified and

classified as acidic, neutral, and alkaline according to their optimum pH (Mao et al., 2000a; Mao

et al., 2000b). Acidic ceramidase is localized in lysosomes and is primarily responsible for

catabolism of ceramide. On the other hand, neutral and alkaline ceramidases have been

implicated in signal transduction and cell regulation (Merrill et al., 1997; Dickson, 1998).

Thus far, two alkaline ceramidases, Ypclp and Ydclp, have been cloned and characterized

in S. cerevisiae (Mao et al., 2000a; Mao et al., 2000b). Although Ypclp catalyzes the

deacylation of phytoceramide to phytosphingosine, Ydclp catalyzes the deacylation of

dyhydroceramide to dyhydrosphingosine. In this dissertation, we have established a connection

between IZHs and the fatty acid, sterol and sphingolipid metabolic pathways.

Structurally, sphingolipids are defined and distinguished by the presence of a sphingoid

base backbone. In mammalian cells, this is usually C18-sphingosine, whereas in yeast cells, this









sphingoid backbone is C18-phytosphingosine (Figurel-8). Other variations, in yeast include C20-

phytosphingosine, C1i-dihydrosphingosine, C20-dihydrosphingosine, and a variety of other

hydroxylated analogs of Cis-phytosphingosine. Collectively, sphingosine, dihydrosphingosine,

phytosphingosine, and related long-chain amino bases are also termed as sphingoid bases. The

next building block in the sphingolipid structure is ceramide, (or phytoceramide in yeast).

Ceramide is derived from sphingosine by the acylation at the 2-amino position by a fatty acid of

varied carbon chain length; with C26 being the most abundant fatty acid found in yeast

ceramides. Ceramide is the backbone of more complex sphingolipids. S. cerevisiae has three

complex sphingolipids, inositol phosphoceramide (IPC), manosylinositol phosphoceramide

(MIPC) and manosyl di-inositolphosphoceramide (M(IP)2C) (Figurel-8). Ceramide is at the

center of the sphingolipid pathway regulating the synthesis of various sphingolipids. In addition

to this, ceramide is considered the point where animal and fungal sphingolipid biosynthesis

begins to diverge.

Summation

This research entails the study of the IZH family of genes in the yeast Saccharomyces

cerevisiae. Investigating the role(s) of this family of genes has been one of our main aims. Based

on our observations, we propose that IZHs have a dual role in metal and lipid metabolic

pathways. Different lines of evidence that support these hypotheses are addressed and discussed

herein. In Chapter 2, we show the transcriptional and post-translational responses of some of the

IZHs by several metals such as zinc, iron, cobalt and nickel. In Chapter 3 we demonstrate that the

expression of some of the IZH genes and proteins are also regulated by exogenous fatty acids at

transcriptional and translational levels, respectively. Genetic and biochemical studies suggest

that IZHs can also be implicated in the sterol metabolic pathway (Chapter 4) as well as in the

sphingoilid pathway (Chapter 5). In Chapter 6, the localization of the proteins Izh2p and Izh3p in









plasma membrane and in the detergent-resistant microdomains termed lipid rafts supports our

hypothesis that some members of the Izh family are implicated in metabolic pathways such as

sterols and sphingolipids. This investigation is just the starting point to understand more

complicated metabolic pathways in higher eukaryotes.

In this dissertation, and according to the yeast nomenclature, a gene is reprented by IZH. A

protein encoded by the IZH gene is called Izhp, or Izh protein. A single mutation of the IZH gene

is referred to as izhA or simply izh, and a wild type strain is in some cases represented like WT.









S[Zn2+]
zinc deficiency


R I Zapip-target genes


Figure 1-1. Zaplp activation is zinc dependent. Under zinc depletion, Zaplp binds to a zinc
response element (ZRE) consensus sequence in the promoter region of target genes
inducing their expression.


.....-.-. ..Cell wall
ET4
R \
Plasma RT1
membrane \ Zn2+ cytoplasm

mitochondria
Golg'
RT3



Zn2+ E

Vacuo e



nucleus


Figure 1-2. A common budding yeast Saccharomyces cerevisiae cell, and some of the most
important and well characterized zinc transporters.















n. m--- 7K a
s. ay.ns- Izh4p
S. casteli 0
S. cwvazzi 0
a --C.otebm 0
S, crevisae 0

E. goossvpJ 0 Izhl p
t- K. Facts O
|C. glraO10
X tacts 0
S. cerevis'a 0 Izh2p
E. Qos^'p 0
C, lu-StaniNe 0
C. afbicansO
-wsemifW 0
Y ipo y ytica o
A. Aumirgaus 0
C, Imilts 8
A, capsulfus 0
C gsab oso Izhi/2p
N. crassa e
-G. zoe W
S, pombe a
C, cERlea 0
P. chrysosporiumn
C. meofarmans 0
U. maydisO
P blakesteeanus 0

C, Imi/s -
A. fumigatus 0
C. globosum 6
N. cressae
G. zeae a
S. pombe 0
R. oryra O t
S-R. Oryae-ab 0
P. blakesleeanus-a
P. blakesBeanus-b g
S. cerevisie 0O
C. lbabr O Izh3p
E. gossypnxe
Iactds 0
C. alfbicans O
C. Iusiteanie o
D. haoenetle
Y lipoayltca 0
R chrysosporit m
-- C. .lnerea a
U. MaydIs O

-D d.iscoidoun-a (q
D, discoideum-b 0


Figure 1-3. Phylogentic analysis of the Izhp family and its closer and more distant homologues
in yeast.










5' 5'
3' 3'




LORE ZRE3'

3' -E 3'
5' 5'


Figure 1-4. Regulatory elements surrounding IZH1, IZH2 and IZH4 genes suggest dual role in
metal and lipid metabolism. In the figure, ZRE stands for zinc responsive element,
LORE is the low oxygen response element, and ORE is oleate response element.


Cytoplasm



SxxxHxnD
PxnGYRxnExxNx3H S H


PEx3PGxnH xxH


Conserved motifs


Figure 1-5. Predicted topology for the Izhp family. Izh are membrane proteins with seven
transmembrane spanning domains and four highly conserved motifs facing the
cytoplasm (black circles in the figure). The C-terminus is proposed to be outside
whereas the N-terminus is inside the cell, specifically in the cytoplasm.














TM1


Ms PAQRAipoR..... L CVB
sc tA p........... PAr CrP
Sc lhZp.............. .IPALOrr
PAQRs LP.OR '............"II >B
Sct l3p ............... z Al F
Ms PJoRJ . . 1 1 r11LGFFLr
,s PAQRnmPR ......... I r .Lp F r rA
MsPAQR&mPRI. LMBPI.. L PTWFL
HP PAQR&mPR,. I. I I L LPL
Ha PAQRS ........... LAMLVLL
Ms PAQRP ....... .. LAALAVL
Ms PAQORlfMMDP-D ...... BHAIJ iFW[ IPS L
M PAQRII'MMDI ...... E FLTVPAT
Cperfrrngwrs (rtsfo mf.. T TGOVVLIT
S yp'rim.nUm ....... y ILIPGT
HemolysinsI sn,,sM ..." ....... ,-sz, cl L
e l.ol ........ .... E, TOLr POT
is cOIus ffsof'D k'. .. GcOA, F
C pwerrngsror m `50r2 1 f AILSI
HISPHCA ... .. TMITPPM
ScYcplp ..... .iiii vrPILSAT
Sc Yflp ........ HI lLKTAr
Ceramidases HiSI-DCaB ........ KI LPFILPP
MsASAM3 ..... I rFIreGr
M dgradn.....P.. P V FLLY6A
M.capuutu ...... PI I prLAPCL
HI As3. ............ TATVtg*TRAQ tLLTL
Ms NAG-5........... .DALTIGrQLLSTLLLCL


TM2 TM3
V~vrcKavr VKNZBLQQaLI B.aRr SHINLLI
AVLCLB II VICH5EFVR. TFS LD!T UIALLII
&ILCLB II TCBSIGVBI .. L. I LD GIALLIK
AFSCLK PICMKHBEFQS FMS LDT UZIBLIB
ArACLI MLRSSLZIATn....L65 LG D GICILIV
CLAPPAa FrMCEQO8AIAWA RLL L COUCLVT?
ALCKL VF rFGISFLALRB..KFACDE ULTILIT
rQCKL rI FCHSEJTCR RIWLD LO ISIG8 L
SCUzLL F FbMSALEAEIM. ICK LD AUNLFbL
ACLrVa C r SMSPSNRs CIc LD ALBLISL
VLLTPA T rSCLSLWLBA. AFF LD BISTTOF
SFTrLB I LLOAKSKrwrB. sFr L %WNvrvi0r
SITrLC LLQS SELSZIr T FIFr osvr
LCGLFPV L ITIMKKSHLRNVEBCLEMI IrrFFIA
LCALFI ZVSMIKKBMLRTAEICFEM D VETYIIA
LLLTL ISIPNEVKR- .....PLRI D SITLLIA
KILLPL r IPHEKRN .....LD WLKD TYLLIJ
LLrLtPL IPROEMI. LR V ATYLTII.
HNVLft I VANVVNEvI. ....PPFRILDI NIiLIA
LVLLS TPIL VMAKEnTRI. -....PLRILD NIIrLIC
LTVVO L T .... ...R iLD PLTCC
TLVOVC l TLKT-......R..P.R LD P IYKC
FBLVOI 1r TLQT HT I LD P LITA
LVVUGI 'lILSr.... LGUI LDI L AVLVLK
NIiTLGL I18r. LOO; LD AILWLLS
VLrUGL Bi LFH.. ... .. TLVY PMPIGPrr
VALVAP II LMP DN.... ATLV PhTVAPM
rFIAY P ACDQPO-AVLCIL. YD LLQ DFLGSGA
TTFTH M ACDQPUIVVPCIM.DTD L CDPLGSLM


TM7
B r LLISTAFP
Bg iP( LAAAF
LFIiPrkOAP
DgLPIMUihGBI
P LMLGBV
LYV AAL
I I V. rSTLOI
SPYuuLOVIl
gI TILIVVMLTWM
.F1 CUI.LAM)
QLFI CIVLGOT
LF LrTPLSIi
LF IFLVLCTL
liP r LISTCTLSL
AR ILFUAFOACI
AIIM LFVAIAAAUI
INE FrVI4GSR&
AII FYLOOGGSY
IN PVYLOOsvc
lI LLGiLL
N FI ZILLGSL
I LTOLOST
ZILTGeMXGTr

ILICL&AILC
BII LGTTFPTYa
S TALCTOLP
A LAALGVTVF
.I, ILLAGBAALL
I SIN LTAGSBVPL


Figure 1-6. Sequence alignment of important conserved regions in PAQRs, hemolysins and

ceramidases. The alignment was performed using CLUXTALX. Predicted

transmembrane segments (TMs) are indicated by solid bars. Highly conserved

regions are highlighted in grey.


Bacterial
ceramidase- M1 3
Ceramidase like NAG-5
sufamily subfami

II


j s rl i V |I
00u 3| I Is I II S


















Figure 1-7. Phylogentic showing the Izhp family, its closer and more distant homologues in
yeast, and other organisms.







Figure 1-7. Phylogentic showing the Jzhp family, its closer and more distant homologues in

yeast, and other organisms.


Progeslird
Adiponectin
receptor
subfamily


,I -7,


Hemolysin 3
subfamily












Serine + Palmitoyl-CoA

LCB1I SPT
LCB2 serinee pamiltoyl transferase)
TSC3
LCB4 Phosphoryl
LCB5 DPLI Ethanolamine
3-keto-dihydrosphingosine Dihydrosphingosine Phyto)sphingosine PHS-Pi +
TSCIO (DHS) (PHS) YSR3 palmitaldehyde
I LCB3


LAGI/LAC1
Ceramide synthase YDC1

Dihydroceramide
(DHC)


YPCi Il LAG1/LAC1/YPCI
Ceramide synthase

(Phyto)ceramide
(PHC)

SCS7(desaturase)
AUR1 (IPC synthase)

Inositol phosphorylceramide
(IPC)


AUR1 (Inositol phosphorylceramide
SCG2 mannosyltransferase)


Mannosylinositol phosphorylceramide
(MIPC)


(Inositolphosphotransferase)


Mannosyldiinositol phosphorylceramide
(M(IP),C)


Figure 1-8. The Sphingolipid biosynthetic pathway in the yeast Saccharomyces cerevisiae.









CHAPTER 2
METALLOREGULATION OF IZHS

Introduction

Elucidating the role of genes of unknown function is sometimes a difficult task; especially

if very few precedents exist that mark the route to follow. This chapter constitutes a first

approach to determine the role of a group of genes of unknown function termed IZHs.

The catalyst for this chapter were pioneering studies conducted by David Eide and co-

workers, in the yeast S. cerevisiae, which revealed the existence of a complex system,

orchestrated by different gene products that regulate the homeostasis of zinc and iron in the cell.

Part of this system includes the IZH family of proteins (Lyons et al., 2000). Two members of the

IZH family, IZH1 and IZH2, were found to be highly induced under zinc deficiency via the

transcription factor Zaplp. Interestingly, the expression oflZH2 and its homologue IZH4 was

induced by excess of zinc in a Zaplp-independent manner.

Overall, the results presented contribute to our understandings about metalloreuglation in

the yeast S. cerevisiae. The fact that IZH2 is positively affected by two opposite effects (zinc

deprivation, and excess of zinc), opened the possibility to explore new avenues in which the

IZHs could function. In fact, excess zinc, cobalt and nickel, as well as iron chelation, can

modulate the expression of genes via the hypoxia sensor Mga2p (Lyons et al., 2004;

Vasconcelles et al., 2001). As mentioned in Chapter 1, under hypoxia (low oxygen), or in the

presence of toxic metals, the transcription factor Mga2p is believed to bind a low oxygen

responsive element (LORE) in the promoter regions of Mga2p-target genes. The presence of

putative LOREs in the promoter regions of ZH2 and IZH4 led us to speculate that these two

genes can be Mga2p-target genes. With this in mind, evidence supporting the regulatory effect

exerted by certain transition metals on the transcriptional response of ZH2 and IZH4 is









presented. Additionally, we show that the response of IZH2 to metals like zinc and iron also

occurs at post-translational level, and that such a response is dependent on the cellular metal

status.

We confirm that the transcriptional response of IZH2 under zinc deficiency is Zap 1-

dependent, and that IZH2 is a Zaplp-target gene (Lyons et al., 2004). Also, we show that IZH2

and IZH4 are Mgalp-target genes (Lyons et al., 2004).

Besides the effect exerted by certain metals on the transcriptional activation of ZH2, we

show that zinc and iron regulate the accumulation of the Izh2 protein. Indeed, under zinc or iron

deficiency, the expression of Izh2p was strongly observed. By contrast, growth in a medium

replete in either iron or zinc decreases the expression of the Izh2 protein. Furthermore, we

present evidence suggesting that Izh2p seems to be ubiquitinated in medium that is iron or zinc-

replete.

Taken together, the results presented in this chapter constitute a pivotal piece of evidence

implicating two members of the IZH family in metalloregulation. Furthermore, our results match

with the idea that in some cases, transcriptional and translational responses are directly related.

Materials and Methods

Yeast Strains and Plasmids

The yeast strains used in this study are listed in Table 2-1. Those strains were obtained

from two different sources, (i) EUROSCARF ( http://web.uni-frankfurt.de/fb 15/mikro/euroscarf)

and (ii) from Dr. David Eide yeast collection (eided@missouri.edu).The IZH promoters were

fused to a lacZ reporter gene as follows. PCR-amplified genomic fragments from approximately

1,000 bp upstream to ATG were inserted by homologous recombination, into the episomal

YEp353 vector, and between the EcoRI and BamHI sites (Lyons et al., 2000). Resultant IZH-

lacZ promoter reporter fusions were used to perform 3-galactosidase assays.









For protein expression, the C-terminus of the Izh2p was tagged with the triple

hemagglutinin epitope (3xHA). The HA-epitope tag is a peptide from human influenza

hemagglutinin protein, which has the amino acid sequence, YPYDVPDYA (Mo et al., 2001).

The tagged construct was generated by Dr. Thomas Lyons, according to published procedures

(MacDiarmid et al., 2002). Briefly, the ZRC1 promoter and open reading frame in the

YCpZRC1-3xHA plasmid were exchanged with those of IZH2. This was accomplished by gap

repair of Age 1-digested YCpZRC1-HA to generate the plZH2-3xHA. This construct has the

IZH2 gene driven by its native promoter and retains the ZRC1 terminator sequence. To provide a

galactose inducible construct, the native IZH2 promoter was exchanged with the GAL] promoter

using gap repair of plZH2-3xHA plasmids previously cut with EcoR1. The resultant plZH2-

3xHA plasmid contained the GAL] promoter to drive protein overproduction. In both cases, the

tagged or untagged protein is overexpressed using the galactose inducible promoter (GAL]).

Yeast Media

In this chapter, 3-galactosidase assays and protein expression were performed using

different types of media, which are briefly described as follows. While all the P-gaslactosidase

assays were performed in a medium that uses glucose as a carbon source, protein expression was

carried out in either a growth medium supplemeted with glucose or galactose. Synthetic medium

supplemented with either glucose (also called dextrose), or galactose, as a carbon source (SD or

SGal, respectively) was used as a medium that contains all metals. To limit either zinc, or iron

availability, or both, chelexed synthetic medium supplemted with either dextrose (CSD) or

galactose (CSGal) was used according to Lyons et al., 2004. Finally, to fully limit availability of

iron, low iron medium (LIM) was used according to Kupchak et al., 2007. LIM was supplement

with an aprropriated carbon source.









Each growth medium was prepared as follows (Apendix A for more details). One liter (1

L) of synthetic medium was prepared by dissolving 1.7 g Yeast Nitrogen Base (YNB) without

amino acids and ammonium sulfate (Fisher). This medium was then supplemented with 5 g of

ammonium sulfate (Fisher), 2% alpha (+) glucose (99%, anhydrous), or 2% D (+) galactose

(Across-Organics), and 0.01% of appropriate amino acids (Sigma). For P-galactosidase assays

under excess of metals like zinc, cobalt, and nickel; synthetic medium (SD medium) was

supplemented with ZnCl2 to a final concentration of 3 mM (excess), whereas CoC12 and NiC12

were added to a final concentration of 400 [LM.

One liter of CSD medium was prepared by dissolving, 20 g of dextrose, 5.1 g of YNB

without divalent cations, amino acids, ammonium sulfate, and phosphates (Qbiogene), and 0.1 g

of appropriate amino acids, into sterile nano-pure water. Chelex-100 ion exchange resin (25 g)

from Sigma was added, and the culture was stirred overnight at 40C. After removal of the resin,

the pH was adjusted to 4.0 with HC1, and the following were added to recommended final

concentration: MnSO4, CuSO4, CaC12, MgSO4, and KPO4 monobasic (Appendix A, for details),

nano-pure water was added to 1 L. This medium is devoid of iron or zinc. To generate medium

that is replete in these metals either zinc or iron was added back to CSD to a final concentration

of 10 [tM repletionn). The solution was then filter-sterilized into polycarbonate flasks. Before

being used, all plastic used for CSD media preparation and cell culturing was washed with

Acationox detergent (Baxter Scientific Products, McGaw Park, IL).

One liter of LIM was prepared by dissolving, 1.7 g of YNB without amino acids and

ammonium sulfate (Fisher), 20 g of glucose (or galactose) (Acros Organics), 20 mL 1.0 M

sodium citrate (pH 4.2), and 5 g of ammonium sulfate. This medium was then supplemented with

0.1 g of appropriate amino acids, as well as 1.0 mM EDTA, at pH 8.0. MnC12 was added back to









LIM to a final concentration of 20 [tM, and ZnSO4 was added to a final concentration of 0.8

tlg/mL (or 5 [tM). Iron deficiency and iron repletion were generated by adding either, 1 [tM or 1

mM FeC13, respectively. The solution was then filter sterilized into polycarbonate flasks.

Yeast Transformations and Assays

Yeast transformations were performed to introduce plasmids into appropriate strains. To

do this, single colonies of each strain were grown for 1 overnight (e.g. 12-18 h) in 1X YPD to

saturation. Aliquots of the overnights were inoculated in fresh 1X-YPD and grown to OD600 of

1.0 to subsequently being used for yeast transformations. Yeast transformations were performed

by using standard procedures and using the lithium acetate method (Gietz and Woods, 1994)

(Appendix B).

Promoter reporter activities were measured by using 3-galactosidase assays, which are

spectrophotometric assays. In this type of assays the enzyme P-galactosidase, which is encoded

by the Escherichia coli lacZ gene hydrolyzes the synthetic chromogenic substrate o-nitrophenyl-

P-D-galactopyranoside (ONPG), generating o-nitrophenol, which is yellow in aqueous solution.

The course of this reaction is followed by monitoring absorbance at 420 nm (Lederberg, 1950).

For yeast, the P-galactosidase assay is performed by permeabilizing the cells with 0.1% sodium

dodecylsulfate (SDS) and chloroform (1:1, v/v), and then by suspending the cells in 3-

galactosidase assay buffer, pH 7.0. After incubation with ONPG for a period of time, the reaction

is terminated with a solution of 1 M Na2CO3 and the OD420 is measured.

Before each assay, cells were inoculated from overnight cultures (OD600 = 3-4) into the

appropriate media to an initial OD600 of 0.1. Cells were grown at 300C to mid-log phase (OD600

of 0.5). P-galactosidase activity was assayed as described by Guarente et al., 1983, and is

expressed in Miller units. The activity was calculated as follows: (A420 x 1000) / min x mL of









culture used x culture A600). Results for experiments are reported are the product of three

independent samples; each experiment was done in triplicate.

Preparation of Microsomes

Microsomes were prepared according to the procedures described by Gable et al., 2000;

Mo et al., 2002 with some minor modifications as follows. Cells in early exponential phase of

growth (OD600 of 0.8) were centrifuged for 5 min, at 3,000 rpm and 4C. Pellets were washed

twice with sterile cold water, and re-suspended at 2 mL/g wet cell weight in mitochondrial

isolation buffer (MIB), which is composed of 0.6 M mannitol, 20.0 mM HEPES-KOH, pH 7.4,

1.0 mM EDTA, pH 7.5, and the protease inhibitors, 1.0 mM phenylmethylsulfonyl fluoride

(PMSF), and 2 [tg/mL of pepstatin A (Sigma) was prepared according to published procedures

(Gitan and Eide, 2000). Glass beads (acid-washed glass beads, from Sigma) were added to just

below the meniscus (approximately 250 [tL), and cells were lysated by six cycles (1 min each

time) of vortexing with cooling on ice (1 min) between each cycle. Unbroken cells, beads, and

debris were removed by centrifugation at 3,000 X g, 4C, for 10 min. The low speed supernatant

was then ultra-centrifuged at 130,000 X g (45,000 rpm), for 90 min using a Beckman 75 Ti rotor,

to provide the microsomal pellet. Microsomal pellet, which is enriched in membrane proteins,

was then resuspended in 200 p.L MIB buffer supplemented with 15% glycerol.

Western Blot Analysis of Protein Expression

Protein concentration was determined by the BCATM protein assay (Pierce) using bovine

serum albumin (BSA) as standard. Samples were suspended to 1 mg/mL in 1X SDS-PAGE

loading buffer, prepared by mixing 0.0625 M Tris-HC1, pH 6.8, 2% SDS, 10% glycerol, 1% 3-

mercaptoethanol, 0.001% bromophenol blue, and sterile water. Protein suspensions were warmed

at 37C for 30 min followed by centrifugation at 12,000 rpm during 50 sec. Equal concentrations









of protein (22 |tg per lane) were loaded onto a 10% SDS-PAGE gel followed by Western blot

analysis.

Western blot was performed following standard procedures (Sambrook and Russell, 2001)

as follows. After separation in an SDS-PAGE gel, proteins were transferred to a polyvinylidene

fluoride membrane (PVDF) (from Millipore) at 80 volts, 40C, and during 1 h using a tank

transfer chamber (Bio Rad). Blots were then washed three times, 5 min each time with 1X Tris-

Buffered Saline solution, (TBS), pH 7.4, (TBS is 20 mM Tris-base, 500 mM NaC1, pH 7.4)

supplemented with 0.05% Tween 20 (Fisher), followed by blocking in IX TBS-Tween 20,

supplemented with 5% nonfat milk, for 1 overnight, at 40C and with constant agitation.

For detecting Izh2p-3xHA, blots were probed for 2 h, with the primary antibody rabbit

polyclonal IgG anti-HA at a dilution of 1/500 (HA-probe (Y-11): sc-805, from Santa Cruz).

After washing three times (5 min each time) with 1X TBS-Tween 20, blots were probed for 1 hr

at room temperature with the secondary antibody, horseradish peroxidase-conjugate goat anti-

rabbit IgG obtained from Santa Cruz (at a dilution of 1/10000, v/v). The bound antibodies were

detected by the ECL Western blotting detection system and using the super signal west pico

chemiluminescence kit (Pierce), and exposing a CL-XPsureTM film (Pierce).

Immunoprecipitation of Izh2p and Western Blot Analysis

The microsomes were solubilized at 1 mg/mL with 2 mM sucrose monolaurate, > 97%

TLC grade, (Fluka) for 20 min at room temperature. After centrifugation at 33,500 rpm for 30

min in a 75 Ti rotor (Beckman Coulter), the supernatant (containing the solubilized microsomes)

was collected. Soluble microsomes (100 [tL) were incubated with 20 p.L of mouse monoclonal

IgG anti-HA with agarose beads-conjugate [HA-probe (F-7): sc7392 AC, Santa Cruz], as the

immunoprecipitated antibody, for one overnight, at 40C and with constant agitation. Suspension









was spun down at 1,000 rpm for 50 sec, and the resultant precipitates were washed three times

with 500 ptL of cold IX Phosphate Buffered Saline (IX PBS), pH 7.4 (Fisher). Proteins were

eluted by re-suspending in 60 ptL 1X SDS-PAGE loading buffer. SDS-PAGE protein separation

and subsequent Western blot analysis were performed in identical fashion as described in former

section.

To investigate the ubiquitination of Izh2p, a blot was probed with a rabbit polyclonal anti-

ubiquitin obtained from Abcam, as primary antibody (1/8000, v/v dilution), for 2 h and at room

temperature followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit

(sc-2004 Santa Cruz) at a dilution of 1/10000, for 1 h at room temperature, as secondary

antibody. Blots were developed in identical fashion as described before.

Results

IZH2 is a Zaplp Target Gene

IZH2 possesses a putative zinc responsive element (ZRE) in its promoter region, which has

the sequence TCCTCTAGGGT. In a wild type strain, the IZH2-lacZ construct yielded 2-fold

more activity under zinc deficiency than under zinc repletion (8.1 0.9 vs. 3.6 0.3, Figure 2-1

A). In a zaplA strain, however, the induction oflZH2-lacZ under zinc deficiency was highly

repressed, whereas a slight induction of the IZH2-lacZ activity was observed under zinc repletion

(2.9 0.0 vs. 5.2 0.2) (Figure 2.1 A). Taking together, our data demonstrate that the IZH2-lacZ

activity is induced by zinc deficiency via Zaplp. Furthermore, under zinc repletion, the IZH2-

lacZ activity is also dependent on the presence of the ZAP] gene. From a different stand point,

we also found that zinc deficiency causes a significant accumulation of the Izh2 protein (Figure

2-1 B), suggesting that zinc deprivation has an post-translational effect on Izh2p. Thus, we can

conclude that IZH2 is a bonafide Zaplp-target gene.









IZH2 and IZH4 Are Part of the Hypoxic Response

To illustrate with examples those genes that can be expressed under hypoxia or conditions

that mimic hypoxia like excess of zinc and iron deficiency, table 2-2 was generated (Lyons et al.,

2004). This table lists genes with an average induction of > 2-fold in cells exposed to 3 mM zinc

(excess) (n=2), and includes known targets of the Mga2p hypoxia sensor, OLE1 and Ty

elements (Vasconcelles et al., 2001; Zhang et al., 1997; Lyons et al., 2004). The remaining genes

are known to be induced by either low pO2 (hypoxia) (Cherry et al., 1998; Kastaniotis and

Zitomer, 2000), or by iron deficiency via the Aftlp iron-responsive transcription factor

(Rutherford et al., 2003). A screen for regulatory elements in the promoters of high zinc-

regulated genes by using RSA-TOOLS, generated probability-based consensus matrices that

matched with two groups of genes. One group has promoters containing the LORE (low oxygen

element). The promoters of the other group have the FeRE (iron responsive element). With these

matrices, 750 bp of the promoters of all genes shown in Table 2-2 were scanned, and found that

most of the 02-regulated promoters contained putative LOREs and that all of the Aftlp-target

promoters contained putative FeREs.

The IZH4 promoter contains a potential LORE sequence between -189 and -197 bp, but

does not contain a FeRE, suggesting that it is a target of Mga2p instead of Aftlp. To address this,

we tested the effects of mga2A and aft]A mutations for their effects on IZH4-lacZ activity.

Figure 2-2 A, shows that although the induction of IZH4-lacZ in response to zinc was still 2-fold

in an aft]A mutant, the basal levels of activity of the reporter construct was increased 5-fold.

Figure 2-2 B confirms that the LORE-IacZ hypoxia reporter is also induced by high zinc in an

Mga2p-dependent fashion (data generated by Brian Kupchak), as well as by aft]A deletion

(recall that LORE is the low oxygen responsive element). Figure 2-2 C shows that basal and









zinc-inducible expression of IZH4-lacZ depends on Mga2p. In addition, other stimuli that are

known to induce the hypoxic response in yeast, such as high Co2+ and Ni2+ (Vasconcelles et al.,

2001), also induce IZH4-lacZ. These responses are not seen in an mga2A strain. The IZH2

promoter also contains a putative LORE consensus sequence between -137 and -145 bp. IZH2-

lacZ is weakly induced by Co2+ and Ni2+ in an Mga2p-dependent manner (Figure 2-2 C), (data

generated by Lisa Regalla). As observed for LORE-lacZ (control), the increase of the IZH2-lacZ

activity under excess of zinc is not maintained in an mga2A mutant, suggesting that Mga2p is

responsible for maintaining elevated expression in high zinc (Figure 2-2 C). Since Mga2p binds

to LORE, the promoter reporter constructs, LORE-lacZ, and OLE]-lacZ were used as positive

controls to see the effect of cobalt on their transcriptional response. Figure 2-2 D confirms that

these reporters are also induced by Co2+via Mga2p.

Regulation of IZH4 by Excess of Several Transitions Metals is only Mga2p-Dependent

MGA2 and its paralog SPT23 show considerable sequence homology, with 43% of the

amino acids being identical and 60% being similar (Jiang et al., 2001). Therefore, function of

Mga2p and Spt23p is in many cases redundant (Zhang et al., 1997). In fact, Zhang et al., 1999

reported that one of the Mga2p-target genes, OLE1 was transcriptionally activated by both

MGA2 and SPT23 under hypoxic conditions.

Because IZH4 was strongly regulated by excess of zinc, cobalt, and nickel via Mga2p, we

investigated if SPT23 had the same effect as MGA2 on the transcriptional response of this gene.

By using P-galactosidase assays, we show that although the IZH4-lacZ activity slightly decreases

in presence of cobalt in a spt23A strain (39.5 + 3.4 for WT vs. 20.9 0.2 for spt23A), no

significant change in the promoter reporter activity was observed under excess of zinc and nickel

in the mutant strain (Figure 2-3). In contrast, the IZH4-lacZ activity was fully repressed in an









mga2A knockout strain under the same conditions. These results indicate that the transcriptional

regulation of IZH4 under hypoxic mimicking-conditions is mainly dependent on Mga2p but not

on Spt23p.

Iron Deficiency Affects the Expression of IZH2 and IZH4

Besides the excess of certain transition metals, iron chelation has a similar regulatory effect

on Mga2p-target genes (Vasconcelles et al., 2001). In order to examine the effect of iron

deficiency on the expression of IZH2 and IZH4, P-galactosidase assays were performed. To do

so, a chelexed synthetic medium (CSD), where the iron concentration was limited to 50 nM (iron

deficiency) or 10 [tM (iron repletion) was used. Figure 2-4 A and B shows that under iron

deficiency Mga2p is required to induce the IZH2-lacZ, IZH4-lacZ, and OLE]-lacZ activities.

However, LORE-lacZ activity was just slightly affected under those conditions, suggesting the

possibility of a different co-activator for Mga2p in the promoter regions of IZH2, IZH4, and

OLE1. Interestingly, although the P-galactosidase activities under the two tested conditions are

similar for the positive controls, the effect of iron deficiency is still more prominent than iron

repletion. These results constitute another piece of evidence that strongly suggests that IZH2 and

IZH4 are Mga2p-target genes.

Since iron deficiency has a dominant effect on the transcription of IZH2, we were

interested in investigating if the accumulation of Izh2 protein was also affected by iron

deficiency. With this in mind, the Izh2p was expressed from the plZH2-3xHA plasmid, in which

the IZH2 gene is driven by its own promoter. Protein expression was performed in CSD medium

supplemented with glucose as carbon source, deficient of iron, or supplemented with 10 [tM

FeC13 (iron repletion). As expected, iron deficiency also induces the accumulation of Izh2p,

(Figure 2-4 C).









Ubiquitination of Izh2p is Dependent on Nutritional Conditions

We tested if Izh2p was post-translationally modified when overexpressed in low iron

medium (LIM). To investigate this, pRS316-GALI-IZH2-3xHA construct was used, which

provides galactose inducible expression. Overexpression of Izh2p was carried out in replete

media supplemented with galactose (SGal). Synthetic medium supplemented with glucose (SD)

was used as control medium to demonstrate specificity of the band for Izh2p. In addition to this,

LIM medium supplemented with galactose or glucose (LIMGal and LIMD, respectively) was

also used to test the effect of iron deprivation and iron repletion, on the expression of Izh2p.

When the Izh2p was overexpressed in synthetic complete medium, or in LIMGal containing 1

mM FeC13 (iron repletion), a group of bands at higher molecular weight than the one

corresponding to the Izh2p molecular weight (36.3 KDa) was seen in the Western blot (Figure 2-

5 A, lanes 4 and 12, respectively). Interestingly, when Izh2p was overexpressed in LIMGal (1

[tM FeC13, iron deficiency), only the band at 36.3 KDa was observed (Figure 2-5 A, lane 8).

Initially, we speculated that those bands at higher molecular weight could be due to the

formation of complexes between Izh2p and other proteins. Alternatively, we envisioned the

possibility that Izh2p is ubiquitinated in media replete of iron or other metals, and that under iron

deprivation (1 ptM FeC13) the ubiquitination of Izh2p does not occur (Figure 2-5 A lane 8). To

test these possibilities, we first immunoprecipitated Izh2p expressed in SGal and in LIMGal (1

[tM FeC13). The Western blot analysis showed a band at a molecular weight between 75 KDa and

50 KDa besides the band at 36.3 KDa (Figure 2-5 B, lane 1). To determine if the band at higher

molecular weight was the result of Izh2p ubiquitination, another Western blot was carried out

and an antibody against the ubiquitin protein was used to investigate ubiquitination. Ubiquitin is

a small and highly conserved eukayotic protein with a molecular weight of about 8.5 KDa (Baker









and Baker, 1987; Peng et al., 2003). Interestinlgy, only the band between 75 KDa and 50 KDa

was observed (Figure 5-2 C, lane 1), suggesting that Izh2p when overexpressed in a synthetic

complete medium is undergoing ubiquitination.

To explore the effect of zinc deficiency and zinc repletion on the overexpression of Izh2p,

Western blot analysis was carried out. In this case, Izh2p was expressed in a chelexed synthetic

medium supplemented with galactose as a carbon source (CSGal), and either no ZnCl2 (zinc

deficiency) orlO [tM ZnCl2 (zinc repletion) was added to the growth medium. Interestingly,

similar results than those obtained with synthetic complete medium and LIMGal (1 mM FeC13)

were obtained (Figure 2-6, A and B). After immunoprecipitation, the band between 75 KDa and

50 KDa was observed only when Izh2p was overexpressed in CSGal (10 [tM ZnCl2) (Figure 2-6

B). These results suggest that the overexpression of Izh2p in a medium replete of zinc also

induces the protein ubiquitination. To confirm this, it is necessary to do a Western blot and use

the anti-Ub antibody to see if the band between 75 KDa and 50 KDa is in fact the result of

ubiquitination.

Discussion

The discovery of the IZH family, along with the discovery that the expression of IZH1

IZH2 and IZH4 is zinc-dependent, opened the possibility that this family of genes is implicated

in zinc homeostasis. This hypothesis was then supported with the finding that IZH1 and IZH2

have a putative zinc responsive element (ZRE) in their promoter regions (Lyons et al., 2000)

(Figure 1-3, panels A and B for details). The presence of a ZRE is necessary and sufficient to

confer Zaplp-regulated expression onto a promoter (Zhao and Eide, 1996a; Zhao and Eide,

1996b). Robust evidence is presented herein, that demonstrates that IZH2 is a bonafide Zap p-









target gene, and, therefore, a potential role for this gene in the metabolism of zinc is envisioned

(Lyons et al., 2004).

On the other hand, the fact that the IZH2 and IZH4 are induced by high zinc implies the

existence of a transcription apparatus that is involved in the modulation of the transcriptional

response of these two genes under excess of metals. Indeed, in the promoter regions of IZH2 and

IZH4 a putative LORE consensus sequence was reported by Lyons et al., 2004, (Figure 1-4

panels B and C, respectively). LORE is a low oxygen response element and the DNA binding

site for the hypoxia sensor Mga2p (Jiang et al., 2001). Besides low pO2 (hypoxia), excess of

metals like zinc, cobalt, or nickel, have been proven to be inducers of the hypoxic response

(Lyons etal., 2004; Vasconcelles etal., 2001; Gong etal., 2001; Rutherford et al., 2003).

High metals are believed to induce Mga2p by displacing iron from important sites. Not

surprisingly low iron also induces Mga2p. Herein, we present evidence that suggests that the

function of some of the IZHs goes beyond a role in zinc metabolism. For example IZH4, and to a

lesser extent, IZH2, are induced under excess of Zn2+, C2+, Ni2+, as well as by iron deficiency,

and deletion of Aftlp, the iron-sensing transcription factor (Figure 2-2 A, B, and C).

Interestingly, we have found that the metalloregulation oflZH2 and IZH4, under the conditions

used, depends on the Mga2p hypoxia-responsive transcription factor, suggesting that IZH2 and

IZH4 are Mga2p-target genes and part of the hypoxic response (results published by Lyons et al.,

2004).

In addition to Mga2p, Spt23p, an Mga2p-related protein, was found to regulate hypoxic

genes (Zhang et al., 1997; Nakagawa et al., 2002). In fact, studies performed by these groups

indicated that both proteins are required for transcription of OLE], a well known hypoxic gene.

During the course of those studies, it was also found that, when synthesized, Spt23p and Mga2p









are dormant proteins anchored through their C-terminal tails on the endoplasmic reticulum or

nuclear envelopes. Under hypoxia, both proteins are ubiquitinated with the subsequent release of

the N-terminal transcription factor domains into the cytosol, where they transcriptionally activate

of OLE]. Although, we tested the effect of the spt23A deletion in the transcriptional response of

IZH4-lacZ, under excess of metals, we did not see any significant effect (Figure 2-3), indicating

that the transcriptional response of ZH4 under the tested conditions is exclusively dependent on

Mga2p.

In Chapter 1, we mentioned that Izhs are homologous with the hemolysin 3 subfamily

(Hly3). In the ca-proteobacterium Azospirillium brasilence, Hly3 genes encode a chimeric protein

with a Hly3-like N-terminus fused to the CheA chemotaxis histidine kinase (Hauwaerts et al.,

2002). The involvement of Hly3-CheA from A. brasilense in hypoxia sensing emphasizes the

importance of our finding that IZH2 and IZH4 are hypoxic genes, and suggests a conservation of

function across species.

Even though our studies regarding transcriptional regulation provides striking information

related to the role exerted by two the IZHs in the metabolism of metals, we were also interested

to explore effect of iron and zinc on Izh2p accumulation. To do this, the expression of Izh2p in

metal replete media (CSD + 10 [LM ZnCl2 or FeC13) versus media that are deficient in either zinc

or iron (CSD ZnCl2 or FeC13) were investigated. Our data, indicate that Izh2p is also induced

under deprivation of either metal (Figure 2-1 B and Figure 2-4 C, respectively), suggesting that

the effect of zinc and iron deficiency goes beyond a simple transcriptional response. In this

scenario, is possible that Izh2p exerts a role directly by scavenging either of these metals to

supply the cell requirements. Alternatively, Izh2p can signal other molecules (e.g. metal

transporters) that will directly uptake the required metal. In this regard, Kupchak et al., 2007









found that overexpression oflZH2 constitutively represses FET3 under iron deficiency. FET3

encodes a high affinity iron transporter (Fet3p), which is implicated in high-affinity iron-uptake

(Rutherford and Bird, 2004).

Another important finding was that when Izh2p was overexpressed in a medium

supplemented with metals and in a medium replete of iron, the protein appears to be

ubiquitinated (Figure 2-5 A, lanes 4 and 12, B and C, lane 1). Ubiquitination is a cellular

mechanism that mediates the sorting of proteins to the ensosomal/vacuolar pathway in response

to nutritional signals (Pizzirusso and Chang, 2004). In this sense, it is plausible that a high

dosage of Izh2p is the driving force for the activation of the ubiquitin pathway, which results in

the trafficking of the protein to the vacuole for further degradation. Another possibility is that

since Izh2p is a membrane protein, its ubiquitination occurs to be removed from the membrane

to further fulfill other roles within the cell.

On the other hand, in an iron limiting medium (LIM, containing 1 [tM FeC13), Izh2p is not

ubiquitinated, suggesting and inherent role for Izh2p under metal deprivation (Figure 2-5 A, lane

8, and B and C, lane 3).

Takeng together, the results presented in this chapter let us envision that Izh2p is directly

involved in the regulation of zinc and iron homeostasis. At transcriptional level, more studies are

required to completely understand the mechanisms by which the responses of ZH2 and IZH4 are

mediated by metal concentrations.









Table 2-1.
Strain

BY4742

DY1457

ZHY6

Y15968

Y14869

Y04438


List of strains used in
Mutation

Wild type

Wild type

zapl

mga2

spt23

aft]


Chapter 2
Source/Derivation

EUROSCARF

David Eide

David Eide

EUROSCARF

EUROSCARF

EUROSCARF


Genotype

MAT a; his3; leu2; ura3; lys2

MAT a; ade6, his3; trpl; leu2;
ura3; canl-100c
MAT a;ade6; his3; trpl;leu2;
ura3;canl-100c
MAT c; his3; leu2;lys2; ura3;
YIR033w::KanMX4
MAT a; his3; leu2;metl5 ura3;
YKL020c::KanMX4
MAT a; his3; leu2;metl5 ura3;
YGL071w::KanMX4










Table 2-2. Genes induced > 2-fold by zinc excess
Group Gene name
Induced by SSN6 deletion or IZH4*
by low oxygen OLEI*
HSP26*
YGLO39w*
ERG3*
PIR3
YOR38w*
HSP30*
COSIO*
IZH2*
NCE103*
AHPl*
YGR161c*
HSPI04*
YOL106wt
Miscellaneous PDR3*
MGA2*
UBS1
HSP1 50
Iron metabolism FIT3f
FIT2f
TAFf
TISl t
ENBl
ARNlf
FTR1*f


Fold induction
6.6
3.4
3.4
2.9
2.8
2.6
2.3
2.3
2.2
2.1
2.0
2.0
2.0
2.0
2.8
2.6
2.3
2.1
2.0
7.6
7.2
6.2
3.6
3.4
3.0
2.4


Iron metabolism FRElT 2.3
SIT1J 2.2
FET3t 2.0
HMX t 2.0
Ty retrotansposons YBLOO5w-A (YBLWTyl-1) 2.1
YER138c (YERCTyl-1) 2.2
YER160c (YERCTy 1-2) 2.3
YHR214c-B (YHRCTyl-1) 2.2
YMLO45w (YMLWTyl-2) 2.6
YBR012w-A/-B (YBRWTyl-2) 2.2/2.2
YCL019w/:r i- (YCLWTy2-1) 2.1/2.4
YJRO26w/27w (YJRWTyl-1)* 2.2/2.5
YJRO28w/:2,' (YJRWTyl-2) 2.2/2.2
YMLO39w/40w (YMLWTyl-1) 2.2/2.6
YMR045c/46c (YMRCTyl-3)* 2.0/2.6
YMR050c/51c (YMRCTyl-4) 2.3/2.6
*Genes in bold have promoters containing putative regulatory elements scoring > 7.0 when using the
LORE or FeRE matrices generated by RSA-TOOLS. *LORE-containing, tFeRE-containing
Tables 2-1 and 2-2 were reproduced, with permission, from Lyons TJ, Villa NY, Regalla, LM, Kupchak
BR, Vagstad A, and Eide DJ (2004) Metalloregulation of yeast steroid receptors homologs. Proc Natl
Acad Sci USA 101: 5506-5511 (Table 2, supplemetary information, and Table 1, page 5508).













A 10


Uti
4)4i~
wre
(Ur~



Ta


B






37.
36.

25.


WT


HA +
Zn2+ -


+ +


Slzh2-3xHAp


WT zaplA

IZH2-lacZ


Figure 2-1. Zinc regulation of IZH2. Panel A shows that the activity of the IZH2-lacZ promoter
reporter is increased by zinc deficiency in a Zaplp-dependent manner (black bars).
On the other hand, the IZH2-lacZ activity is repressed under zinc repletion (10 JM
ZnC12) (grey bars). Panel B is a Western blot showing that the expression of Izh2p
under zinc deficiency is also induced.


















S g WT a WT m82 WTaft
LR--kKZ Z LLORE--cZ

S120 80
100
35 60

S^60 40

20 1

WT oga2 WT mga2 I /
H4rCZ H2-1 V WT mga2 WT mga2
LORE-lacZ OLE1-acZ

Figure 2-2. Transcriptional regulation of IZH2 and IZH4 by different metals. Panel A shows the
effect of aftlA mutation on IZH4-lacZ activity. B, effect of zinc excess, Mga2p, and
Aftlp on LORE-IacZ activity. In panels A and B, grey bars show activity in + zinc
(10 |JM ZnC12), white bars show activity in + + zinc (3 mM ZnC12). C shows the
Mga2p dependence of IZH4-lacZ and IZH2-lacZ activities in cells exposed to 10 |tM
ZnCl2 (grey bars), 3 mM ZnCl2 (white bars), 400 LM Co2+ (hatched bars), or 400 CtM
Ni2+ (black bars). D, shows the effect of Mga2p + zinc (10 |JM ZnCl2, grey bars), and
400 [tM Co2+ (hatched bars) on LORE-IacZ and OLEI-lacZ activities. Panels A, B,
and C were reproduced, with permission, from Lyons TJ, Villa NY, Regalla, LM,
Kupchak BR, Vagstad A, and Eide DJ (2004) Metalloregulation of yeast steroid
receptors homologs. Proc NatlAcad Sci USA 101: 5506-5511 (Figure 3, page 5508).













60 I "IIL Io
3 mM Zn2+
>% 0 13 400 gM Co2+
> 400 gpM Ni2+

~. 40

S 30
0=
c- 20


10


WT spt23A mga2A
IZH4-lacZ

Figure 2-3. Transcriptional activation of IZH4-lacZ in cells exposed to excess of different metals
is dependent on the presence of MGA2 but independent on SPT23.














* s
en,

mt*


WT mga2A
IZH2-lacZ


IZH4-1acZ


30 40

25 B
S 30O
20

20
10
o
5 10

0 I I
WT mga2A WTf mga2A

LORE-lacZ OLEI-lacZ


WT


HA +

Fe3 -


37.0
36.3


25.0


- +

+ +


, lIzh2-3xHAp


Figure 2-4. Iron regulation of IZH2 and IZH4. A and B show the effect of iron deficiency, and
the presence of Mga2p, on the transcriptional regulation of IZH2-lacZ, IZH4-lacZ,
LORE-lacZ, and OLE]-lacZ activities. C is a Western blot showing that the
expression of Izh2p is positively modulated by iron deficiency. In the panels
corresponding to P-galactosidase activities, gray bars show activity in + iron (10 [tM
FeC13, iron repletion), and black bars show activity in iron (no FeC13, iron
deficiency).


-r












A


KDa
150,
100.
75'
50,
37.
lzh2-3xHAp 136.3.


1 i-M Fe3+

SD SGal LIMD LIMGal

12 3 4 5 6 7 8


1 mM Fe3+

LIMD LIMGal

9 10 11 12


1= Vector
2 = lzh2-3xHA
3 = Vector
4 = lzh2-3xHA
5 = Vector
6 = lzh2-3xHA
7 = Vector
8 = lzh2-3xHA
9 = Vector
10= Izh2-3xHA
11 = Vector
12= Izh2-3xHA


LIMGal
B SGal 1 gM Fe3.
1 2 34




Izh2p
Ubiquitination?

Izh2-3xHApp


LIMGal
SGal 1 [pM Fe3+
1 9 A A


1 = Izh2-3xHA
2 = Vector


Figure 2-5. Post-translational effect of iron deficiency on the overexpression of Izh2p. Figure
shows Western blot analysis of Izh2p under different nutritional conditions. A shows
the Izh2-3xHA protein expression in different growth media (e.g. SD, SGal, LIMD,
and LIMgal). B is aWestern blot of immunoprecipitated samples. C shows the
ubiquitination of Izh2p. In panels A and B, an antibody against the HA epitope tag
was used to identify Izh2-3xHAp. In panel C, an antibody against ubiquitin was used
to determine protein ubiquitination. In the figure, SD and SGal stands for synthetic
medium, supplemented with glucose and galactose, respectively. LIMD and LIMGal
represent low iron media, supplemented with glucose and galactose, respectively.
Iron deficiency is 1 [LM FeC13 and 1 mM FeC13 is iron repletion.











A Zn2+
HA +
KDa
150
100
75
50
37
Izh2-3xHAp *36.3


- + +


Zn2+ -
HA +
KDa
150
100
75
50

37
Izh2-3xHAp* 36.3


Figure 2-6. Zinc-dependence on the translational response for Izh2p. A is a Western blot
showing the overexpression of Izh2p in cells exposed to zinc deficiency (no ZnCl2
added to the growth medium) and + zinc repletion (10 [LM ZnCl2). B shows a
Western blot for the Izh2-3xHA immunoprecipitates. Izh2p was overexpressed in
chelexed synthetic medium supplemented with galactose (CSGal). In the figure +
HA and HA represent HA-tagged and untagged Izh2protein, respectively.


- +
- +









CHAPTER 3
DUAL REGULATION OF THE IZHS BY METALS AND FATTY ACIDS: THE FIRST LINE
OF EVIDENCE IMPLICATING THE IZH FAMILY IN LIPID METABOLISM

Introduction

Saccharomyces cerevisiae is able to survive on a wide range of growth media due to its

ability to activate pathways that enable the utilization of fermentable and non-fermentable carbon

sources. Fatty acids are one of those nutrients that tightly regulate gene expression when used as

a sole carbon source (Choi et al., 1996; DeRusso et al., 1999; Black et al., 2000; Kandasamy et

al., 2004).

In the S. cerevisiae genome, a myriad of genes of known and unknown function have been

found to be activated or repressed, at transcriptional and translational levels, when cells are

grown in media supplemented with fatty acids. Perhaps one of the most interesting examples that

illustrate the regulatory effect exerted by fatty acids is the oleate-dependent activation of genes

encoding proteins implicated in the fatty acid P-oxidation pathway and peroxisomal proliferation

(Hiltunen et al., 2003). In S. cerevisisae, peroxisomal P-oxidation is the only means to catabolize

long chain fatty acids (Luo et al., 1996). Oleate, a very abundant fatty acid in yeast cells, is a cis-

A-9 monounsaturated fatty acid containing an acyl chain of 18 carbons in length, (Vasconcelles

et al., 2001).

Despite the positive effect exerted by fatty acids in the transcription of specific genes,

other genes are transcriptionally repressed in response to changes in a carbon source. One of

those genes that illustrate such an opposite effect is OLE1. This gene encodes A-9 fatty acid

desaturase, an enzyme involved in the formation of unsaturated fatty acids. In presence of oleate,

the expression of OLE1 is repressed. Conversely, transcription of this gene is induced when cells

are grown in the presence of saturated fatty acids (McDonough et al., 1992). Interestingly, the









hypoxia sensors MGA2 and SPT23 have been implicated in the transcription of OLE1

(Kandasamy et al., 2004). Jiang et al., 2002, reported that in S. cerevisiae the presence of the

LORE (low oxygen response element) is important for the transcriptional regulation of target

genes via Mga2p, and that certain lipids like unsaturated fatty acids can repress the LORE-

dependent induction of Mag2p targes like OLE1. Thus, hypoxia and unsaturated fatty acids work

in opposing manners (Kwast et al., 1999; McDonough et al., 1992; Nakagawa et al., 2001;

Vasconcelles et al., 2001).

In the promoter regions of genes regulated by certain fatty acids, a consensus sequence

termed as an Oleate Response Element (ORE) has been identified (Kos et al., 1995). This

sequence is the binding site for the transcription factors Oaflp and Pip2p. Upon addition of

oleate, these two proteins bind to ORE as a heterodimeric complex, and mediate oleate-

dependent transcriptional activation (Karpichev and Small, 1998; Baumgartner et al., 1999).

Interestingly, three of the four IZH genes, (IZH1, IZH2, and IZH4), contain putative ORE

sequences in their promoter regions (Figure 1-4, panels A, B, and C for details).

In this chapter, we present some evidences that suggest other roles for the IZHs besides

their implication in the metabolism of metals. First of all, we show that IZH2-lacZ promoter

reporter fusion responds independently to both zinc and the addition of myristate. Second, we

demonstrate a dual regulatory effect exerted by metals and fatty acids. For example, we found

that certain fatty acids and metals like cobalt when added to the same growth medium produce an

additive effect on the induction of IZH2-lacZ and IZH4-lacZ activities. Even more interesting is

the finding that these regulatory effects occur at transcriptional level via the hypoxic sensor

Mga2p. This last result illuminates the idea that diverse metabolic pathways like metals and

lipids can converge to the point where genes like the IZHs, are up-regulated.









Also, we show that supplementation of growth medium with fatty acids as a sole carbon

source, elicits the transcriptional and post-translational response of ZHs. In this regard, one of

the most promising results is the strong transcriptional and post-translational induction of IZH2

by myristate (a saturated fatty acid with a chain of 14 carbons in length). Surprisingly, the

transcriptional induction oflZH2-lacZ occurs via Oaflp/Pip2p. This finding was unexpected,

since these two transcription factors are known to be activated by oleate but not by myristate.

Furtheremore, we also show that Oaflp and Pip2p are required to maintain the weak but still

measurable IZH2-lacZ activity in presence of oleate.

Overall, the regulatory effects of different fatty acids on the transcription of the IZHs, is

investigated. Furtheremore, a dual regulatory effect of metals and fatty acids on the trnacription

of IZH2 and IZH4 is established during this study. Taken together the results presented herein,

suggest a dual implication of ZHs in metals and lipid metabolism.

Materials and Methods

Yeast Strains and Plasmids

Yeast strains used in this chapter are listed in table 3-1. As mentioned elsewhere, the IZH-

lacZ fusions were generated by gap repair of the plasmid vector YEp353 (Lyons et al., 2000).

Briefly, PCR products were generated from genomic DNA that contained 1,000 bp of the target

promoter sequence flanked by regions of vector homology. These fragments were gel-purified

(Promega) and co-transformed into the BY4742 wild type strain with EcoRI- BamHI-digested

YEp353; transformants were selected for Ura+ prototrophy. Plasmids were then transferred to

Escherichia coli and confirmed by sequencing. Although the IZH2 Open Reading Frame (ORF)

has two ATG codons, only the fusion of the second in-frame ATG of in the IZH2 (ORF) to lacZ

resulted in a functional promoter-reporter construct (IZH2-lacZ).









Biochemical Assays

Yeast transformations were performed according to standard procedures, in identical

fashion as described in Chapter 2.

Promoter reporter activities were measured by P-galactosidase assays (Chapter 2). Before

each P-galactosidase assay, cells were grown aerobically during one overnight in synthetic

medium containing dextrose (SD medium). When required, and unless otherwise indicated, the

SD medium was supplemented with 1 mM of the respective fatty acid, and 0.5% Tergitol-NP40

(Sigma). To limit zinc availability, chelexed-synthetic medium (CSD) was used. CSD was

prepared in identical fashion as described in Chapter 2 (Apendix A for more details). When

myristate was used along with zinc, a 37.5% (w/v) stock solution of the fatty acid was dissolved

in 50% EtOH, 25% Tween-40 and then added to the CSD growth medium to a final

concentration of 0.375% (Lyons et al., 2004).

Protein expression, immunoprecipitation, and Western blot analysis were performed as

described in Chapter 2.

Results

Fatty Acids Exert a Regulatory Effect on the Expression of IZHs

Previously Karpichev et al., 2002 reported that the expression of IZH2 was induced by

exogenous myristate. In that publication, they also reported that addition of monounsaturated

fatty acids like oleate induced the Oaflp/Pip2p-dependent regulatory effect of target genes. As

mentioned before, putative ORE sequences are present in the IZH2 (-159 to -167 bp), IZH1 (-302

to -328 bp), and IZH4 (-204 to -263 bp) promoters (Karpichev et al., 2002). Herein, we have

confirmed that IZH2-lacZ responds independently to both, zinc and the addition of exogenous

myristate (Figure 3-1) (Lyons et al., 2004).









We have also found that while IZHl-lacZ and IZH3-lacZ respond to exogenous oleate

(C18:1), stearate (C18:0), and palmitate (C16:0) (Figures 3-2 A and C, respectively), the IZH4-

lacZ reporter responds to the addition of palmitate (Figure 3-2 D), and the transcriptional

activation of the IZH2-lacZ reporter was exclusively observed in presence of myristate (Figure 3-

2 B). Interestingly, we also found that the transcriptional regulation oflZH2 by myristate and

oleate is Oaflp/Pip2p-dependent (Figures 3-3 A and B, respectively). These results suggest that

the Oaflp/Pip2p complex is not exclusively activated by oleate, and that myristate can also

mediate the activation of this transcription factor complex.

The Expression of the Izh Proteins is Regulated by Fatty Acids

Due to the effect exerted by several fatty acids in the transcriptional response of the IZH

gene family, we decided to investigate if this effect also exists at level of protein accumulation.

To do this, 3xHA epitope-tagged IZH constructs under the control of their own promoter or a

galactose inducible promoter (for protein overexpression) were used. A polyclonal antibody

against the HA tag, probe Y-l 1 was used to recognize each of the Izhl-3xHA fusion proteins in

the Western blots. Cells expressing untagged Izh proteins or the empty plasmid vector

(pRS316-GAL]), were used as negative controls. Interestingly, we did not see any change in the

expression profile of Izhlp when the IZH1 contained its own promoter (Figure 3-4 A). However,

when Izhlp was overexpressed in galactose, a slightly induction of Izhlp expression, upon

addition of plamitate, was observed (Figure 3-4 B). Likewise, Izh2p was induced by myristate

(Figures 3-4 C and D). Also, the expression of Izh3p was induced only when the cells were

spiked with oleate for 2 h (Figure 3-4 F). In contrast, the expression of Izh3p was fully repressed

upon incubation with oleate for 12 h (Figure 3-4 E). This could be attributed to toxic effects of

oleate during long periods of incubation. To detect Izh3-3xHAp, the protein had to be

concentrated by immunopreciopitation. Immunoprecipitation was done in identical fashion as









explained in Chapter 2 and using a mouse monoclonal anti-HA conjugated with agarose beads to

pull down the 3xHA-tagged Izh3p. Immunoprecipitates were then detected by Western blot. For

Izh4p, addition of plamitate did not produce any significant effect on the levels of accumulation

of Izh4p (Figure 3-4 G).

In general, the results obtained with the Western blots indicate that the effect of the

exogenous fatty acids goes beyond a mere transcriptional response, and that certain fatty acids

constitute a sufficient stimulus to induce the expression of the majority of the IZH gene products.

Taken together, these results represent additional evidence suggesting a strong implication of

IZHs in lipid metabolism.

Transcriptional Regulation of IZH2 and IZH4 in Presence of Metals and Fatty Acids
Occurs via Mga2p.

In the former chapter, the Mga2p-dependent regulation of ZH2-lacZ and IZH4-lacZ

activities by excess of metals was reported. Nakagawa et al., 2001; Vasconcelles et al., 2001,

reported that OLE1 has an LORE in its promoter region and that its expression was induced in

response to excess of cobalt and saturated fatty acids via Mga2p. In that study, the expression of

OLE1 was also found to be repressed by unsaturated fatty acids like oleate (C18:1) and

palmitoleate (C16:1).

Like OLEI, IZH2 and IZH4 contain in their promoter regions a low oxygen responsive

element (LORE) and an oleate responsive element (ORE) (Karpichev et al., 2002; Lyons et al.,

2004). Therefore, it is plausible that under similar stimuli, IZH2 and IZH4 respond like OLE1.

To investigate if the expression of IZH2 and IZH4 upon the simultaneous addition of Co2+and

myristate, as well as Co2+ and oleate were Mga2p-dependent, we performed reporter gene assays.

Surprisingly, we found that the simultaneous addition of Co2+ and myristate produced an additive

effect on the IZH2-lacZ activity (Figure 3-5 A). On the other hand, the inducible effect of Co2+









on the IZH4-lacZ reporter activity was partially repressed upon addition of myrsistate (Figure 3-

5 B). The transcriptional response of the promoter-reporter fusions analyzed in this study, were

Mga2p-dependent (Figure 3-5 A and B).

In addition to this, we also investigated the effect of adding oleate and cobalt to the same

growth medium. IZH2 and IZH4 respond similarly to OLE1 under these external stimuli and this

response is Mga2p dependent. In effect, oleate repressed the Co2+-dependent induction of the

IZH2-lacZ reporter (Figure 3-6 A). Moreover, these effects were even more stringent when the

MGA2 gene was knocked out. Similarly, the inducible effect of Co2+ in the IZH4-lacZ reporter

activity was also fully repressed by oleate (Figure 3-6 B). These transcriptional responses were

also confirmed for the positive controls LORE-lacZ and OLE]-lacZ (Figure 3-6 C and D,

respectively). Overall, our results suggest a connection between the hypoxia and the fatty acid

metabolic pathways. In this scenario, it is totally feasible that the hypoxia pathway controls the

levels of unsaturated fatty acids in S. cerevisiae, by regulating the expression of three genes

OLE1, IZH2, and IZH4.

Discussion

Saccharomyces cerevisiae cells can grow on a variety of carbon sources, including fatty

acids. When used as a sole carbon and energy source, fatty acids elicit the transcriptional and

translational up-regulation of different genes. Herein, we report that IZHs respond to exogenous

fatty acids at the transcriptional and post-translational levels, suggesting a role for the IZHs in

lipid metabolism.

Also, we demonstrate that zinc deficiency and the addition of myristate acid have an

additive effect on the induction of the IZH2-lacZ activity and that response to each stimulus is

independent of each other. The transcriptional response of IZH2 under zinc deficiency is Zap 1-

dependent, whereas the induction of the gene by myristate appears to be dependent on the









presence of the transcription factors Oaflp/Pip2p. Although oleate did strongly induce IZH2-

lacZ activity, per se, single mutations of OAF1 and PIP2 produce repression on the IZH2-lacZ

activity, indicating that both transcription factors are required for activation.

Another interesting finding was that oleate overrides the cobalt-induction of IZH2-lacZ and

IZH4-lacZ activities, and that such a repressing effect is Mga2p dependent. Furthermore,

myristate and cobalt have a strong additive effect on the IZH2-lacZ activity. These results

suggest a critical role for IZH2 and IZH4 under diverse stimuli such as hypoxia and exogenous

fatty acids.

The results presented in this chapter, constitute a first piece of evidence that reveal the

promiscuity of this family of yeast proteins. Their dual regulation by metals and fatty acids

makes of these proteins an attractive target of research. Understanding how such divergent

metabolic pathways can transcriptionally and post-translationally affect a family of proteins is

vital because it can contribute to the elucidation of how these metabolic pathways are tightly

regulated. Undoubtedly, alternative hypotheses need to be addressed to decipher this metabolic

puzzle.

For future work, it is imperative to investigate the mechanism by which some of the IZH

genes can respond to different nutritional stimuli. A first experiment that can illuminate the

results presented in this chapter is a DNA microarray experiment using the nutritional conditions

reported herein. With this type of experiment we can have a global idea about which genes and

which metabolic pathways are affected under hypoxic conditions and the use of fatty acids as a

sole carbon source. Microarray data can then be compared to the P-galactosidase data reported in

this chapter. Furthermore, Western blots can be used to explore the post-translational response of

the IZH and other genes found by DNA microarrays.









List of strains used in
Mutation

Wild type

oafl (yafl)

pip2 (oaf2)

mga2


Chapter 3
Source/Derivation

EUROSCARF

EUROSCARF

EUROSCARF

EUROSCARF


Table 3-1.
Strain

BY4742

Y10355

Y11660

Y15968


Genotype

MAT a; his3; leu2; ura3; lys2

MAT a; his3; leu2; lys2; ura3;
YAL051w::KanMX4
MAT u; his3; leu2; lys2 ura3;;
YOR363c::KanMX4
MAT c; his3; leu2;lys2; ura3;
YIR033w::KanMX4












100-
U-
S4Ju
S50.


ro

& Zapl+ +

C. C14:0 + +


Figure 3-1. Zaplp-dependent regulation of IZH2-lacZ. The IZH2-lacZ reporter responds to both
zinc and exogenous myristate (C14:0). Black bars show reporter activity in -zinc,
and gray bars show reporter activity in + zinc (10 [LM ZnCl2). In this, and all other
figures showing lacZ data, a representative experiment performed in triplicate is
shown and the error bars represent standard deviation. Figure reproduced, with
permission from Lyons TJ, Villa NY, Regalla, LM, Kupchak BR, Vagstad A, and
Eide DJ (2004) Metalloregulation of yeast steroid receptors homologs. Proc Natl
AcadSci USA 101: 5506-5511 (Figure2 D, page 5507).










140 250
4 A no fatty acids
; 120 myritate
SE2:p palmitate 200


80 150




h -50
20

0 0
IZH1-lacZ IZH3-lacZ

80 3.0
B no fatty acids
Z, b myristate
'Izz= palmitate 26
4, 60 state
U in oleale

'- 02
40- -1.5
0=
m 20i
S0.5


IZH2-lacZ IZH4-lacZ

Figure 3-2. Transcriptional regulation of IZHs by exogenous fatty acids. A, ZHI-lacZ and
IZH3-lacZ activities are slightly induced by addition of palmitate (C16:0), stearate
(C18:0) and oleate (C18:1). B, shows that while the IZH2-lacZ activity is highly
induced by myristate (C14:0), (panel B-left), the IZH4-lacZ activity is induced by
palmitate (C16:0), (panel B-right).









160
140


BT


no fatty acids
m + oleate


A no fatty acidj
m + my ristate











WT oaflA pip2A


IZH2-4acZ


Figure 3-3. Oaflp/Pip2p dependence of the IZH2-lacZ activity in presence and in absence of
myristate (C14:0) and oleate (C18:1), (panels A and B, respectively).


WT oaflA pip 2A


120
100
80

60
40
20
0


I


-40


-30


-20


10


0












A Native promoter C

HA + + HA +
C16:0 + + C14:0 +

36.5 Izh1-3xHAp KDa

36.3

B
Overexpressor


HA + +
C16:0 + +


36.5- Izhl-3xHAp


dative promoter

+


Izh2-3xHAp


D Overexpressor

HA + +
C14:0 + +
KDa I


E Native promoter Overexpressor
Izh3-3xHA lzh3 Vector lzh3-3xHA

C18:1 + + -
112 2 -Time of incubation
KDa h
100

75 .

6:1.3 zh3-3xHAp


Native promoter
Izh3-3xHA Izh3

C18:1 + + -
KDa 12 2
KDa


63.3 '


Native promoter

Izh4-3xHA Izh4

C16:0 +
12 +- Time of incubation

36.6 Izh4-3xHAp


Time of incubation
/h

*Izh3-3xHAp


Figure 3-4. Post-translational response of the Izhp family upon addition several fatty acids.
Panels A and B show the effect of palmitate (C 16:0) in the expression of Izhlp. The
effect exerted by C16:0 is remarkable when Izhlp was overexpressed, panel B. C and
D, the expression of Izh2p is highly induced upon addition of myristate (C14:0). E
and F, time-dependence of Izh3p expression upon addition of oleate (C18:1). G, no
effect of C16:0 on the expression Izh4p was observed.











350 40
A B SD medu
300 Con
,and + C14:0
4
"5 2i50 + C 140 + Co1: 30


St 250 -
m .20
m43
= 150

m 100 -
0 10

50-

0 0
WT mga2A WT mga2A

EH2-IacZ IZH4-acZ

Figure 3-5. Dual regulation of IZH2 and IZH4 by exogenous myristate (C14:0) and cobalt. A
and B show the Mga2p dependence of IZH2-lacZ and IZH4-lacZ activities in cells
exposed to 400 [tM Co2+, 1 mM myristate, and the addition of both 400 [LM CoC12,
and 1 mM C14:0. A shows that exogenous C14:0 has an additive effect on the Co2+_
dependent induction of IZH2-lacZ activity. B, the addition of C14:0 partially
represses the inductive effect of Co2+on the IZH4-lacZ activity.










100


*s
M -

o =2
0-


a


0J




200



c-
150
- g




St50
o =
m "
"5 100
0-s



n 50


WT mga2A WT mga2A

IZH2-tacZ IZH4-lacZ

1 II..


C SD medium
-+ Co2+
+ C18:1
+ C18:1 + Coa2














WT mga2A

LORE-lacZ


WT mga2A

OLEI-lacZ


Figure 3-6. Regulation of IZH2 and IZH4 by exogenous oleate (C18:1) and cobalt. A, Mga2p
dependence of IZH2-lacZ activity in cells exposed to 1 mM C18:1. Panels A, B, C,
and D, show the repressive effect of 1 mM C18:1 in the Co2+-dependent induction of
the promoter -reporter activities. Mga2p-independence of the IZH4-lacZ, LORE-
lacZ, and OLE]-lacZ activities upon addition of C18:1 is shown in panels B, C, and
D, respectively.


30



20



10



0




160

140

120

100

80

60

- 40

20

0









CHAPTER 4
NYSTATIN-RESISTANCE OF IZH3A, IS ASSOCIATED TO ALTERATIONS IN THE
ERGOSTEROL CONTENT

Introduction

Sterols are essential structural and regulatory components of eukaryotic cell membranes

(Figure 4-1) (Veen et al., 2003; Mullner et al., 2005). Although cholesterol is the main sterol in

mammals, ergosterol is the most abundant sterol in fungal membranes (Reiner et al., 2006).

In yeast, sterols are found in two main forms: (i) free sterols and (ii) steryl esters. Free

sterols accumulate in the plasma membranes where their main role is to maintain membrane

integrity and fluidity (Veen et al., 2003). Steryl esters, on the other hand, are found in lipid

particles, where they serve as storage reservoirs or as intermediates in intracellular transport

(Zinser et al., 1993, Yang et al., 1996; Valachovi6 et al., 2001). When free sterols are required,

esterified sterols are hydrolyzed producing free sterols and fatty acids, which are then mobilized

to plasma membranes to fulfill the membrane requirements (Koffel et al., 2006).

Despite its structural role, ergosterol has been recently implicated in regulation of cell cycle

progression, cell polarization and membrane function during mating, endocytosis and vacuolar

fusion as well as protein sorting along the secretary pathway and signal transduction (Reiner et

al., 2006).

Because sterols are required to maintain the fluidity and integrity of the fungal membranes,

the sterol pathway has emerged as a potential target for the development of antifungal drugs

(Carrillo-Mufioz et al., 2006). The reason for this is the specificity of some antifungals toward

distinct sterols. For example, some antifungals like azoles inhibit enzymes at specific steps of the

sterol pathway (Gachotte et al., 1997), and others like nystatin and amphotericin B exert their

action by interacting directly with sterols like ergosterol (Sharma, 2006).









Currently fungal infection is one of the major causes of mortality in immunocompromised

patients such as those with AIDS. In addition, treatments of chemotherapy, organ transplantation,

and environmental stresses like UV radiation are also potential causes of fungal infection

(Kontoyiannis et al., 2002). Members of the Candida spp family, in particular, are the major

cause of virulence (Mukhopadhyay et al., 2002). Besides azoles, polyene antifungals like

amphotericin B and nystatin have been widely used in the treatment of fungal infections

(Dupont, 2006).

Although S. cerevisiae is a non-pathogenic fungus, its genetic similarity to Candida spp

makes it a safe model system to investigate the mechanisms that govern polyene effects.

Nystatin, a polyene antifungal (Figure 4-2 C), isolated from Streptomyces noursei (Marini

et al., 1960), selectively targets ergosterol in the fungal plasma membrane (Lees et al., 1995)

generating pores in the plasma membrane through which the leakage of nutrients occurs and the

concomitant cell death (Lampen et al., 1962) (Figure 4-2 panels A and B). Despite the variety of

studies performed, the mechanism by which nystatin exerts its fungicidal effect still remains

unclear.

Karpichev et al., 2002 reported that mutation of the fungal gene IZH2 (izh2A) caused

resistance to nystatin (or a normal growth in presence of nystatin). Nystatin resistance has been

associated with mutations that lead to changes in the sterol composition or the sterol content in

the cell membranes (Lampen et al., 1960; Hapala, et al., 2005). Although we could not confirm

the results reported by Karpichev et al., 2002, we found that the mutation of the IZH3 gene

(izh3A) was consistently resistant to nystatin.

In this chapter, we report for the first time the identification and initial characterization of

izh3A as a novel nystatin resistant mutant. As a first approach to this discovery, phenotypic









studies revealed that izh3A grows better than wild type and the single mutations izhIA, izh2A,

and izh4A in a medium supplemented with nystatin. Second, complementary studies where the

IZH3 gene was re-introduced into the izh3A mutant, rescued the nystatin-dependent growth

defects. Finally, by using different analytical techniques, we demonstrate that izh3A shows

alterations in the sterol composition; particularly, we have discovered that this mutant has less

free ergosterol than wild type, suggesting that the IZH3 gene could be involved in the sterol

biosynthetic pathway.

Even though nystatin requires ergosterol for toxicity (Bhuiyan et al., 1999), recent studies

have revealed that sphingolipids can also be targets for the nystatin action (Leber et al., 1997).

Phenotypic studies conducted to see the dual effect of nystatin and certain sphingoid bases in the

growth pattern of the wild type strain BY4742 and the single and multiple izhA mutants suggest

that sphingoid bases can ameliorate the toxic effects of nystatin. This observation also opens the

possibility that the IZH3 gene could be implicated in related sterol metabolic pathways such as

the sphingolipid biosynthetic pathway.

Materials and Methods

Yeast Strains and Reagents

The strains used in this chapter are described in Table 4-1. Deletions were generated by Dr.

Thomas Lyons (during his post-doctoral fellow) using PCR-based gene disruption using short

flanking homology, according to Wach et al., 1994. Multiple, mutations were generated from

heterozygous quadruple knockout strain engineering by successive rounds of mating and

sporulation. The kanMX4 (kanamycin) markers in the izh2, izh3, and izh4 strains were replaced

with the hphMX4 (hygromycin), natMX4 (nourseothricin), and ura3MX4(URA ) cassettes,

respectively (Goldstein and McCusker, 1999).









Nystain (Nys), sterylamine, and ergosterol, lanosterol, and cholesterol were obtained from

Sigma. The sphingolipids Cis-phytosphingosine (Cis-PHS) and Cis-dihydrosphingosine (Cls-

DHS), C2-phytoceramide (C2-PHC), C16-phytoceramide (C16-PHC), and Cis-pytoceramide (Cls-

PHC) were obtained from Avanti Polar Lipids. The solvents for GC-MS or HPLC-MS analysis

were from Burdick & Johnson.

Yeast Transformations

Yeast transformations were performed as described before. In this case, the centromeric

plasmid vector pRS315-CEN (empty vector), or the plasmid vector expressing IZH3 via its own

promoter were introduced into the izh3A mutant. Likewise, the pRS316-GAL] (empty vector) or

the plasmid vector expressing IZH3, were transformed into izh3A. Finally, the pRS316-GAL1

and the plasmid expressing each of the IZHs were also transformed into the wild type strain

BY4742.

Yeast Growth Media and Conditions

Cells were grown aerobically, during one overnight in liquid synthetic media, prepared

with 0.67% Yeast Nitrogen Base without amino acids and ammonium sulfate (Fisher), and

supplemented with 0.5% ammonium sulfate, 2% glucose and appropriate amino acids at a final

concentration of 0.01% (SD medium). For gene overexpression, glucose was replaced with 2%

galactose as carbon source (SGal medium). For growth in solid media, the SD- or the SGal-

medium was supplemented with 1X bacto agar (Fisher). Cell growth in liquid media was

monitored using a UV-VIS 170-2525 SmartSpec Plus spectrophotometer (Bio-Rad).

For ultraviolet spectrometric, or gas chromatography mass spectrometric analysis (GC-

MS) of total sterols, cells (50 mL liquid culture) containing the WT strain, or the izh3A mutant

were grown to stationary phase (OD600 of 3.0-4.0), followed by centrifugation at 3000 rpm, 4C









for 5 min. Pellets were washed twice with cold sterile nano-pure water, and their net wet weight

was determined. When used, 5 U/mL of nystatin was added to the growth media at a very early

OD600 (e.g. OD600 of 0.1). Once again, cells were grown to stationary phase (OD600 of 3.0-4.0),

followed by harvesting in identical fashion as described just before.

For high performance liquid chromatography-mass spectrometric analysis (HPLC-MS) of

total and free sterols, cells (5 mL) where the izh3A strain was transformed with the empty vector

pRS315-CEN(same than izh3A strain), or the pRS315-CEN expressing IZH3 (same than WT),

were grown for one overnight, to saturation. Fresh liquid media (50 mL) were inoculated with

aliquots of the overnights and grown to logarithmic phase (OD600 of 1.0). Cells were then

harvested by centrifugation at 3,000 rpm, 4C and for 5 min. Resultant pellets were washed twice

with cold sterile water, and freeze dried by using a lyophilizer. Dried cells were further used for

sterol extractions.

Phenotypic Studies

Cells were grown overnight to saturation (OD600 of 3.0) at 300C, in synthetic medium

supplemented with glucose as a carbon source. Cells were then serially diluted at OD600 = 1.0,

0.1, and 0.01, and aliquots of 5 ptL of these dilutions were plated on synthetic medium agar

plates supplemented with 5 U/mL, or 10 U/mL nystatin dissolved in DMSO. Nystatin solutions

were always prepared and immediately used to avoid degradation.

For the phenotypic studies in presence of sphingolipids, +/- 12.5 [LM C18-PHS, and C1s-

DHS sphingoid base-like stearylamine (dissolved in 100% ethanol), +/- 0.5 [LM C2-PHC, C16-

PHC and C1i-PHC (dissolved in a mixture of cholorform : methanol at a ratio 1:1, v/v) were used

to plate 5 p.L of each serial dilution. Plates were incubated in the dark, at 300C for 3 days; and

observed for growth.









Complementation Studies

The izh3A mutant strain, containing the kanMX4 cassette was transformed with the

centromeric plasmid vector pRS315 (pRS315-CEN) or with the pRS315-CEN plasmid,

expressing the IZH3 via its native promoter. To see the effect of overexpressing IZH3 in

presence of nystatin, the mutant izh3A was also transformed with both the pRS316 plasmid

containing the strongly inducible GAL] promoter (pRS316-GAL1), or with the pRS316-GAL]

plasmid expressing IZH3, in a medium supplemented with galactose.

Sterol Extractions

For UV and GC-MS analysis, sterols were extracted according to the protocol described by

Arthington-Skaggs et al., 1999. Briefly, pellets were suspended in 3 mL of 25% alcoholic

potassium hydroxide solution (25 g of KOH and 35 mL of sterile nano-pure water, and brought

to 100 mL with 100% ethanol), and mixed by vortexing for 1 min. Cells suspensions were

transferred to 13 X 100 mm borosilicate glass tubes with screw-caps, and incubated in the dark

in 85C for 2 h. Following incubation, tubes were allowed to cool at room temperature. Sterols

were then extracted with 4 mL of a mixture of sterile nano-pure water and n-heptane (1:3, v/v)

followed by vigorous vortex mixing during 3 min. The heptane layer was transferred to new

sterile 13 X 100 mm borosilicate glass test tubes with screw-cap. The obtained sterol extracts

were used for UV spectrophotometric analysis.

Extractions of total and free ergosterol were performed adapting the protocols described by

Bailey and Parks, 1975; Megumi et al., 2005. For semi-quantitative sterol analysis of total and

free ergosterol, 0.026 moless of the internal standard cholesterol (e.g. 3 [tl 8.7 mM solution of

cholesterol prepared in ethyl acetate) were added to dried cells before extraction. Samples were

briefly dried under a stream of N2, and followed by sterol extraction. On the other hand, for









quantitation, 0.625 [tM cholesterol was added to the samples after extraction and just before

injection into the HPLC-MS instrument in order to compensate for variations in ionization

efficiency and stability of the samples.

Total sterol extraction, was performed as follows. Cells were suspended in 1 mL absolute

ethanol followed by addition of 6 mL of methanol and 0.4 g of KOH. Saponification of the

mixtures was carried out by heating at 750C for 40 min. After the mixture was cooled down at

room temperature, 2 mL of sterile water was added, and the sterols were extracted twice with 2

mL of petroleum ether, each time. The fractions of petroleum ether were collected and

evaporated to dryness under a stream of N2, and then weighed.

For free ergosterol extraction, dried cells were suspended in 400 ptL of DMSO, and heated

for 1 h at 100C. After cooling at room temperature, the mixture was mixed with 3 mL of sterile

H20 and then extracted three times with 2 mL of petroleum ether. After each extraction,

suspensions were briefly centrifuged to help layer separation. Extracts containing the free

ergosterol, (enriched in the petroleum ether layer) were combined and evaporated to dryness

under a stream of N2.

Sterol Analysis by Ultraviolet Spectroscopy

For qualitative and semi-quantitative analysis of ergosterol and A5, 7, 22, 24(28)-

dehydroergosterol [4(28)-DHE], total sterol extracts were scanned spectrophotometrically

between 190 and 310 nm using a UV-visible CARY Varian spectrophotometer. Quantitation of

the ergosterol and 24(28)-DHE contents was performed according to the method described by

Breivik and Owades, 1957, applying the equations: % ergosterol + % 24(28)-DHE = (A281 / 290)

/ pellet weight. % 24(28)-DHE = (A230 / 518) / pellet weight. % ergosterol = (% ergosterol + %

24(28)-DHE) % 24(28)-DHE, where 290 and 518 are the E values (in percentages per









centimeter) determined for crystalline ergosterol and 24(28)-DHE, respectively (Breivik and

Owades, 1957). Results are reported as the percentage of each sterol per wet weight of cells

Analysis of Sterols by Gas Chromatography-Mass Spectrometry (GC-MS)

Before GC-MS analysis, sterol extracts as well as ergosterol, lanosterol and cholesterol

were converted into their more volatile trimethylsilyl (TMS) ether counterparts following the

procedure described by Gerst et al., 1997 with some modifications. Briefly, non-saponifiable

lipid extracts (100 [tL) were transferred into glass screw-cap GC vials, and spiked with 5 pL of a

2.58 nM solution of cholesterol. After vortexing thoroughly, suspensions were dried under a

stream of N2. Dried samples, and standards were treated with 200 p.L of a N,O-

bis(trimethylsilyl)-trifluoroacetamine (BSTFA) with 1% trimetylchlorosilane (TMCS), (Fluka).

The silylation reactions were carried out at 450C, in the dark, for 1 h. Silylated sterols were

identified by GC-MS, by comparing their retention times with the silylated standards. This was

performed by Dr. Cristina Dancel at the Mass Spectrometry Laboratory, department of

Chemistry, University of Florida)

Tandem GC-MS was done on a Thermo Scientific Trace DSQ instrument, equipped with

an Rtx-5MS column (15 m x 0.25 mm i.d. x 0.25 ptm d.f.). 2 [tl of each solution was introduced

into the capillary column at 1-min splitless mode, with helium carrier gas flowing at a rate of 0.7

mL/min. The injector was kept at 2250C while the transfer line and ion source were at 2000C.

The GC column was maintained at 2000C for 2 min and then heated to 2800C at 200C/min. Mass

spectra were generated via electron ionization (EI) and compared against those in the NIST El

Spectral Library (Appendix C). The silylated derivatives of standard ergosterol and lanosterol

were analyzed to validate retention times and to generate a calibration curve with cholesterol as

the internal standard.









The calibration curve was generated by plotting the peak area ratio of the standards

ergosterol and lanosterol to the peak area of cholesterol against the concentration in picomolar

(pM) of each standard. The concentrations of ergosterol and lanosterol in the samples were

determined from the calibration curve, and expressed in picomolar (pM).

Analysis of Total and Free Ergosterol by High Performance Liquid Chromatography-
Atmospheric Pressure Chemical Ionization- Mass Spectrometry (HPLC-APCI-MS)

HPLC-APCI-MS analysis of sterols was done (in conjunction with Dr. Cristina Dancel at

the Mass Spectrometry laboratory, department of Chemistry, University of Florida), using an

Agilent 1200 Series HPLC System equipped with a Phenomenex Luna C18 column (150 x 2.0

mm, 5[t). Sterol extracts were dissolved in 9:1 isopropanol-hexane and 5 ptL of each solution was

injected. Methanol was used as the mobile phase in isocratic mode and at a flow rate of 0.4

mL/min. Ions were obtained by atmospheric chemical ionization (APCI) and detected with an

Agilent 6210 time-of-flight (TOF) mass spectrometer. For semi-quantitation, and quantitation

analyses, a stock solution of 25 [tM ergosterol was prepared in 9:1, v/v isopropanol : hexane, and

diluted with methanol before injection. Semi-quantifitation of total and free ergosterol was based

on comparing the peak areas of the sterol chromatograms as follows. The ratio of peak area for

ergosterol to peak area for cholesterol was calculated, and normalizing to OD600 of 1.0. Free

ergosterol quantitation was based on the response factor method and using cholesterol as internal

standard.

Results

IZH Genes Affect the Tolerance to the Antifungal Nystatin

We have observed that single and multiple deletions of the IZH genes grow with wild type

characteristics (Figure 4-3 A) indicating that none of these genes is required for viability.

However, under exposure to the antifungal drug nystatin, defects in the growth were observed for









the wild type BY4742 and for all the izhA mutant strains except for izh3A, which displayed a

remarkably resistant phenotype under these conditions (Figure 4-3).

In order to investigate if the resistant phenotype observed for izh3A was not due to side

mutations during the period of incubation with nystatin, complementation studies were

performed. If izh3A is in fact resistant to nystatin, we would expect that when the IZH3 gene is

re-introduced into the izh3 mutant strain, the sensitivity to nystatin must rescue. In order to

investigate this, the centromeric plasmid pRS315-CEN (empty vector), and the pRS315-CEN

plasmid vector containing the IZH3 gene driven by its own promoter were introduced into the

izh3A strain by yeast transformation. Likewise, the effect of overexpressing IZH3 gene in

presence of nystatin was also investigated by introducing the pRS316-GAL] plasmid vector or

the pRS316-GAL] containing the IZH3 gene into the izh3A by the same procedure. In both cases,

IZH3 restored the wild type nystatin-growth defect in the resistant strain (Figures 4-3 B and 4-3

C). In addition, when the all the IZH1, IZH2, andlZH4 genes and the vector control (pRS316-

GALl) were overexpressed in a medium supplemented with 2.5 and 5 U/mL nystatin, the cells

grew better. Interestingly under these conditions IZH3 showed defects in its growth. At a higher

concentration of nystatin (e.g. 10 U/mL) cells did not survive (Figure 4-3 D). These latter results

confirm that the presence of the IZH3 gene produces sensitivity to nystatin and that the presence

of IZH1, IZH2, and IZH4 has the opposite effect conferring resistance to nystatin.

Another important aspect was investigating if the resistance of izh3A to nystatin occurred

at a particular stage of growth, or if it was independent on the state of growth. In yeast, a growth

curve has different stages, which can be monitored by measuring the optical density of the

growth cultures (OD600) (Figure 4-4 A). To investigate if the effect of nystatin was dependent of

the cell growth stage, phenotypic studies of WT and izh3A were analyzed in presence of nystatin,









at different OD600 [e.g. 0.25 and 0.5 (logarithmic phase) and 3.4 (stationary phase)].

Interestingly, izh3A always showed resistance to nystatin independently of the stage of growth

(Figure 4-4 B).

Nystatin Induces Alterations in the Total Sterol Composition of Wild Type and izh3A

Because nystatin preferentially targets sterols in the plasma membrane, we envisioned the

possibility that cells harboring the izh3A mutation shows alterations in the sterol composition or

the sterol content, and therefore that the IZH3 gene is required to maintain the appropriate levels

of sterols in the membranes. We explored this possibility by determining the sterol content in

both wild type and izh3A. To do this, ultraviolet spectrophotometry was used as a first approach.

This technique takes advantage of the unique spectral absorption patterns of ergosterol and its

precursor 24(28)-DHE. In fact, both sterols have a characteristic UV spectrum with three peaks

at 271, 281, and 293 nm, with a maximum at 281 nm (Figure 4-5 A). In addition, 24(28)-DHE

has a side-chain diene that results in absorptions at 230, and 235 nm (Figure 4-5 B). The UV

spectra of the sterol extracts of wild type and the izh3A indicate that these strains have the same

sterol profile. In addition a peak at 205 nm was also identified, which corresponds to squalene

(Figures 4-5 A and B).

In absence of nystatin, the content of ergosterol and 24(28)-DHE was the same (Figure 4-6

A), although, the levels of 24(28)-DHE were very low (< 0.2%). Interestingly, upon addition of

of nystatin, 24(28)-DHE seems to be accumulated (Figure 4-6 B). On the other hand no

significant difference was observed for the levels of ergosterol (Figure 4-6 B). These results

suggest that nystatin induces the production of precursors of ergosterol. Unfortunately, UV-VIS

did not allow us to identify sterols other rather than squalene, ergosterol and 24(28)-DHE.









Gas Chromatography-Mass Spectrometric (GC-MS) Analysis Revealed not Significant
Differences in the Basal Levels of Total Sterols for Wild Type and izh3A

Even though, UV spectrophotometry has been widely used for the analysis of sterols that

contain the A5,7- diene systems, identification of other sterols is outside the capability of this

technique (Woods, 1971). Therefore, GC-MS was used as an alternative method not only to

validate the UV-spectrophotometric results but also to characterize other sterols and to quantify

them by using standards. Before analysis, samples had to be derivatized by silylation with

BSTFA containing 1.0% TMCS. The resultant silylated ethers were then suitable for electron

ionization and mass spectrometric analysis. A calibration curves using ergosterol and lanosterol

allowed to quantitfy the content of these sterols in the samples analyzed (Figure 4-7 A)

(Appendix C). Interestingly, the data generated with this technique suggest that both wild type

and izh3A have the same basal levels of ergosterol and lanosterol (Figure 4-7 B). Since 24(28)-

DHE is not commertially available, we could not quantify this sterol in our samples.

Alterations in the Free Ergosterol Content Were Observed for the Mutant izh3A

Ergosterol can be present in two forms, forming esters with fatty acids (steryl ergosterol)

and as free ergosterol. As mentioned elsewhere in this chapter, free ergosterol is primarily found

in plasma membrane where it plays a structural role, contributing to maintain the integrity of the

plasma membrane (Veen et al., 2003). To investigate the total and free ergosterol content in wild

type and the mutant izh3A, HPLC-APCI-MS was used. One advantage of using HPLC-APCI-MS

over GC-MS is that the first one does not require pre-treatment of the samples (or derivatization),

which is time consuming, and can affect the results if the dreivatixation is incomplete. The use of

HLPC-MS let us establish significant conclusions regarding the ergosterol content of

izh3A versus that one of wild type. First, we confirmed that the total ergosterol content was









similar for both samples (Figure 4-8 A).This observation was in agreement with the results

obtained with UV-spectrophotometry and GC-MS analysis.

Because ergosterol is the most abundant sterol in yeast, and a well known-target for

nystatin, we hypothesize that the izh3A's plasma membrane has alterations in the ergosterol

content. Plasma membrane has been proposed to be the place where nystatin makes a first

contact (Sharma, 2006). Since free ergosterol is predominantly found in plasma membrane, we

reasoned that izh3A could have less free ergosterol than wild type; and consequently nystatin

would have less chance to be deleterous for the mutant yeast membrane. To test this hypothesis,

we first semi-quantifed the free ergosterol content of izh3A and wild type, by using HPLC-APCI-

MS. As we expected, the content of free ergosterol in the mutant was dramatically diminished

(Figure 4-8 B).

In an effort to quantify free ergosterol in both WT (pRS315-CEN-IZH3) and the mutant

izh3A (pRS315-CEN transformed with a izh3A strain) single standard quantitation method was

used. To do this, the peak area for ergosterol's m/z 379.3, was normalized to the peak area

corresponding to cholesterol at m/z 369.3 (internal standard). Concentration of ergosterol in each

sample was calculated using a 3.75-tlM standard solution, and normalized to OD600 (For each

experiment OD600 was 1.0, see materials and methods for more details). Free ergosterol content

is reported in [tM. This method allowed us to quantify free ergosterol and demonstrate that izh3A

has altered sterol composition compared to wild type, (Figure 4-8 C) (Appendix D). Our results

not only support our hypothesis, but also, suggest an implication of ZH3 in the sterol

biosynthetic pathway.









Addition of Certain Sphingolipids Ameliorate the Aberrant Nystatin Effects on Wild Type
and izhA Mutants

In addition to sterols, sphingolipids are other important components of plasma membranes

(Eisenkolb et al., 2002). Kerridge, 1986 and Leber et al., 1997 suggested that besides sterols,

certain sphingolipids like the mannosyl-diionosytolphosphorylceramide, the most abundant

sphingolipid if S. cerevisiae, are also be targets for nystatin. With this in mind, we investigated

the effect on the growth of wild type and the single, and multiple izhA mutations upon the

simultaneous addition of nystatin and sphingolipids to the growth culture. To do this, phenotypic

studies in solid agar-syntheric medium were conducted. Interestingly, the sphingoid bases Cls-

PHS and Cis-DHS were able to alleviate the toxic effects of nystatin (Figure 4-9). In fact, in a

medium supplemented with any of the sphingoid bases (e.g. Cis-PHS or Cis-DHS) and nystatin,

the growth of wild ype and the single and multiple izhA mutations was normal (Figure 4-9). On

the other hand, ceramides and stearylamine (a sphingolipid-like compound) failed to alleviate the

toxic effects of nystatin (Figure 4-10 A and B, respectively). Taken together, these results

confirm our idea that sphingolipids and sterols like ergosterol could be potential ligands for

nystatin.

Discussion

In this chapter, we have identified izh3A as a novel nystatin-resistant mutant. Even more

interesting, we have discovered this phenotype is associated with alterations in the sterol content

for the mutant strain. This finding suggests a potential implication oflZH3 gene in sterol

metabolism.

We first, have shown that the growth of izh3A, upon addition of nystatin, was normal

compared to the defects in the growth observed for the wild type strain and the other single and

multiple mutation strains (Figure 4-3 A). The discovery that IZH3 rescues the sensitivity of the









cell to nystatin, suggests that the observed nystatin resistant phenotype for izh3A is not due to

side-mutations and that is in fact, the mutation oflZH3, per se, which produces such a phenotype

(Figure 4-3 B, C, and D). Additionally, we found that the overexpression of ZH], IZH2, and

IZH4, but not IZH3 ameliorate the toxic effect of nystatin (in a concentration-dependent

manner), with the concomitant resistant phenotype (Figure 4-3 D). Another important discovery

was that the resistant of izh3A to nystain is independent on the growth stage (Figure 4-4 B).

Based on this result we could speculate that the izh3A plasma membrane has a lipidic

composition that is perhaps different from the other izhA mutants and the wild type. Thus,

experiments that elucidate the lipidic composition of the izh3A plasma membrane can generate

valuable information that contributes to understand the izh3A resistant phenotype.

Because nystatin preferentially interacts with ergosterol (Ghannoum and Rice, 1999;

Sharma, 2006), this lipid became the first candidate to investigate. Also, we contemplated the

possibility that IZH3 could have a role in the sterol biosynthetic pathway. It is known that upon

mutation, genes that are implicated in the sterol pathway have alterations in the sterol

composition or sterol content (Arthinggton-Skaggs et al., 1996; Baudry et al., 2001). Therefore,

as a first approach to justify the observed phenotypes, we used different analytical techniques

like UV-spectrophotometry, and gas-chromatography/mass spectrometry (GC-MS), to analyze

total sterol composition and content in wild type and izh3A. No differences in the total sterol

profile and content of both samples were detected. However, upon addition of nystatin,

alterations in the sterol composition of both strains were observed by UV-spectrophotometry.

For instance, while an increase in the levels of the intermediate 24(28)-DHE was detected no

significant changes in the ergosterol content were observed with all of the techniques used during

the analysis. This result confirms the specificity between nystatin and ergosterol.









Free ergosterol, and esterified ergosterol accounts for total ergosterol in yeast. Free

ergosterol is accumulated at high concentration in plasma membrane (Zinser et al., 1993), being

a suitable target for nystatin. We hypothesized that izh3A is resistant to nystatin due to alterations

in the free ergosterol content. We explored this possibility using HPLC-MS analysis of free

ergosterol versus total ergosterol. The advantage of using HPLC-MS is that it does not require

derivatization like the GC-MS technique. Interestingly, we found that izh3A is more deficient in

free ergosterol than its wild type counterpart. This result truly explains the resistance phenotypes

observed by this mutant. On the other hand, the levels of total ergosterol appear almost identical

for both the mutant and wild type. It is quite possible that izh3A has more esterified ergosterol

than wild type. Therefore, the presence of IZH3 is necessary to maintain the levels of free

ergosterol in the plasma membranes.

Besides sterols, the effects of antifungals like azoles are also associated with defects in the

sphingolipid content. For example, genes involved on the synthesis of phytosphingosine (SUR2,

or LCB1), are down-regulated by azole drugs (Veen and Lang, 2005). Our observations that

sphingoid bases reconstitute the normal growth of wild type and mutants seem reasonable. We

do not discard the the possibility that once added to the growth media, the sphingoid bases are

embedded in the plasma membrane protecting it against the toxic effects of nystatin. It is also

possible that when added simultaneously to the cells, nystatin competes for a place in plasma

membrane with the sphingoid bases. Therefore, experiments conducted to test these possibilities

are necessary.

Based on our results, it is important to investigate the transcriptional response of the IZH3

gene in order to have a better idea about the role, if any, that this gene plays in the sterol

pathway. Overall, the results presented in this chapter open an avenue regarding the role of IZH3









that demands further investigation. For example, the steryl ester composition of WT and izh3A

must be investigated. It is plausible, that izh3A has more steryl esters than the WT strain, which

could explain why the mutant strain has less free ergosterol available for the nystatin action.









Table 4-1. Sources and genotypes of the strains used in Chapter 4


Mutation


BY4742

TLY9

TLY50

Y01578

TLY46

TLY20


TLY41



TLY45


Strain


izhlizh2izh3izh4 Thomas Lyons


Genotype


Source/Derivation

EUROSCARF

Thomas Lyons

Thomas Lyons

Thomas Lyons

Thomas Lyons

Thomas Lyons


Thomas Lyons



Thomas Lyons


Wild type

izh]

izh2

izh3

izh4

izhlizh2


izh2izh4



izhlizh2izh4


MAT a; his3; leu2; ura3; lys2

MAT a; his3;leu2; ura3;lys2;
izhl::kanMX4
MAT a; his3; leu2; ura3; lys2;
izh2::hphMX4
MAT a; his3; leu2; ura3;
metl5; lys2; izh3::KanMX4
MAT c; his3; leu2; ura3;
lys2; izh4::ura3MAX4
MAT c; his3; leu2; ura3;
lys2; izhl::kanMX4; izh2::
hphMX4
MAT a; his3; leu2; ura3;lys2;
izh2::hphMX4; izh4::
ura3MAX4

MAT a; his3; leu2; ura3;lys2;
izhl::KanMX4; izh2::
hphMX4; izh4::ura3MX4


MAT c; his3; leu2; ura3;
lys2; izhl::KanMX4; izh2::
hphMX4; izh3:: natMX4;
izh4::ura3MX4


TLY23




















CH3
Lanosterol


Squalene


CH2


CH3


CH3


24(28)-Dehydroergosterol Ergosterol


Cholesterol

Figure 4-1. Chemical structures of the sterols analyzed in this chapter.





Plasma
membrane
I1SS


Mil
0 W 1
94


Nutrients
Ny.


Nys Plasma
membrane


SOH
COOH


Figure 4-2. Model proposed for the nystatin action in the yeast plasma membrane. A shows an
intact yeast plasma membrane. B the interaction between nystatin and ergosterol
produces the formation of holes in the plasma membrane with the consequent leakage
of nutrients. C shows the chemical structure of nystatin.


(INyS)


,snuPtl
If;""""'










0 UlmL Nuv 1Q UlmL Nuv


WT

izhlA

izh2A

izh3A

izh4A





D


pRS316-GAL1
pRS316-GAL1-IZH1
pRS316-GAL2-IZH2
pRS316-GAL I-ZH3
pRS316-GAL1-lZH4


O UlmL 2.5 U/mL
N Nys


WT

pRS315-izh3A

pRS315-lZH3-
izh3A


5 U/mL 10 U/mL


Figure 4-3. Nystatin-dependent phenotypes. A shows the growth of wild type and the izhA
mutant strains on synthetic medium agar plates supplemented with +/- nystatin. B
and C illustrate the complementation of the nystatin-dependent phenotype by the
pRS315-CENplasmid and pRS316-GAL], respectively. In B, the single copy plasmid
(pRS315), or pRS315 expressing IZH3 was transformed into the izh3A strain. In
panel C, the plasmid containing the pRS316-GAL], or expressing IZH3, was
transformed into the izh3A strain. D shows the effect of different concentration of
nystatin onto the growth of cells overexpressing IZHs. In this case the pRS316-GAL1
containing each IZH was tested. When CEN plasmid was used plates were
supplemented with 2% glucose. For gene overexpression, 2% galactose was used.











A
1.4 -- BY4742WT

izh3A
1.2


1.0 -
Stationary
phase
o 0.8
(D
o 0.6 Logarithmic
phase

0.4 -
Lag
phase
0.2 -

F without nystatin
0.0 .
0 10 20 30 40 50 60

Time / h


B No
Nystatin 10 U/ml Nytstain

ODo 0.50 0.25 0.50 3.50


WT-1
Izh3A-1
WT-2

zh3A-2

WT-3
zh3A-3


Figure 4-4. Effect of nystatin on izh3A and the wild type strain BY4742 at different stages of
growth. Panel A shows a typic growth curve generated to illustrate the different
growth stages of both WT and izh3A. B shows that izh3A is remarkable resistant to
nystatin regardless its growth stage.


7.


















0.8


O 0.6
0
,,
0.4


0.2


0.0


200 220 240 260 280 300 320
X/ nm


200 220 240 260 280 300 320
X/ nm


Figure 4-5. Ultraviolet spectrophotometric characterization of total A5-7 sterols in wild type
strain and izh3A. A represents the UV spectra for WT and izh3A obtained in absence
of nystatin. B shows the UV spectra for WT and izh3A, when the cells are exposed to
5 U/mL of nystatin.



1.0 1.4
A wT B
SH izh3A 1.2
0.8
o 1.0
0.6 .
o 0.8

0.4 0.6

0.4
0.2
0.2

0.0 0.0
Ergosterol 24(28)-DHE Ergosterol 24(28)-DHE

(-) nystatin (+) nystatin
Figure 4-6. Semi-quantitation of total ergosterol and 24(28)-DHE content in wild type and izh3A
by UV-spectrophotometry. A shows the basal levels of ergosterol and 24(28)-DHE.
B shows the levels of both sterols in cells exposed to 5 U/mL of nystatin. In the
figure, the content of each sterol is expressed as the percentage of total ergosterol per
wet weight of cells.


0.6



S0.4
0
0
U)
S0.2



0.0













Sterol TMS Calibration Curves C 100-
1.2


y- y=0.007x-0.0286 8
R'=0.9902
y=0.0047x-0.0283 0
o l R'=0.9978 U
UE 0.3 40


Concentration pM50 100 150 200 20

0 -
Ergosterol Lanosterol

Figure 4-7. Quantitation of basal levels of total ergosterol and lanosterol of WT and izh3A by
GC-MS. In the figure, A shows a calibration curve generated with the standards
ergosterol, lanosterol, and cholesterol (internal standard). B shows the concentration
(pM) of total ergosterol and lanoterol derived form the calibration curve. Data are the
mean + standard deviation of three experiments.













3.5 10
o 0
o A I B
o 3.0
0 0 8
O 2.5 o


2.0 6

1.5 _
0 4
1.0 -

M m 2
S0.5 -

0.0 0
WT izh3A WT izh3A

12
C izh3A
.5 WT

I-



0



0

"2 8
0


WT izh3A


Figure 4-8. Total and free ergosterol content on WT and izh3A. The figure shows the semi-
quantitative analysis of total ergosterol, A and free ergosterol B, using HPLC-APCI-
MS. C shows the quantitation analysis of free ergosterol for WT and izh3A by using
HPLC-APCI-MS. Data shown in each panel represent the mean standard deviation
of two independent experiments.









10 UlmL Nys 10 U/mL Nys


12.5 MM + 12.5 MM 12.5 0M
Control 10 UlmL Nys C1,-PHS C18-PHS C10"DHS
WT
izh A]
izh2 2A
izh3A
izh4A
izh12A
izh24A
izh124A
izhl1234A


+ 12.5 pM
C,8-DHS


Figure 4-9. Sphingoid bases override the toxic effects of nystatin. To generate the figure, 5 jtl of
10-fold OD600 serial dilutions were spotted on agar-simthetic medium (e.g. from the
left to the right OD600 of 1.0, 0.1, and 0.01, respectively) and incubated for 3 days at
30C.












0.5 pM
Control C2"PHC


0.5 pM
C16-PHC


0.5 pM 10U/mL
C,,1PHC Nys


10 U/mL
Nys +
12.5 pM 12.5 iM
Control Sterylamine Sterylamine


WT
izh A
izh2A
izh3A
izh4A
izh 2A
izh24A
izh124A
izh 234A


Figure 4-10. Effect of ceramides and stearylamine in cells exposed to nystatin. The figure
shows that contrary to the effect of sphingoid bases, ceramides and the sphingoid base
homolog stearylamine, are unable to ameliorate the toxic effect of nystatin.


10 UImL
Nys +
12.5 pM
C.-PHC


10 U/mL
Nys +
12.5 pM
C..-PHC


WT
izhlA
izh2A
izh3A
izh4A
izhl2A
izh24A
izh124A
izh1234A


10 U/m L
Nys +
12.5 pM
C,,-PHC









CHAPTER 5
POTENTIAL IMPLICATION OF IZHS IN THE SPHINGOLIPID BIOSYNTHETIC
PATHWAY

Introduction

Sphingolipids are a group of ubiquitous lipids found in all eukaryotic cells where they

comprise 10% to 20% of the total lipid species found in membranes (Smith et al., 1974).

Structurally, sphingolipids are formed by three elements; the sphingoid base backbone (or long

chain base), a very long chain fatty acid, and a head group. In yeast, fatty acids with a chain of

26 carbons in length, is common (Lester and Dickson, 1993). The yeast Saccharomyces

cerevisiae, has only three complex sphingolipids named inositol phosphoryl ceramide (IPC),

mannosyl inositol phosphorylceramide (MIPC), and mannosyl diinositolphosphoryl ceramide,

(M(IP)2C) (Dickson, 1998). The yeast S. cerevisiae has served as unique model to dissect and

understand metabolic pathways and has emerged as a scaffold upon which sphingolipid

metabolism and function can be elucidated.

Originally, sphingolipids were considered only as structural components whose main role

was to protect the cell surface against potential harsh and hostile environments (Hannun and

Bell, 1989; Meer et al., 2002). In fact, in plasma membranes, sphingolipids are found tightly

packed with sterols forming microdomains termed "lipid rafts" (Toulmay et al., 2007). Recent

studies have demonstrated that the role of the sphingolipids goes beyond a simple structural role,

and that they can also play roles as second messengers implicated in signal transduction. In this

sense, sphingolipids can mediate cellular differentiation and apoptosis (Merrill et al., 1997;

Edsall et al., 1997; Hannun and Obeid, 2002; Dickson and Lester, 2002).

Multiple sequence alignments as well as phylogenetic tree analysis have revealed that the

Izhp family shares distant homology with a group of enzymes known as alkaline ceramidases.

Ceramidases hydrolyze the amide linkage of ceramide to generate a free sphingoid base and the









corresponding fatty acid (Figure 5-1). In yeast, two alkaline ceramidases (Ypclp and Ydclp)

have been characterized (Mao et al., 2000a; Mao et al., 200b). Interestingly, Ypclp and Ydclp

also catalyze the reverse ceramidase reaction with the concomitant formation of phytoceramide

and dihydroceramide from free sphingoid bases and fatty acids (Mao et al., 2000a; Mao et al.,

2000b; Vallee et al., 2005), (Figure 5-2). This latter reaction is usually catalyzed by two

ceramide synthases called Laglp and Laclp (Vallee et al., 2005).

Different observations have highlighted the possibility that the IZHs could be implicated in

the sphingolipid biosynthetic pathway. Herein, we present evidence that indicates that the

overexpression of the IZHs results in an increase in the levels of sphingoid bases and certain

ceramides. However, in vitro ceramidase assays suggest, that Izhs are not alkaline ceramidases.

Alternatively, we investigate the possibility that the IZHs may modulate the levels of

sphingolipids by affecting the de novo sphingolipid biosynthesis. As is well known, sphingoid

bases can be synthesized de novo as follows. In this case, serine is condensed with palmitoyl-

CoA via serine palmitoyl transferase (SPT), to form 3-keto-dehydrosphingosine, which by

successive reactions is converted to the sphingoid bases dehydrosphingosine and

phytosphingosine (Perry, 2002; Menalindo et al., 2003; Cowart and Obeid, 2007) (Figure 5-3 A).

In an effort to investigate if the IZHs could be implicated in de novo sphingolipid

biosynthesis, three different experimental approaches are explored. First, qualitative analysis of

radioactive sphingolipids indicates that the levels of sphingoid bases produced under IZH

overexpression are bigger than those produced by the vector control, but similar to the levels

produced when YPC1 is overexpressed. Second, the use of myriocin, an inhibitor that blocks de

novo biosynthesis of sphingoid bases via serine palmitoyl transferase, significantly reduces the

levels of sphingoid bases produced when the Izhs are overexpressed. However, the effects of









myriocin on the levels of sphingoid bases found when YPC1 was overexpressed and those in the

vector control are barely affected. Finally, the effect of fumonisin B1 (FBi), an inhibitor that

inhibits ceramide synthase, was investigated. Our results indicate that after treatment with this

drug, an accumulation of sphingoid bases in all the samples analyzed is produced, suggesting that

the role of Izhs is more related to the de novo sphingolid biosynthesis.

Although, still preliminary, our results suggest a connection between the levels of

sphingolipids (specifically sphingoid bases and ceramides) and the high dosage oflZHs in the

cell.

Materials and Methods

Strains Plasmids and Yeast Transformations

For the studies described in this chapter, the strains and their respective genotypes are

described in table 5-1. The YPC1 and IZH1-4 open reading frames were placed under the control

of galactose inducible GAL] promoter, by using the plasmid vector pRS316, which contained the

URA selection marker gene. The pRS316-GAL1 (empty vector) and the vector containing the

YPC1 and each of the IZHs (pRS316-GAL1-YPC1 and, pRS316-GAL]- IZH1-4, respectively)

were transformed with the wild type strain BY4742, or with the double mutant ypclAydclA

(Gietz and Woods, 1994). The double mutant strain ypclAydclA was kindly provided by Dr.

Howard Riezman (howard.riezman@biochem.unige.ch).

Yeast Growth Conditions

Unless otherwise stated, the compounds used for these studies were dissolved in either

100% ethanol, or DMSO. When mentioned, the detergent Tergitol NP-40 was used to facilitate

the uptake of certain sphingolipids by the cells. At low percentage, this detergent does not have

any significant effect on the growth of cells. The inhibitors, myriocin and fumonisin B1 (FBi)

were obtained from Sigma.









For gene overexpression, cells lacking YPC1 and YDC1 or wild type cells were

transformed in identical fashion as mentioned elsewhere in this dissertation. When used, N-

acetylphytosphingosine (or C2-PHC), (Avanti Polar Lipids), was prepared in 100% ethanol. Cells

in early logarithmic phase, (e.g. OD600 of 0.2) were spiked with C2-PHC to a final concentration

of 278 nM, and grown to OD600 was 0.8 (usually for 1 h).

When fumonisin B1 (FBi) was used, cells were prepared according to the procedure

described by Wu et al., 1995 with some minor modifications. Cells were grown at 300C in 30 mL

of uridine-limited synthetic medium (SGal-Uri), supplemented with 0.005% Tergitol NP-40

(Sigma); in the absence or in presence FB1 to 100 [tM. Cells were grown to OD600 of 0.8 and

then harvested to collect pellets.

Likewise, when myriocin was used, cells were grown in 30 mL of synthetic medium

(SGal-Uri), in the absence and in presence of 1.0 [LM myriocin were grown to OD600 of 0.8. Cells

were harvested and resultant pellets were washed twice with cool sterile water and used for total

lipid extraction.

In Vitro Ceramidase Assay

Microsomes were isolated from the yeast cells according to Mao et al., 2000a; Mao et al.,

2000b with some minor modifications as follows. Cells were suspended in 0.5 mL of lysis buffer

A (20 mM Tris-HC1, pH 7.4, 1.0 mM EDTA, and the protease inhibitor mix form Sigma). Acid-

washed glass beads were added to just below the meniscus (1/3 beads per every 1/3 of cell

pellets). Cells were homogenized three times (3 min for 30 sec), and at 40C using a Mini-bead-

beater-8 (Biospec Products) set at the maximum speed. The cells were chilled on ice for 1 min

between homogenizations. The resultant supernatant was transferred to a new tube after

centrifugation at 2,000 rpm for 10 min. Unbroken cells and cell debris were removed by









centrifugation at 4,000 rpm for 10 min. To pellet the membrane fraction, the supernatant was

centrifuged at 40,000 rpm for 40 min at 40C. The membrane fraction was rinsed gently with the

lysis buffer A and suspended in 100 ptL of the same buffer A. Protein concentration was

determined by the BCA kit (Pierce), and using BSA as standard.

Ceramidase activity was determined by the release of NBD-fatty acid from fluorescent

substrates, NBD-C12-ceramide, NBD-C12-dihydroceramide (Matreya) and NBD-C12-

phytoceramide (kindly provided by Dr Yusuf A. Hannun, hannun@musc.edu) described by Mao

et al., 2003 with some modifications. Briefly, 20 ptL of microsomes (containing 10 |tg of protein)

in buffer B (25 mM Tris-HC1, 0.5 mM CaCl2at three different pHs, 8.6, 9.4, and 7.0) was mixed

with 20 ptL of each substrate in buffer B with 0.4% Nonidet P-40 in a 1.5 mL microfuge tube.

After incubation at 300C for 60 min, the reactions were stopped by boiling for 5 min, and dried in

a heating block at 800C during 20 min in the dark. The C12-NBD-dodecanoic acid, (generously

provided by Dr. Yusuf. A. Hannun) was used as marker for the completion of the ceramidase

reaction. Additionally, this fluorescent fatty acid in one of the products released from the

catalytic breakdown ofNBD-C12-sphingolipids by the ceramidases. Reaction mixtures were

dissolved in 30 ptL of chloroform : methanol (2:1, v/v). 25 ptL of each sample, the substrates and

the NBD-dodecanoic acid was applied onto a silica gel 60A TLC plate (Whatman) and resolved

by the solvent system composed by chloroform : methanol: 4.2 N ammonium hydroxide

(90:30:0.5, v/v/v). Fluorescent lipids were detected by scanning the TLC plate in a StormT 860

chromatoscanner (Molecular Dinamics), operated in the fluorescent mode, with an excitation

wavelength of 450 nm and an emission of wavelength of 525 nm.









Phenotypic Studies

Yeast cells where the YPC1 and YDC1 genes were mutated (ypclAydclA), and containing

the pRS316-GAL] (empty vector) or overexpressing each IZH gene or YPC1 were first grown to

stationary phase. After spinning down, pellets were suspended in uridine-limiting synthetic

medium supplemented with galactose, and appropriated amino acids. Aliquots (5 [tL) of the

overnight were plated at OD600 of 1.0, 0.1, and 0.01, onto solid-agar uridine-limiting synthetic

medium supplemented galactose, and appropriate amino acids, with or without myriocin. When

used, myriocin was prepared in DMSO (Sigma), and added fresh to solid-agar medium to

concentrations of 0.1 [LM and 1.0 [LM. Plates were incubated at 300C during 3 days, and followed

by growth analysis.

Total Lipid Extraction

Unless otherwise stated, all reagents used during this procedure were of high quality and

the solvents used were HPLC grade. Total lipid extraction was performed according to the Bligh-

Dyer method (Bligh and Dyer, 1959), with some modifications. Briefly, pellets were suspended

in 0.8 mL of water, and 3.0 mL of a mixture of methanol : chloroform (2:1, v/v). After vortexing

thoroughly, suspensions were left during 1 overnight, at 40C to permeate the cell wall and the

yeast plasma membrane. Cell debris was discarded by centrifugation at 3,000 rpm forl0 min, and

the supernatant was transferred to a sterile 13 X 10 mm glass screw capped borosilicated tubes

(Fisher). Supernatant was then mixed with 1.0 mL of chloroform and 1.0 mL of water and

vortexed vigorously for 1 min. The resultant suspension was settled down during 30 min at room

temperature, followed by centrifugation at 3,000 rpm for 10 min to help the separation of two

phases. The aqueous phase was aspirated, and the organic one was transferred to a new 13 X 10

mm glass screw capped borosilicated tube, and dried under a stream of N2. Dried fraction was









suspended in 300 ptL chloroform. One-third of this suspension (100 [tL) was dried and used to

determine phospholipids (this procedure is described in the next section). The remaining two-

third fraction, (200 [tL) were suspended in 800 ptL of a 0.125 M methanolic KOH solution and

incubated during 75 min in a 37C water bath to hydrolyze the acyl glycerolipids. After cooling

at room temperature, 1.4 mL of chloroform and 200 ptL 0.3 M HC1 were added to the mixture,

followed by the addition of 400 ptL of a solution of 1.0 M NaCl in 5% glycerol. Lipids were

extracted by vortexing vigorously during 3 min. Centrifugation at 3,000 rpm, during 5 min

allowed the formation of two phases. Again, the aqueous phase was aspirated and the organic

one was washed with 1 mL of neutralized water (prepared by mixing 1.0 M ammonium

hydroxide and water at a ratio of 1:300, v/v). After aspirating the aqueous phase, the organic

phase, which is enriched of sphingolipids was dried under a stream of N2, and stored at -20C

until HLPC analysis.

Phospholipid Determination

Phospholipid determination from the lipid extracts was performed, with some

modifications according to Merrill et al., 1988. Briefly, standards ranging from 0-80 nmol of

N2HPO4 (e.g. 0, 5, 10, 20, 40, 60, 80 ptL 1.0 mM NaHPO4) and one-third of the dried lipid

fraction (as described in former section) were mixed in 600 ptL of ashing buffer prepared by

mixing 10 N H2SO4, 70% perchloric acid, and water (1:9:40, v/v/v), and heated at 1600C during

1 overnight (18 h). After cooling at room temperature, the lipid fraction was mixed, by

vortexing, with 900 ptL of sterile water, 500 ptL of 0.9% (w/v) ammonium molybdate (Fisher)

and 200 ptL of a fresh solution of 9%, w/v L-ascorbic acid (Sigma). Standards and samples were

incubated during 30 min in a 45C- water bath. Absorbances were measured at 820 nm using a

Safire Xluor4D microplate reader (version 4.50). A standard calibration curve was generated by









plotting the nmol of the standards against their absorbance at 820 nm. The obtained curve was

used to calculate the nmol of phospholipids present in each sample.

High-Performance Liquid Chromatography Analysis of O-Phthalaldehyde-Sphingoid Base
Derivatives

Standards used in this section were obtained from Avanti Polar Lipids. Extracts containing

sphingolipids and the standards D-eryhtro-C18-DHS, D-ribo-C18-PHS, C20-PHS, and L-threo-

C18-DHS, were derived according to Merrill et al., 2000 with some minor modifications, and by

using the fluorescent reagent O-phthaladehyde (OPA), (Sigma). The following protocol was

performed by Charlene Alford, (Medical University of South Carolina, MUSC) who also ran the

samples in the HPLC instrument. The two-third dried lipid fractions were first dissolved in 100

tL of methanol. Fifty-[tL (50 [tL) of the standards and samples dissolved in methanol were

mixed with 50 jtL of the OPA reagent for derivatization. After thoroughly mixing, the mixtures

were incubated for 20 min under darkness, and at room temperature. The samples and the

standards were then centrifuged briefly to clarify and kept at 40C until HPLC analysis. The OPA

reagent was prepared by mixing 5 mg of OPA in 100 ptL 100% ethanol, with 5 p.L of 2-

mercaptoethanol and 9.9 mL of a solution of 3%, w/v boric acid (H3BO3), which was prepared in

water, and adjusting its pH to 10.5 with KOH. Before HPLC analysis samples and standards

were spiked with 25 pmol of the internal standard L-threo-C-i-DHS prepared in methanol (L-

threo-C1i-DHS is a non natural sphingoid base). Fluorescent derivatives were analyzed using a

Ci8 analytical Ultrasphere column with an internal diameter of 46 mm, and a length of 250 mm.

The HPLC (Breeze system Waters binary HPLC, model 1525) was coupled with a Shimadzu

RF-551 spectrofluoremetric detector (Beckman Coulter). The solvent system was methanol : 5

mM potassium phosphate, pH 7.0, (90:10, v/v). The OPA derivatives were detected using the

spectrofluoremetric detector, with an excitation wavelength of 345 nm and an emission









wavelength of 455 nm. The sphingoid bases were identified by comparing their elution profile

with those of the standards.

Analysis of Radiolabeled Sphingolipids by One-Dimensional Thin Layer Chromatography

As describe before, yeast cells where the YPC1 and YDC1 genes were mutated

(ypclAydclA), and containing the pRS316-GAL] (empty vector) or the pRS316-GAL],

expressing each IZH gene or YPC1 were first grown to stationary phase. After spinning down,

pellets were suspended in uridine-limiting synthetic medium supplemented with galactose. Fresh

synthetic medium supplemented with 2% galactose and appropriate amino acids was inoculated

with aliquots of the overnights to an OD600 of 0.1. Cells were to an OD600 of 0.5. Aliquots of 3.0

mL of each culture was transferred to 50 mL capped Falcon tubes and spiked with 150 ptL 1.0

mCi/mL [3H]serine (American Radiolabelled Chemicals, ART-246) during 40 min at 300C.

Subsequently, 1.0 mL of each culture was transferred to 13 X 10 mm glass screw capped

borosilicate tubes and spun down at 3,000 rpm, 4C for 5 min. After aspirating the supernatant,

the pellet was washed with cold sterile water, and immediately used for lipid extraction.

Lipid extraction was performed according to the Mandala procedure (Mandala et al., 1995).

Briefly, pellets were suspended by vortexing in 1.0 mL of the Mandala reagent [95% ethanol :

water : diethylether : Pyridine : NH40H, (15:15:5:1:0.018, v/v/v/v/v)]. Suspensions were then

incubated in a 60C- shaking water bath for 15 min. After centrifugation at 3,000 rpm, 10C for 5

min, the supernatant was pipeted off and saved. Pellets were re-suspended again in 500 ptL of the

Mandala reagent, and extraction was repeated one more time. The two supernatants were

combined and dried under a stream of N2. Dried samples were then suspended in 50 ptL of a

mixture of chloroform : methanol : water, (1:2:0.1, v/v/v) and vortexed hard for 15-20 sec.









Sphingolipids were analyzed by one-dimensional chromatography (TLC), on Whatman Partisil

LK6D silica gel 60 A, 20 X 20 cm size thin layer thickness 250 |tm plates, (Whatman). To do

this, aliquots of 25 p.L of each suspension and 10 ptL of the standards phytoceramide (PHC),

dihydroceramide (DHC), phytosphingosine (PHS), dihydrosphingosine (DHS),

phosphatidylserine (PS), phosphatidylinositol (PI), (type of standards) were applied to the TLC

pate, which was previously treated with 150 mL acetone for 45 min and equilibrated for 3 h in

100 mL of the solvent mixture choloroform : methanol : 4.2 N ammonium hydroxide, (9:7:2,

v/v/v). Lipids were resolved by using the same mixture of solvents. When the level of solvent

mixture reached 1 cm form the top edge, the plate was taken out the chamber and allowed to dry

at room temperature. The non-radioactive standards were visualized in iodine vapor. Radioactive

bands on the TLC plate were visualized by autoradiography after treatment with EN 3HANCE

(NEN Life Science) and exposure to a tritium screen. Radiolabelled lipids were compared with

the standards.

Analysis of Sphingolipids by Electrospray-Ionization Tandem Mass Spectrometry (ESI-
MS/MS)

All the experiments described in this section were performed using the strain ypclAydcIA

Internal standards were either prepared by the Lipidomics Core, at MUSC, or obtained from

Avanti Polar Lipids or Matreya. All solvents used were obtained from Fisher.

Sphingolipid analysis was performed by HPLC-Tandem mass spectrometry

(HPLC/MS/MS). High performance liquid chromatography (HPLC) was performed in a

"Surveyor" equipped with a quaternary HPLC pump, "Surveyor" an autosampler, and a BDS

Hypersil column (C8 150 x 3.2 mm; 3 |tm particle size), (Phenomenex). The HPLC system was

coupled with a triple quadrupole mass spectrometer equipped with a Thermo Finigan, PE Sciex









Electrospray Ion Source (EIS), a syringe pump, and syringes (5 [tL-1 tL) and a nitrogen

generator (Parker Hannifin Corp).

Cells containing the plasmid vector (pRS316-GAL]) or the vector containing each of the

genes YPC1, IZH2, and IZH3 (pRS316-GAL1-YPC1, pRS316-GALI-IZH2, pRS316-GAL1-

IZH3) were grown in synthetic media supplemented with galactose to OD600 = 0.8. Sample and

standards preparations were carried out in the lipid analysis were prepared by the Lipidomics

Core, at the Medical University of South Carolina (MUSC). Total lipid extractions were

performed according to Bielawski et al., 2006 with some modifications. A mixture of the

following internal standards was used, 1.0 [tM solution containing C17-sphingosine, C17-

sphingosine-l-P, 17C16-Ceramide, and 18C17-Ceramide and C6-phytoceramide in methanol were

used. Pellets were first spiked with 50 ptL of the internal standards solutions, and then mixed by

vortexing with 2.0 mL of the extraction mixture iso-propanol : water : ethanol : pyridine : 25%

ammonia, (5.0:1.5:1.0:0.2:0.04, v/v/v/v/v). Extracts were incubated at 600C for 15 min and then

vortexed thoroughly. Suspensions were centrifuged for 5 min at 3000 rpm, and the supernatant

was collected. Pellets were suspended in another 2 mL of the extraction mixture and the

procedure was repeated. The two supematants were combines and dried under a stream of N2.

Before analysis, extracts were suspended in 150 ptL of mobile phase (1.0 mM ammonium

format in methanol containing 0.2% formic acid), and vortexed. Extracts were centrifuges at

4,000 rpm for 5 min, and the supernatant was transferred to an autosampler HPLC vial with 200

tL insert. 20 ptL of this supernatant was injected on the HPLC system.

For quantitation, calibration curves for each of the internal standards were prepared as

follows. Cells were substituted with bovine serum albumin (BSA). This artificial matrix was

spiked with a known amount of the target analyte (sphingolipid of interest), and a constant









amount of the corresponding internal standard. The above described extraction procedure was

followed.

Standard extracts were analyzed by HPLC/MS/MS system in positive MRM mode

(Multiple Reaction Monitoring). Calibration curves were generated by plotting the peak area

ratio of analyte (sphingolipid of interest) / internal standard, against the concentration in pM of

each sphingolipid of interest. For quatitation, the peak areas were determined using extracted ion

chromatograms. The amount of each sphingolipid detected was normalized to the OD600 of each

sample; and the results are reported in pmol lipid/OD600.

Results

Overexpression of IZHs Produces Increase in the Levels of Free Sphingoid Bases

Ypclp and Ydclp degrade ceramides to produce sphingoid bases and fatty acids (Mao et

al., 2000a; Mao et al., 2000b). Because the Izhs have distant similarity with the yeast alkaline

ceramidases, we questioned whether the IZHs could be implicated in the sphingolipid metabolic

pathway. With this in mind, we first characterized and quantified the levels of sphingoid bases in

cells overexpressing the IZH genes. To do this, HPLC analysis of fluorescent sphingolipid

derivatives was performed. In a first trial, the pRS316-GAL] (empty vector) or the vector

expressing IZH2, IZH3, and YPC1 were transformed with a BY4742 wild type strain. Cis-PHS

and C18-DHS (the two major sphingoid bases found in yeast) were detected by HPLC. Not

surprisingly, an increase in the levels of these sphingoid bases was observed when IZH2 and

IZH3 were overexpressed. But even more prominent was the fact that the overexpression of

IZH3 produces more sphingoid bases than YPC1 (Figure 5-4). Although at first glance, we could

envision the possibility that IZHs encode proteins that play similar functions to ceramidases, we

wanted to make sure that the results obtained were not because we were using a wild type strain,

which contain the ceramidase genes YPC1 and YDC1. Therefore, we decided to investigate the









levels of sphingoid bases when the IZHs and YPC1 were overexpressed in the double mutant

strain ypclAydclA. With this in mind, two different experiments were performed. First, the basal

levels of sphingoid bases were investigated by HPLC. The results obtained indicated that the

levels of the sphingoid bases C18-PHS, C20-PHS, and C1i-DHS were increased, (Figure 5-5 A, B,

C). To investigate if an external stimulus like the addition of a ceramide could activate the

production of sphingoid bases in the analyzed samples, the effect of C2-PHC was monitored by

HPLC. Although C2-PHC is a non natural ceramide, it has the advantage that is less hydrophobic

that the natural C26-PHC. Therefore, it can easily be delivered into the cells. When used, C2-PHC

was added to a final concentration (278 nM) for a short period of time (e.g. 1 h) to avoid possible

artifacts. An increase in the levels of sphingoid bases was observed for the IZHs and YPC1

(Figure 5-5 D, E, and F).

Taken together, these results truly represent a first approach, and the first piece of evidence

suggesting a possible implication of IZHs in the sphingolipid metabolic pathway.

In Vitro Ceramidase Assays Suggest that Izhs May not be Alkaline Ceramidases

There are two ways to synthesize sphingoid bases. First, it can proceed by de novo

sphingolipid biosynthesis, a process that starts with the condensation of serine and a fatty acid

which is usually palmitic acid through the action of serine palmitoyl transferase (SPT). Second,

sphingoid bases can be synthesized from hydrolysis of ceramide by the action of ceramidases

(Figure 5-6 A). To investigate if the IZHs encode ceramidases, the vector pRS316 containing the

GAL] inducible promoter (pRS316-GAL]) and the vector expressing the YPC1 and the IZHs

(e.g. pRS316-GALI-YPC and pRS316-GALI-IZHs, respectively) were transformed with the

yeast double mutant strain ypclAydclA. Protein expression was induced in 2% galactose. To do

this, microsomes were prepared, as described under materials and methods, and assayed for









ceramidase activity at different pHs (pH 8.6, pH 9.4, and pH 7.0). Flurorescent ceramides, N-

C12:0-NBD-phytoceramide, and N-C12:0-NBD-dihydroceramide, were used as substrates for

the ceramidases, and the N-C12:0-NBD-dodecanoic acid was used as a maker for the ceramidase

reaction product (Figure 5-6). TLC analysis was performed to identify the florescent products.

We did not observe any significant difference between the empty vector and the Izhs in terms of

the intensity of the band corresponding to the fluorescent fatty acid (Figure 5-7 A, B, C, and D).

This suggests that Izhs are not ceramidases; however, it is possible that the substrates used for

this assay were not appropriate. The fact that this band, only appears when YPC]was

overexpressed at alkaline pH (e.g. pH 9.4), indicates that under the conditions used only Ypclp

is acting as a ceramidase.

Each experiment was carried out in duplicate. Although, it is well known that the optimal

pH for the alkaline ceramidase activity is in a range of 8 to 9, we wanted to investigate if the pH

had any effect on the enzymatic reaction. In this assay, the catalytic action of the ceramidase

produces the release of the fluorescent fatty acid. It is important to remember that C26-PHC or

C26-DHC, are the natural substrates for alkaline ceramidases. However, fluorescent C26-

ceramides are not commercially available. Thus, to do these experiments, we had to use artificial

florescent substrates. This could be one of the reasons by which we did not obtain the expected

results.

The preliminary data presented in this section represent a valuable piece of information

that suggests that Izhs may not be ceramidases. However, additional studies need to be

conducted to rule out this idea.









Thin Layer Chromatography of Radioactive Sphingolipids Reveals Similar Sphingolipid
Profiles for YPC1 and the IZHs

The altered levels of sphingoid bases observed when the IZHs were overexpressed may be

due to a role for IZH genes in de novo sphingolipid biosynthesis. To test this, cells were labeled

with [3H]serine during 40 min. In yeast, serine and palmitic acid are the two precursors of de

novo sphingolipid biosynthesis (Cowart and Obeid, 2007) (Figure 5-3 A). After addition of

[3H]serine to the growth medium, we expected its rapid incorporation into the cells followed by

the efficient biosynthesis of labeled sphingolipids. Interestingly, when we analyzed the

sphingolipid profiles by TLC, an increase on the levels ofPHS, DHS and other complex

sphingolipids were observed when the IZHs and YPC1 were overexpressed compared to the

vector control (Figure 5-8). These results suggest that de novo biosynthesis of sphingoid bases is

induced under IZH and YPC1 overexpression, supporting our hypothesis about the implication of

the IZHs in this part of the biosynthetic pathway.

Fumonisin B1 Induces the Acummulation of Sphingoid Bases in YPC1, IZH2, and IZH3

Fumonisin B1 is a mycotoxin that bears structural similarities to sphingoid bases (Wu et

al., 1995), (Figure 5-3 B). It has been shown that in mammalian cells, as well as in yeast, FB1

promotes the accumulation of sphingoid bases by inhibiting the activity of the ceramide

synthases Laclp/Laglp (Merrill et al., 1993; Wu et al., 1995) (Figure 5-3 A). To continue

investigating the implication of Izhs in de novo biosynthesis of sphingoid bases, we decided to

use FBi. When used, FB1 was added to a final concentration of 100 [LM to cells where the genes

YPC1 and YDC1 were mutated and containing pRS316-GAL1 (empty vector), or the vector

containing YPC1, IZH2 or IZH3. Once exposed to FB1, cells were harvested and total lipids were

extracted, and analyzed by HPLC as described under materials and methods. Some observations

that are worth mentioning are the following: (i) the addition of FB 1 resulted in the accumulation









of sphingoid bases such as C18-PHS, C20-PHS, and C18-DHS (Figure 5-9 A, B, C), (ii) The vector

control accumulated more C18-PHS and C20-PHS than YPCI, IZH2 and IZH3 (Figure 5-9 A and

B, respectively). This result suggests that in YPCI, IZH2 and IZH3, the synthesis of such as

sphingoid bases is somehow blocked under treatment with FB1.

Myriocin Inhibits the Increase in the Levels of Sphingolipid Biosynthesis Mediated by
IZH2 and IZH3

Myiocin is another potent inhibitor of de novo sphingolipid biosynthetic pathway, which

inhibits serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme of the sphingolipid

pathway (Figure 5-3 A and B) (Fujita et al., 1994). We reasoned that if the IZHs are implicated

in the synthesis of sphingolipids de novo, the inhibition of SPT would suppress the increase of

sphingoid bases produced when the IZHs are overexpressed. To test this, we first performed

phenotypic studies to determine the minimal inhibitory concentration of myriocin. These studies

indicated that 1 tlM myriocin had an inhibitory effect in the growth but, at that concentration, it

was not lethal for the cells (Figure 5-10 A). Therefore, cells grown in liquid medium were treated

with 1 tlM myriocin for 30 min. HPLC analysis only allowed the identification of C18-PHS,

which is one of the most abundant sphingoid bases in yeast. Interestingly, upon addition of

myriocin, the levels of C18-PHS were noticeably reduced when IZH2 and IZH3 were

overexpressed, compared to those detected under overespression of YPC1 and the empty vector

(Figure 5-10 B). Therefore, our results buttress our hypothesis that IZHs contribute to modulate

de novo sphingolipids biosynthesis.

Finally, to analyze the effect of overexpression in the production of ceramide, HPLC-

Tandem mass spectrometric analysis was performed. Our results indicate that the overexpression

of IZHs also has an effect in the synthesis of C26-PHC and C26-DHC, which are the most

abundant ceramides in yeast (Figure 5-11, A and B). These latter results not only support our









hypothesis about the implication of the IZH genes in de novo sphingolipid biosynthesis, but also

suggest that the IZH genes can be implicated in the synthesis of complex sphingolipids like

ceramides.

Taken together, the results presented in this chapter indicate that an increase in the dosage

of the IZH genes results in consistent with increases in the levels of sphingoid bases and

ceramides. In this sense, we envisioned the possibility that IZHs can be implicated in the

biosynthesis of this sphingolipids de novo.

Discussion

The primary objective of this chapter was to establish a connection between the the IZHs

and the sphingolipid biosynthetic pathway. Particularly, we set out to determine if the IZH family

plays role(s) in the sphingolipid pathway. To this end, biochemical studies were performed. One

first observation that suggests an implication oflZHs in the sphingolipid pathway was increased

levels of certain sphingolipids detected when the IZHs were overexpressed. To explain this

observation, two different possibilities were explored. First of all, the IZH genes encode alkaline

ceramidases. As mentioned before, ceramidases are enzymes that catalyze the deacylation of

ceramide to produce a sphingoid base and a fatty acid. Our hypothesis regarding the role of Izhs

as possible ceramidases was generated based on the fact that the IZHs might encode membrane

proteins that have distant similarities with this group of enzymes. To test that hypothesis, HPLC

analysis was first performed to measure the levels of sphingoid bases. Data generated by this

means indicate that under overexpressing conditions, IZHs increase the levels of sphingoid bases.

As a second approach, in vitro ceramidase assays were performed. However, our data suggest

that under our experimental conditions, Izhs do not appear to be alkaline ceramidases, per se.

However, it is imperative to do more studies that help test this hypothesis.









Although our ceramidase assays did not support our original hypothesis regarding the role

of Izhs as ceramidases, they illuminated the possibility that Izhs can modulate the de novo

synthesis of sphingolipids. Sphingoid bases can be synthesized by two different mechanisms.

First, complex sphingolipids can be converted to free sphingoid bases and fatty acids through the

action of alkaline ceramidases (Mao et al., 2000a; Mao et al., 2000b). Second, free sphingoid

bases can be synthesized de novo starting with the condensation of serine and palmitoyl-CoA

(Cowart and Obeid, 2007) (Figure 1-8 for details). To investigate a possible role oflZHs in the

de novo sphingolipid biosynthesis we generated radiolabeled sphingolipids by treating the cells

with [3H]serine. The data generated with this type of experiments revealed a moderate but still

measurable increase in the levels of PHS and DHS when the IZHs were overexpressed (Figure 5-

8). As a second approach, we used myriocin and fumonisin B1 two drugs that inhibit different

satges of de novo biosynthesis of sphingolipids (Figure 5-3 A). When myriocin was used, a

significant decrease in the levels of sphingoid bases was observed in cells overexpressing IZH2

and IZH3 (Figure 5-10 B). This result suggests the possibility that IZHs encode proteins directly

implicated in de novo sphingoid base biosynthesis. On the other hand, an accumulation of

sphingoid bases was observed upon treatment with fumonisin B1, suggesting that IZHs can also

mediate the synthesis of more complex sphingolipids, (Figure 5-9 A and B).

Our current data support but do not absolutely prove the implication of IZHs in

sphingolipid biosynthesis. However, different possibilities are envisioned. First of all, the IZH

genes could modulate the synthesis of sphingoid bases and complex sphingolipids. From this

standpoint, it is possible that these genes exert a regulatory role, modulating other genes that are

directly implicated in the de novo production of sphingoid bases. On the other hand, is plausible

that IZHs are directly implicated in the synthesis of sphingolipids, perhaps forming complexes









with other proteins. It is also entirely possible that IZHs exert a regulatory role, modulating other

genes that are directly implicated in the de novo production of sphingoid bases.

Future studies are required to clarify the mechanism by which IZHs modulate the

production of sphingoid bases and ceramides. Among the myriad of possibilities, the following

experiments are imperative. To really prove if IZHs encode alkaline ceramidases more a

sensitive assay is required. In this regard the use of radioactive ceramide substrates like

[3H]phytoceramide and [3H]dehydroceramide could be used. Following a similar procedure than

that one described in the in vitro ceramidase can be followed. Besides qualitative analysis,

quantitation of the levels of radiolabeled ceramides and sphingoid bases can be performed by

using a scintillation counter. To continue investigating the implication of IZHs in the de novo

biosynthesis of sphingolipids, qualitative and quantitative TLC analysis of radiolabeled

sphingolipids is also proposed. The post-translational effect on the Izhs, produced upon addition

of inhibitors like myriocin and fumonisin B1 can also be explored. To do this Western blot

analysis is an appropriate technique to be used.









Table 5-1. Strains and genotyes in Chapter 5
Strain Mutation Source/Derivation


Wild type

ypclAydclA


Wild type


EUROSCARF

Howard Reizman


Howard Reizman


MAT ua; his3; leu2; ura3; lys2

MAT a; ypcl::LEU2; ydcl::
TRP1; ade2; his3;leu2; trpl;
ura3;canl-l00c
MAT a; ade2; his3;leu2; trp]
ura3;canl-l00c


BY4742

ypclydcl


W303-1A


Genotype









0


H2N 3-Keto-dihydrosphingosine
(KDS)

OH


H2N Dihydrosphingosine
C18-DHS

OH


H2N OH
Phytosphingosine
C18-PHS

OH

HO -
H2N OH
Phytosphingosine
C20-PHS



NH2

Sterylamine (sphingoid
base-like compound)


OH

H



H
C26-Dihydroceramide
(C26-DHC)

H

OHNH




C26-Phytoceramide
(C26-PHC)

H
H
OHNH


C2-Phytoceramide to
(C2-PHC)

H
H

----r-------K0


C26-Dihydroceramide
(C26-DHC)

Figure 5-1. Chemical structures of the sphingoid bases, ceramides, and sterylamine a structural
sphingoid base homolog.


















(Phyto)ceramide


Ceramide synthases
Lag1p, Lac1p, Liplp,,
Ydclp, Ypclp )


Alkaline ceramidases
Ydclp, Ypclp
Izhl-4p ?


(Phyto)sphingosine


Fatty acid OH

Figure 5-2. Synthesis and hydrolysis of the yeast ceramides. The alkaline ceramidases Ydclp
and Ypclp catalyze the hydrolysis of the yeast ceramides (dihydroceramide and
phytoceramide, respectively) to produce the sphingoid base and a fatty acid. On the
other hand, the ceramide synthases Laglp, Laclp, Liplp, Ydclp and Ypclp catalyze
the reverse alkaline ceramidase reaction. Ydclp and Ypclp are implicated in both
reactions. The Izhl-4 proteins are proposed to be alkaline ceramidases.










0
CoA-S- +
Palmitoyl-CoA


Serine palmitoyltransferase
LCB1, LCB2, TSC3 Myriocin




H2 3-Keto-dihydrosphingosine

TSC10


OH
H"SI

H2N Dihydrosphingosine


FB1 -ICeramide synthase Alkaline
LAG1, LAC1, ceramidase
LIP1, YDC1 YDC1

OH


HN
H 2 Dihydroceramide

o 23


)H


UR2

H2N OH Phytosphingosine


FB1 Ceramide synthase Alkaline
LAG1, LAC1, Iceramidase
LIP1, YPC1 YPC1

OH


H Phytoceramide
HN OH Phytoceramide

o^


B
OR QH OH
OH NH2

COOH 3 OR H3 H NH3+

O R = COCH2CH(COOH)CH2COOH

Myriocin Fumonisin B1 (FB1)


Figure 5-3. Overview of de novo biosynthetic pathway in yeast. In scheme A, straight arrows
represent the stages of de novo sphingolipid biosynthesis with the respective genes
enzymes and their inhibitors; represents the blockage of sphingolipid biosynthesis
produced by myriocin and fumonisin B1 (FBi). B shows the chemical structures of
the mycotoxins, myriocin and and fumonisin B1 (FBi).


HO H
NH2
orirno


0


Serinel












o Vector
I- YYPC1
20 EZZ IZH2
c- IZH3
0
15 -

0
C
10 -



o 5
E


0
C18-PHS C18-DHS

Figure 5-4. Overexpression of IZH2 and IZH3 produces an increase in the basal levels of the
sphingoid bases C18-PHS and C18-DHS. The empty vector pRS316-GAL] (Vector),
or the vector containing either of the YPC1, IZH2, or IZH3 genes (e.g. pRS316-
GAL1-YPC1, pRS316-GALI-IZH2 and pRS316-GALI-IZH 3) were transformed with
the the BY4742 wild type strain. The figure shows the basal levels of C18-PHS and
C18-DHS. Data represent the mean + standard deviation of three independent
experiments. PHS, phytosphingosine; DHS, dihydrosphingosine; C18- represents the
number of carbon atoms of the sphingoid base backbone.











A


C20-PHS


E


B












to


C,8-PHS


C20-PHS


Cgs-DHS


Figure 5-5. Overexpression of YPC1 and IZHs induces the increase in the levels of C18-PHS,
C20-PHS, and C18-DHS. Total lipids were extracted from the double mutant strain
ypclAydclA, containing the empty vector pRS316-GALI (Vector), or expressing the
YPCI and IZHI-4 (pRS316-GALI-YPCI and pRS316-GALI-IZHI-4, respectively).
A, B, and C show the basal levels of sphingoid bases. D, E, and F show the levels of
sphingoid bases after spiking with 278 nM C2-PHC.


20 -
2A




0.
E I -
g.

C0
05-


oja -


.3
4


2

E


~ 25

o 2.0
C S-



1.5


"A L



0.0



C18-DHS


C18-PHS


3.5

S3.0-


S 2.05-


~ LS



S0.5-










OH

HO
HN OH
(CH3)7 H

O
-ooN N-C12:0-NBD-phytoceramide
NO2
OH

HO

HNr (CH3)7 H


S-N
N N-C12:0-NBD-dihydroceramide
NO2



HOOC (CH3)7 H



'NON N-C12:0-NBD-dodecanoic acid

NO2


Figure 5-6. The fluorescent ceramide substrates and the fatty acid product used during the in
vitro ceramidase assays. The scheme shows the substrates for alkaline ceramidases
N-C 12:0-NBD-phytoceramide and N-C 12:0-NBD-dehydroceramide, and N-C12:0-
NBD-dodecanoic acid, the ceramidase reaction product.














A




C12-NBD-PHC








C,2-NBD-FA
by-product ?


C12-NBD-FA -







C -






C,0-NBD-DHC -


pH 8.6


FA Vector Ypcl Izhl Izh2 Izh3 Izh4


pH 9.4

C12-NBD-DHC C,1-NBD-PHC



#%t 'g'


-C,2-NBD-PHC


B





C12-NBD-DHC










C,-NBD-FA -


pH 8.6

FA Vector Ypcl Izhl Izh2 Izh3 Izh4


D pH 7.0
C12-NBD-DHC C12-NBD-PHC





L..... MMlAd
.L.. .l ,. ,..Ju~ ll*Wr lIYjr


c,.-o-oNDc-m MMMMMM


-:r Ma l q C12-BD-PHC


C,,-NBD-FA
to M M W 1W-C,-NED-PA


-C,,-NBD-FA


-A* a


Figure 5-7. TLC analysis of in vitro ceramidase activity at different pHs. In panels A and B, the
empty vector (pRS316-GAL]) or the vector expressing Ypclp and Izhl-4p, were
assayed for ceramidase activity toward the fluorescent substrates C12-NBD-PHC (left)
and C12-NBD-DHC (right), at pH 8.6. Panels C and D, the empty vector and Ypclp,
Izh2p, and Izh3p were tested for ceramidase activity at pH 9.4 and 7.0, respectively.
All panels show the TLC analysis the alkaline ceramidase reaction. In the figure, C12-
NBD-FA represents the fluorescent dodecanoic acid, PHC is phytoceramide and
DHC is dihydroceramide.


-'' "'"'









.4000 C9 0 ?


DHC '
PHC *


DHS
PE
PHS
PC

PS


IPC
MIPC

M(IP)2C-


Origin


Figure 5-8. TLC-autoradiograph of radiolabeled lipids shows increased levels of PHS and DHS
when IZHs and YPC1 are overexpressed. Cells were labeled with [3H]serine and their
total lipids were extracted as described under materials and methods. Radiolabeled
lipids were resolved by TLC. Most of the sphingolipids were identified according to
authentic standards like PHS, phystosphingoisine; PHC, phytocceramide; DHS,
dihydrosphingosine; DHC, dihydroceramide; PE, phosphatidylethanolamine; and
IPC, inositol phosphoceramide.


F

i'


:e
44t


I


- *
























+ FB1


C18-PHS


-FB1


120

100

80

60

40

20

0


- FB1


C20-PHS


+ FB1


C18-DHS

Figure 5-9. Effect of fumonisin B1 on the production of sphingoid bases. The figure shows the
HPLC analysis of fluorescent sphingoid bases. Data are the mean + standard
deviation of two experiments. FB1, fumonisin B1; PHS, phystosphingosine, DHS,
dihydrosphingosine; C8i- or C20-, are number of carbon atoms of each sphingolipid
backbone.


120

100

80

60

40

20

0


- FB1


+ FB1










Control
(0 pM myriocin)


pRS316-GAL1

pRS316-GAL1-YPC1

pRS316-GAL1-IZH1

pRS316-GAL1-IZH2

pRS316-GAL1-IZH3

pRS316-GAL1-IZH4



B


10
aQ.



0

o
I 5
a.


Vector


0.1 pM
myriocin


1.0 piM
nyriocin


YPC1 IZH2 IZH3


C18-PHS

Figure 5-10. Effect of myriocin on the growth and the sphingolipid biosynthesis of IZH2 and
IZH3. A shows the phenotypic response of vector control, IZHs and YPC1 in presence
of two different concentrations of myriocin. B shows the quantitation of C18-PHS in
cells gown in +/- myriocin by HPLC. The results shows a significant decrease on
levels of C18-PHS is observed for IZH2 and IZH3 upon addition of 1.0 [tM myriocin.
























C26-PHC


20
18
16
14
12
10
8
6
4
2
0


Figure 5-11. Overexpression of IZH2 and IZH3 produces an increase in the levels of C26-PHC
and C26-DHC. The figure shows mass spectrometric analysis of the sphingolipids;
C26-PHC; and C26-DHC; C26, a fatty acid with 26 carbon atoms. Data represent the
mean + standard deviation of two independent experiments.


C26-DHC


A

_



n^


L 1L L









CHAPTER 6
CELLULAR LOCALIZATION OF THE Izh2 AND Izh3 PROTEINS BY MEMBRANE
FRACTIONATION

Introduction

The IZH genes encode proteins with seven transmembrane domains and little amino acid

conservation in the loop regions on the extracellular face of the membranes where external

ligands might make contact. In previous chapters, we demonstrated that certain nutritional

conditions can affect the expression of these proteins, as well as their transcriptional response.

Despite the progress reached in this regard, many gaps concerning the functions) of the Izhp

remain to be uncovered.

Perhaps one of the most important aspects that needs to be explored in order to gain more

insights regarding the role(s) exerted by proteins is to determine their cellular localization. With

this in mind, our aim was to investigate the localization of Izh2p and Izh3p. As we have reported

before, the expression of these two proteins is regulated by certain fatty acids (Chapter 3). In

addition to this, when IZH2 and IZH3 are overexpressed, the sphingolipid content is altered

(Chapter 5). The fact that sphingolipids are found in plasma membranes led us to hypothesize

that the IZH2 and IZH3 products are in plasma membrane possibly associated with lipid

microdomains termed lipid rafts.

Lipid rafts are formed by the lateral association of sphingolipids and sterols (e.g. ergosterol

in yeast, and cholesterol in mammals) (Dickson and Lester, 2002; Bagnat et al., 2000; G6mez-

Mount6n et al., 2004). In addition to lipids, rafts also contain proteins (Grossmann et al., 2006)

(Figure 6-1). Because of their composition, rafts were originally implicated in maintaining the

optimal fluidity and integrity of plasma membranes (Grossmann et al., 2007). Nevertheless,

recent studies have indicated that lipid rafts are also implicated in intracellular protein trafficking

and signaling processes (Dupre and Haguenauer-Tsapis, 2003). Other studies have also revealed









the specific lipid raft association of GPI-anchored proteins (Moffett et al., 2000) as well as

transmembrane proteins whose transmembrane domains are thought to have certain affinity for

rafts (Simons et al., 1997).

One interesting characteristic that has allowed the isolation and analysis of lipid rafts is

their low density and insolubility in the mild nonionic detergent 1% Triton X100, at 4 C (Pike,

2003; Grossmann et al., 2006) Therefore, we took advantage of these properties to isolate lipid

rafts from the rest of the yeast membranes.

To our knowledge, there is only one report published by Narasimhan et al., 2005,

suggesting the presence of Izh2p in plasma membranes. In this chapter, we have broadened the

scope of that finding by localizing Izh2p and the paralogous Izh3p in plasma membrane and

within lipid rafts. In fact, we present evidence that suggests Izh2p and Izh3p are co-localized in

plasma membrane and lipid rafts. In addition to this, our results suggest that a fraction of these

two proteins can eventually be dissociated from lipid rafts. In this scenario, Umebayashi et al.,

2003 have proposed that lipid rafts serve as sorting platforms for proteins.

Although still preliminary, the data presented herein, constitute an important framework

that strongly contributes to elucidate the functions) of the Izhs. Our findings certainly illuminate

our original premise, implicating Izh2p and Izh3p in different lipid biosynthetic pathways.

Materials and Methods

Plasmids and Yeast transformations

For this study, the wild type strain BY4742 (mating type a obtained from EUROSCARF),

(http://web.uni-frankfurt.de/fbl5/mikro/euroscarf/) was used. Plasmids used in this study were

generated by Dr. Thomas Lyons as described in Chapter 2. For the experiments described in this

chapter, we used plasmids where the IZH2 and IZH3 genes had their own promoters. The









plasmids plZH2-3xHA and plZH3-3xHA were introduced into the wild type strain BY4742 as

described in Chapter 2.

Growth Conditions

Cells containing the plZH2-3xHA and plZH3-3xHA plasmids were grown in identical

fashion as described in Chapter 2. Cells were grown to logarithmic phase (OD600 of 0.8)

followed by harvesting at 3,000 rpm, for 5 min at 40C. Pellets were washed twice with cold

sterile nano pure water. Resultant pellets were then used to isolate Izh2-3xHA and Izh3-3xHA

proteins.

Isolation Purification and Characterization of Yeast Plasma Membranes

Unless otherwise indicated, the entire procedure was carried out at 40C. Reagents used

were of high quality and obtained from Fisher or Sigma, Protease inhibitors and glass beads were

obtained from Sigma. The TED buffer (10 mM Tris adjusted to pH 7.5 with HC1, 0.2 mM

EDTA, pH 7.5, and 2 mM dithiothreitol, DTT) is present in all the sucrose and glycerol

solutions. Pellets were re-suspended manually with a plastic stirring rod. Plasma membrane was

isolated using a combined method of differential and density gradient centrifugations according

to Serrano, 1988, and with some minor modifications as follows. Portions of 25 g of cells (fresh

wet weight) were diluted with nano pure water to 80 mL. Aliquots of 20 mL of the diluted cells

were transferred to 50 mL sterile polypropylene centrifuge tubes, (Fisher), and suspended with

lysis buffer, (0.5 M Tris, pH 8.5; 6 mM EDTA, pH 8.0; 0.6 mM phenylmethyl-sulfonyl fluoride

(PMSF), 2 [tg/mL pepstatin A), and 15 mL of glass beads-acid washed, (Sigma). Cells were

lysed by vortexing 10 times, one minute each time and with repeated incubations on ice for 4

min between each vortexing. Lysates were transferred to a thick wall 25 X 89 mm polycarbonate

tube (from Beckman coulter), and centrifuged at 1,000 rpm, 4C during 10 min in a Sorvall SS34

rotor to remove unbroken cells and debris. An aliquot of the low speed supernatant (lysate









fraction or Lys) was saved for further SDS-PAGE and western blot analysis, and the rest of the

supernatant was further centrifuged for 20 min at 20,000 rpm in a Sorvall SS34 rotor. An aliquot

of this supernatant (Cytosolic fraction or Cyt) was saved for SDS-PAGE and Western blot

analysis. The 20,000 rpm pellet, which is enriched in plasma membranes, was re-suspended with

7.5 mL of 20% glycerol, and protease inhibitors. Suspensions were homogenized by hand,

applying 10 strokes in a 10 mL Potter-Elvehj em homogenizer. Ultra clearTM ultracentrifuge tubes

(25 X 89 mm) from Beckman were used to prepare a discontinuous gradient made of 8 mL 53%

(w/w) sucrose and 16 mL 43% (w/w) sucrose. The homogenates were then applied to the

gradient and centrifugated for 6 h at 25,000 rpm in a Beckman SW 28 rotor. Plasma membranes

were recovered at the 43/53 interface. The band was collected with a Pasteur pipette, diluted with

4 volumes of water, and pelleted by centrifugation at 35,000 rpm for 20 min, using a Beckman

70.1 Ti rotor. Protein concentration of all the protein fractions was determined by the Markwell

assay (Marwell et al., 1981), and using lyophilized bovine serum albumin (Bio Rad) as standard.

Analysis of each protein fraction (100 |tg of protein) was performed by SDS-PAGE and

western blot analysis. To do this, a 10% SDS-PAGE gel was run overnight at 45 volts, at room

temperature, and then transferred to a nitrocellulose membrane (Bio Rad) by applying 200 mA

for 2 h, and at 40C. The Izh2- and Izh3-3xHA-tagged proteins were analyzed incubating the

membranes during 1 overnight, at 40C with a 1/1000 and 1/500 (v/v) dilution, respectively of the

primary antibody rabbit polyclonal anti-HA, (HA-probe (Y-11)sc-805, from Santa Cruz),

followed by incubation during 1 h, at room temperature with the secondary antibody goat anti

rabbit-Horse Radish Peroxidase-conjugate, (HRP-) (Santa Cruz) at a dilution of 1/10000, v/v.

Likewise, the mouse monoclonal [40B7] to the plasma membrane marker Pmalp (Abcam),

(1/8000, v/v dilution), was used as primary antibody. Pmalp is a proton-pumping H -ATPase









that is an abundant and very long lived protein of the yeast plasma membrane. Pmap associates

with lipid rafts (detergent-resistant membrane domains) (Gaigg et al., 2006). Immunoreactive

proteins were visualized in an X-ray film (Pierce) and by using the pico super signal ECL

detection kit (Pierce).

Isolation of Lipid Rafts from Yeast Plasma Membrane: Procedure 1

Once prepared, the Izh2p and Izh3p plasma membrane fractions were used for the isolation

of lipid rafts according to Kumar et al., 2004 as follows. Purified yeast plasma membranes (1

mg) were extracted in MBS buffer (25 mM MES and 150 mM NaC1, pH 6.5) containing 0.2%

TX100 obtained from Fisher, and supplemented with protease inhibitor mix (Sigma). The

samples were mixed end-over-end in a rotator for 20 min at 40C, and then homogenized by hand,

with 10 strokes of a Dounce homogenizer. The homogenizer was rinsed with 500 ptL of MBS-

0.2% Triton and then combined with the 1 mL extract. These extracts were mixed with 1.5 mL of

80% sucrose (w/v) in MBS. Homogenates (now in 40% sucrose) were placed at the bottom of 14

X 89 mm ultra clearTM ultracentrifuge tubes (Beckman Coulter), and overlaid with 6 mL of 30%

sucrose and 3 mL of 5% sucrose (in MBS). After centrifugation at 240,000 X g (37,400 rpm) in a

Beckman SW41 rotor for 18 h, 1.0 mL fractions were collected by upward displacement using

60% (w/v) sucrose solution (prepared in the MBS buffer) as the displacement fluid at 40C.

Protease inhibitor mix (Sigma) was added to each fraction. Protein was precipitated by adding

500 [tl of 30% ice- cold trichloroacetic acid (TCA), followed by incubation at 40C for 30 min.

The protein precipitate was collected at 13,200 rpm at 40C, for 10 min, after which it was washed

two times with 500 p.L of ice-cold acetone. The precipitate was then dissolved in 120 pL 1X

SDS-loading buffer, and the protein (100 [tg) was resolved on a 10% SDS-PAGE gel under

reducing conditions. Gels were then transferred onto nitrocellulose membranes (Bio Rad) for









protein analysis. After transfer, nitrocellulose membranes were stained with a solution of 10%

(v/v) amido black to visualize protein bands. The amido black solution was prepared mixing 1

volume of amido black stock, [(0.1% naptol blue black, 50% methanol, 10% glacial acetic acid,

and 40% nano pure water), and 9 volumes of amido black destain, (50% methanol, 10% glacial

acetic acid, and 40% ultra pure water)].

Western blot analysis was performed in identical fashion as described in former section. A

rabbit polyclonal IgG antibody against the HA-tag (Santa Cruz) was used at a dilution of 1/1000

(v/v) for Izh2-3xHAp and at a dilution of 1/500 (v/v) for Izh3-3xHAp. Membranes were then

incubated with the secondary antibody goat anti-rabbit IgG HRP-conjugate (Santa Cruz) used at

a dilution of 1/10000 (v/v). Pmalp, an associated yeast lipid raft protein was used as a marker of

the lipid raft fraction (Bagnat et al., 2000). This protein was identified with a 1/8000 (v/v)

dilution of a mouse polyclonal IgG anti Pmalp (primary antibody) obtained from Abcam, and

followed by incubation with 1/10000 (v/v) dilution of goat anti mouse IgG HRP-conjugate

(secondary antibody) obtained from Bio Rad. Likewise, the vacuolar alkaline phosphatase (V-

ALP), a marker for plasma membrane proteins that are not associated with lipid rafts (Bagnat et

al., 2000; Nothwehr et al., 1995) was identified with a 1/100 (v/v) dilution of a mouse

monoclonal IgG anti-alkaline phosphatase (Molecular Probes), followed by incubation with

1/10000 (v/v) dilution of goat anti mouse IgG HRP-conjugate (secondary antibody) obtained

from Bio Rad. Immunoreactive proteins were visualized as described before.

Isolation of Lipid Rafts from Total Membranes: Procedure 2

In this procedure, lipid rafts were isolated from total membranes according to Kibler et al.,

1996 with some modifications as follows. Fresh pellets (20 g of wet weight) were washed twice

with cold sterile water, and then re-suspended in a total volume of 100 mL of ice-cold lysis

buffer (20 mM triethylethanolamine pH 7.2, 0.3 M sorbitol, 1 mM EDTA, 0.1 mM PMSF, 0.7









mM pepstatin A, and 2 mL of protease inhibitor cocktail (Sigma). Portions of 20 mL of cell

suspensions were mixed with 15 mL of acid washed glass beads (Sigma) and lysed 10 times by

vigorous vortexing, 1 min each time and with repeated incubations on ice for 2-3 min between

each vortexing. Unbroken cells were removed by a 1,000 rpm spin for 10 min, and using a

Sorvall SS34 rotor. The resultant supernatant (total cell lysate fraction) was centrifuged for 1 h to

pellet cellular membranes and using a Beckman SW28 rotor at 27,500 rpm. The supernatant

(Cytosolic fraction or Cyt) was discarded, and the resultant pellet is referred as total membrane

fraction (TM).

Pellets corresponding to the total membrane fraction was suspended in 3 mL MBS buffer

(25 mM Mes and 150 mM NaC1, pH 6.5), supplemented with protease inhibitor mixture from

Sigma. Suspensions were homogenized by hand applying 10 strokes in a 10 mL Potter-Elvehjem

homogenizer. 1.0 mL of the homogenate was transferred to a 1.5 mL eppendorftube and treated

with cold 1% TX100, followed by mixing end-over-end in a rotator for 30 min at 40C. The

extracts were brought to 1.5 mL by adding 500 p.L of a solution of cold MBS-1% Triton. These

extracts were then mixed with 1.5 mL of 80% sucrose (w/v) in MBS.

Homogenates (now in 40% sucrose) were placed at the bottom of 14 X 89 mm ultra

clearTM ultracentrifuge tubes (Beckman Coulter); and overlaid with 6 mL of 30% sucrose and 3

mL of 5% sucrose (in MBS, pH 6.5). After centrifugation in a Beckman SW41 rotor at 240,000

X g (37,400 rpm) at 40C for 18 h, 12 fractions of 1.0 mL each one were collected by upward

displacement using 60% (w/v) sucrose solution (prepared in the MBS buffer, pH 6.5) as the

displacement fluid. Protease inhibitor mixture (1/1000, v/v dilution) from Sigma, was added to

each fraction.









Proteins were precipitated by adding 500 ptL of 30% ice- cold trichloroacetic acid (TCA),

followed by incubation at 40C for 30 min. Precipitates were collected at 13,200 rpm, 4C for 10

min. Precipitates were thoroughly washed twice with 500 ptL of ice-cold acetone. Resultant

protein pellets were suspended in 120 p.L of loading buffer prepared by mixing 1 part of sample

dilution buffer (2X SDB) consisting of SDS, DTT, bromophenol blue, glycerol, and Tris-base,

pH 6.8 from Fluka) with 1 part of 0.1% SDS and 1% of P-mercaptoethanol (f3ME). After

determination of total membrane protein concentrations by using the Markwell assay, 1 mg of

each protein was suspended in 2X SDB and 2% 3ME (1:1, v/v). Proteins were then analyzed by

10% SDS-PAGE and Western blotting in identical fashion as described before (procedure 1).

Results

Izh2p and Izh3p Were Found Enriched in Plasma Membrane

After synthesis occurs in the endoplasmic reticulum, some sphingolipids are trafficked to

the Golgi apparatus becoming structurally more complex, whereas others directly can be sorted

to the plasma membrane to serve as structural and signaling molecules. Because Izh2p and Izh3p

are predicted to be involved in the sphingolipid metabolic pathway, our initial rationale was that

these two proteins could be embedded in the plasma membrane, modulating role(s) in the

sphingolipid pathway. So far, however, there is just one report suggesting that Izh2p is a plasma

membrane protein (Narasimhan et al., 2005), and no precedent exists regarding the localization

of Izh3p. Therefore, our aim was to investigate if Izh2p and Izh3p are in fact plasma membrane

proteins. To do this, a standard procedure developed by Serrano, 1988 to isolate and purify

plasma membrane from yeast cells was followed. This method, which is based on differential

centrifugation of discontinuous sucrose gradients allowed us to detected Izh2p and Izh3p tagged

with the HA-epiptope in plasma membranes. Isolation and purification of plasma membrane was









carried out by conducting two independent experiments. Yeast cells were first lysed, and an

aliquot of the resultant lysis fraction (Lys in Figures 6-2 and 6-3) was used for SDS-PAGE and

Western blot analysis. The rest of this fraction was centrifuged at 20,000 rpm to generate a pellet

enriched in plasma membranes, and a supernatant composed mainly of soluble proteins (Cyt in

Figures 6-2 and 6-3). The 20,000 rpm pellet was homogenized and then applied to a sucrose

gradient and centrifuged at 25,000 rpm, which allowed the isolation of the pure plasma

membranes at the interface of the 43% and 53% sucrose gradient (PM in Figures 6-2 and 6-3). A

layer at the top of the 43% sucrose gradient was also isolated and designated as the microsomal

fraction (M, in Figure 6-3) because it contains proteins from different organelles like

mitochondria, ER, Golgi, vacuole, etc (Serrano 1988).

The purity of the resulting plasma membrane isolate was tested by Western blotting, and

using the yeast proton-ATPase protein, Pmalp as a marker. Pmalp is an appropriate marker, not

only because it is an essential protein in the in plasma membrane where comprises > 25% of the

total protein at steady state (Lee et al., 2002), but also due to the commercial availability of

antibodies against it. On the other hand, the presence of the HA-tagged Izh2p and Izh3p was

tested by using an antibody against the HA epitope tag. Our results suggest that both proteins

Izh2p and Izh3p are in plasma membrane. However, Izh3p showed a particular pattern of

migration with bands at higher molecular weight than that one predicted by its molecular weight

(at z 63.0 KDa), (e.g. a band close to 97.4 KDa and another one close to 66.2 KDa ) (Figure 6-2

B, and F). We could not determine the identity of these bands. However, we speculate that are

the results of a post-tranlational modification for the Izh3p, like glycosilation. Finally, a band

observed just below 63.0 KDa (Figure 6-2 F) might correspond to degradated protein.Thus, more









experiments need to be conducted to elucidate the identity of those un-expected bands, and to

improve the results presented in this study.

Izh2p and Izh3p Are Associated with Lipid Rafts

To address the possibility that Izh2p and Izh3p are localized in lipid rafts, we first adapted

a well established experimental procedure used to isolate and characterize lipid rafts from

mammalian cells (Kumar et al., 2004). In our case, we first isolated and purified plasma

membrane fractions as described previously in materials and methods, followed by treatment

with cold 1% TX100, and detergent-lysate homogenizing. Homogenized samples were then

adjusted to 40% sucrose and loaded to the bottom of a step-density gradient (40%, 30%, 5%

sucrose) followed by ultracentrifugation. A successful isolation of lipid rafts produces a low

density layer floating at the top of the step-sucrose gradient. Although we could not detect such a

layer, 12 fractions (1 mL each one) were collected and analyzed by SDS-PAGE and Western blot

(Figure 6-3). Usually, fractions 1-5 correspond to lipid rafts if any, and fractions 6-12 contains

membranes proteins that have been dissociated from membranes by treatment with cold TX100

(Bagnat et al., 2000; Kumar et al., 2004).

The results obtained suggest, at first glance, that Izh2p (Figure 6-3 A, top panel) and Izh3p

(Figure 6-3 A Bottom panel) are fully soluble under the conditions used in this procedure (Figure

6-3, fractions 10 and 11 of the sucrose gradient), and that although localized in plasma

membrane, these proteins are not present in lipid rafts at all. We confirmed that fractions 10-11

were indeed soluble due to the identification of V-ALP in such fractions (Figure 6-3 B, bottom

panel). At this point is pertinent to remember that V-ALP (a vacuolar membrane protein) was

used as a marker for membrane proteins that are soluble upon treatment with TX100 (Bagnat et

al., 2000; Nothwehr et al., 1995). An interesting aspect that is necessary to mention is the

absence of Pmalp from lipid rafts and its detection in the soluble fraction (Figure 6-3 A, top









panel). Besides being a plasma membrane protein, Pmalp has also been co-localized in lipid

rafts. Pmalp has been found in association with lipid rafts (Bagnat et al., 2000; Lauwers et al.,

2006). Therefore, our results suggest a possible disruption of lipid rafts under the conditions

used. In fact, it has been reported that the extent of physical manipulations of detergent lysates,

in some cases, disrupt lipid rafts and thus their components (Pike, 2003).

In an effort to solve experimental problems associated with the procedure 1, a second

procedure, based on the isolation of lipid rafts from total membranes, was conducted. To do so,

yeast cells were lysed and then centrifuged at 100,000 X g (25,700 rpm) to obtain the total

membranes. These membranes were homogenized by hand as described in materials and

methods, followed by incubation in cold 1% TX100 for 30 min and further ultracentrifugation at

240,000 X g (37,400 rpm). A thick layer floating at the top of the sucrose-step gradient was

observed. Twelve fractions of 1 mL each one were collected as described under materials and

methods and analyzed by SDS-PAGE and Western blot. This purification scheme allowed us to

identify Izh2p and Izh3p in lipid rafts by their distribution along the density gradient (Figure 6-4,

panels A and B, respectively). The successful isolation of lipid rafts was tested by the presence

of Pmalp in fraction 4 of the gradient (Figure 6-4 C, top panel). Interestingly, Izh2p, Izh3p, and

the marker Pmalp were also observed in the soluble fractions of the gradient suggesting that

these proteins although associated, are not permanent residents of lipid rafts. However, more

experiments need to be performed to prove this hypothesis. Finally, as expected the V-ALP

protein was localized in the soluble fractions of the sucrose gradient (Figure 6-4 C, bottom

panel).

Discussion

The aim of this study was to investigate the co-localization of Izh2p and Izh3p in plasma

membranes and lipid rafts. By using differential centrifugation of discontinuous sucrose









gradients, we were able to isolate and partially purify plasma membrane fractions. In addition to

this, we isolated lipid rafts from total membranes by flotation and after treatment with 1% Triton

X100 at 40C.

Although Izh2p and Izh3p were localized in plasma membrane, both proteins were also co-

localized in other fractions. For instance, the first experiment shown in Figure 6-2, panels A, B,

and C, clearly shows that Izh2p is mainly enriched in plasma membrane. However, Izh3p was

compartmentalized in the microsomal and plasma membrane fractions. In addition to this, other

bands, at higher molecular weights than the predicted one (at z 63.0 KDa), were also observed.

One possible explanation for this migration pattern is that Izh3p could be undergoing post-

translational modifications, which yield a protein with a higher molecular weight. Future

experiments are required in order to test this hypothesis.

A second experiment was performed to confirm the results obtained in the first one. This

experiment showed that Izh2p was also co-localized in the microsomal fraction (Figure 6-2 D).

Izh3p showed more than one band, confirming the results obtained in the first experiment

(Figure 6-2 F). The result obtained for Izh2p, suggests an incomplete purification of plasma

membranes. This result was confirmed with the co-localization of Pmalp in plasma membrane

and microsomal fraction (Figure 6-2 E). This is attributed to contamination generated during the

isolation of plasma membrane from the sucrose gradient.

Although two procedures were followed to isolate lipid rafts, only one of them was

effective. In a first approach, plasma membranes were isolated and purified as described under

materials and methods. After that, we tried to isolate lipid rafts, from purified plasma

membranes, by treatment with cold TX100, followed by homogenizing. This procedure failed to









yield lipid rafts (Figure 6-3). We attributed this to possible disruption of lipid rafts during the

treatment of the samples with detergent and followed by homogenizing.

In a second approach, lipid rafts were successfully isolated from total membranes by

flotation. This procedure allowed us to identify Izh2p and Izh3p in the detergent resistant

fractions (Figure 6-4), suggesting that these proteins are associated with lipid rafts. Interetingly,

we also found that Izh2p, Izh3p, and the Pmalp (used as positive control) are fractionated with

detergent sensitive membranes (Figure 6-4, fractions 8-11). One possible reason that would

support our results is an inherent sensitivity of the tested proteins to the TX100 extraction. In

fact, Umebayaski, 2003 has reported that certain lipid raft proteins are resistant to solubilization

by detergents like CHAPS, but are highly sensitive to the treatment with TX100. Regarding

Pmalp, Bagnat et al., 2000 have argued the possibility that the lack of association of this protein

to lipid rafts arises from the pelleting step, just before the floatation in 60% sucrose. When total

membranes are pelleted from the cleared lysate, the pellet likely contains the remainder of cell

walls, debris, and cytoskeletal elements that result in the trapping of material, that otherwise

would have a lower density. Likewise, studies performed by Bagnat et al., 2001; Lawrens and

Andre, 2006 have indicated that, upon treatment with TX100, Pmalp can dissociate from lipid

rafts and missort to the vacuole for further degradation.

Overall, the results obtained are consistent with the hypothesis that Izh2p and Izh3p

proteins are colocalized with sterols and sphingolipids. The fact that the overexpression oflZH2

and IZH3 induces an increase in the levels of sphingoid bases (Chapter 5), and the localization of

these two IZH gene products in plasma membrane and lipid rafts constitute additional and robust

evidence indicating a potential role of the Izhp family in sphingolipid metabolism.









The results presented in this chapter are very valuable since thery have started to shed light

into the cellular localization of two members of the Izh family. In addition to this, we

demonstrate that the metodologies used to isolate plasma membrane and lipid rafts are powerful

approaches to study membrane proteins.














Plasma


Figure 6-1. Lipid composition of the yeast lipid rafts. In the figure sterols and sphingolipds
associate to form detergent-resistant microdomains or lipid rafts. Lipids rafts are in
plasma membrane where membrane proteins are also localized. In the figure black
circles (A, B, C, and D) are highly conserved motifs of a membrane protein with
seven tansmembrane domains.











Lvs Cvt PM


* Izh2-3xHAp


KDa
97.4

66.2
63.0


* Izh3-3xHAp


a Pmalp


Lys Cyt M PM
KDa
36.3
31 ..
SLys Cyt M PM
105
97.4


M PM


* Izh2-3xHAp



* Pmalp


Figure 6-2. Localization of Izh2p and Izh3p in plasma membrane. Two independent
experiments are shown in this figure. The first one is represented by panels A, B, and
C. A shows that Izh2p is mainly localized in plasma membrane. B shows that Izh3p,
although localized in plasma membrane, it is also co-localized in the cytosolic
fraction (Cyt). C shows that the plasma membrane marker Pmalp is localized in
lysate (Lys) and plasma membrane (PM) fractions, but not in the soluble cytosolic
fraction (Cyt). In a second experiment, D shows that Izh2p is co-localized in the
microsomal and the plasma membrane fractions. E confirms that Pmalp is mainly
localized in plasma membrane. F, Izh3p is co-localized in all the isolated fractions.
Figure shows Western blots. HA-tagged Izh2p and Izh3p were recognized with anti-
HA antibody. Pmalp was recognized with an anti-Pmalp antibody.


Lys Cyt


rF


4 Izh3-3xHAp









Fraction number


KDa
36.3

31.0


63.0








B

KDa
105.0
97.4

66.2

97.4

70.0
66.2


PM 1 2 3 4 5 6 7 8 9 10 11 12











Gradient: Top Bottom



Fraction number

PM 1 2 3 4 5 6 7 8 9 10 11 12

i--


Gradient:


Top


* Pmal






* V-ALP


Bottom


Figure 6-3. Izh2p and Izh3p are dissociated from lipid rafts prepared from plasma membranes.
Panel A-top shows that although Izh2p is present in plasma membrane, it was fully
solubilized under treatment with cold TX100. Likewise Izh3p was primarily detected
in the soluble fractions of the sucrose gradient panel A-bottom. Panel B-top shows
that the plasma membrane marker Pmalp although enriched in this fraction, it also
undergoes dissociation of lipid rafts under our experimental conditions. V-ALP
(Vacuolar Alkaline Phosphatase), a soluble protein under treatment with cold TX100,
was recovered from fractions 10 and 11 (panel B-bottom). To generate the panels
shown in the figure, 12 fractions were collected from the top of the step sucrose
gradient. Proteins were TCA-precipitated and analyzed by Western blot, using with
anti-HA, anti-Pmalp, and anti-V-ALP antibodies. PM stands for plasma membrane.


--


^












Fraction number

Lipid rafts Soluble fractions
TM 1 2 3 4 5 6 7 8 9 10 11 12


a% akd&^ I lZh24wYHAp


Bottom


Fraction number

Lipid rafts Soluble fractions

1 2 3 4 5 6 7 8 9 10 11 12


a


Bottom


Fraction number


Lipid rafts
KDa TM 1 2 3 4 5
1050
662 .
I


Lipid rafts
KDa TM 1 2 3 4 5


Gradient Top


Soluble fractions
6 7 8 9 10 11 12
SPmalp




Soluble fractions
6 7 8 9 10 11 12

L V-ALP


Bottom


Figure 6-4. Localization of Izh2p and Izh3p in lipid rafts prepared from total membranes. Panel
A from top to the bottom, shows that Izh2p and Izh3p are distributed between lipid
raft and soluble fractions indicating that these proteins are associated to lipid rafts but
are not permanent residents of these membrane microdomains. In a similar way,
Pmalp was barely localized in lipid raft, as well as in soluble fractions (panel B-top),
indicating that this proteins is also undergoing dissociation from lipid rafts. On the
other hand, V-ALP was strictly recovered in the lowermost fractions, corresponding
to soluble proteins (panel B-bottom). In the figure, proteins shown in each Western
blot were recognized using specific antibodies against each of the proteins analyzed.
In the figure TM, stands for total membrane.


3633


Gradient


KDa
97A
86.2
63.3
Gradient









APPENDIX A
YEAST GROWTH MEDIA AND PROCEDURE

Yeast Peptone Dextrose (1X-YPD)

5 g/L yeast extract [Fisher]
10 20 g/L peptone [Fisher]
2%, w/v alpha (+) glucose (99%, anhydrous) [Across Organics]
Nano pure water to 1 L
Before being used, the solution was sterilized

Synthetic Medium Supplemented with Dextrose (SD)

1.7 g/L YNB without amino acids and ammonium sulfate [Fisher]
2%, w/v alpha (+) glucose (99%, anhydrous) [Across Organics]
5 g/L ammonium sulfate [Fisher]
0.01%, w/v appropriate amino acids [Sigma-Aldrich]
Nano pure water to 1 L
Before being used, the solution was sterilized

Synthetic Medium Supplemented with Galactose (SGal)

1.7 g/L YNB without amino acids and ammonium sulfate [Fisher]
2%, w/v D (+) galactose [Across Organics]
5 g/L ammonium sulfate [Fisher]
0.01%, w/v appropriate amino acids [Sigma-Aldrich]
Sterile nano pure water

Chelexed Synthetic Medium Supplemented with Dextrose (CSD)

5.1 g/L YNB without divalent cations, amino acids, ammonium sulfate, and phosphates
[Qbiogene]

2%, w/v alpha (+) glucose (99%, anhydrous) [Across Organics]

5 g/L ammonium sulfate [Fisher]

0.01%, w/v appropriate amino acids [Sigma-Aldrich]

Sterile water to 850 mL

The mixture was stirred for 1 overnight at 40C with 25 g Chelex-100 ion exchange resin
[Sigma]

The resin was removed and then the pH was adjusted to 4.0 with HCI









The mixture was supplemented with 10 mL 100g/L potassium phosphate monobasic
(KH2PO4),

24 pL 100 mM manganese sulfate (MnSO4), 10 pL 4 g/L copper sulfate (CuSO4), 1 mL
100 g/L calcium chloride (CaCl2), and 1 mL 500 g/L magnesium sulfate (MgSO4).

Either of the following solutions was added to the indicated final concentration only if the
medium did not have restriction of each metal:

1.2 [tM iron chloride (FeC13) final concentration

2.2 [tM zinc chloride (ZnCl2) final concentration

Sterile nano pure water was added to complete 1 L


For either zinc or iron deficiency ZnCl2 or FeC13 was not added back to CSD. For a mdium

replete of zinc or iron 10 [tM of either ZnCl2 or FeC13 was adde d back to the growth medium.

The solution was then filter-sterilized into polycarbonate flasks. All plastic used for CSD media

preparation and cell culturing was washed with Acationox detergent (Baxter Scientific Products,

McGaw Park, IL) before being used.


Chelexed Synthetic Medium Supplemented with Galactose (CSGal)

5.1 g/L YNB without divalent cations, amino acids, ammonium sulfate, and phosphates
[Qbiogene]

2%, w/v D (+) galactose [Across Organics]

5 g/L ammonium sulfate [Fisher]

0.01%, w/v appropriate amino acids [Sigma-Aldrich]

Sterile water to 850 mL

The mixture was stirred for 1 overnight at 40C with 25 g Chelex-100 ion exchange resin
[Sigma]

The resin was removed and then the pH was adjusted to 4.0 with HC1

The mixture was supplemented with 10 mL 100g/L potassium phosphate monobasic
(KH2PO4),









24 pL 100 mM manganese sulfate (MnSO4), 10 [tL 4 g/L copper sulfate (CuSO4), 1 mL
100 g/L calcium chloride (CaC12), 1 mL 500 g/L magnesium sulfate (MgSO4).

Either of the following solutions was added to the indicated final concentration, only if
the medium did not have restriction of each metal:

1.2 [tM iron chloride (FeC13) final concentration

2.2 [tM zinc chloride (ZnCl2) final concentration

Sterile nano pure water was added to complete 1 L.

Either, zinc or iron was added back to CSGal to a final concentration of 50 nM

(deficiency), and 10 [tM repletionn). The solution was then filter-sterilized into polycarbonate

flasks. All plastic used for CSD media preparation and cell culturing was washed with Acationox

detergent (Baxter Scientific Products, McGaw Park, IL) before use.


Low Iron Medium Supplemented with Dextrose (LIMD)

1.7 g/L of YNB without amino acids and ammonium sulfate [Fisher]

2%, w/v alpha (+) glucose (99%, anhydrous) [Across Organics]

5 g/L ammonium sulfate [Fisher]

0.01%, w/v appropriate amino acids [Sigma-Aldrich]

20 mL 1.0 M sodium citrate, pH 4.2 (sodium citrate was obtained from Fisher]

1 mM, (final concentration) ofEDTA, pH 8.0 [EDTA was obtained from Sigma]

MnC12 was added back to LIM to a final concentration of 20 [tM

ZnSO4 was added to a final concentration of 0.8 [tg/mL, (5 ptM)

Iron deficiency, or iron repletion were generated by adding either, 1 ptM or 1 mM FeC13,
respectively. Sterile nano pure water was added to 1 L

The solution was then filter sterilized into polycarbonate flasks.









Low Iron Medium Supplemented with Galactose (LIMGal)

1.7 g/L of YNB without amino acids and ammonium sulfate [Fisher]

2%, w/v D (+) galactose [Across Organics]

5 g/L ammonium sulfate [Fisher]

0.01%, w/v appropriate amino acids [Sigma-Aldrich]

20 mL 1.0 M sodium citrate, pH 4.2 (sodium citrate was obtained from Fisher]

1 mM (final concentration) ofEDTA, pH 8.0 [EDTA was obtained from Sigma]

MnC12 was added back to LIM to a final concentration of 20 [tM

ZnSO4 was added to a final concentration of 0.8 [tg/mL, (5 [tM)

Iron deficiency, or iron repletion were generated by adding either, 1 [tM or 1 mM FeC13,
respectively. Sterile nano pure water was added to 1 L

The solution was then filter sterilized into polycarbonate flasks.









APPENDIX B
YEAST TRANSFORMATION

Protocol

Single colonies of a S. cerevisiae yeast strain (e.g. the BY4742 wild type or the double

mutant ypclAydclA strain) were grown until stationary phase (OD600 of 3) during one overnight

(betweeenl2-18 h) in 1X-Yeast Peptone Dextrose medium (1X-YPD) at 30C and with constant

agitation at 250 rpm. Fresh YPD (5 mL) was inoculated with the overnight culture and cells were

then grown to logarithmic phase (OD600 of 1.0). Cells were harvested at 3,500 rpm for 5 min and

resultant pellets were washed with 5 mL of LiTE solution. The suspension was spun down and

most of the supernatant was discarded (50 ptL of cell suspension in LiTE solution is enough for

each transformation).

Yeast transformation was performed as follows. Cell suspension (50 tiL) was aliquoted in

a 1.5 mL eppendorf tube and mixed with 400 |tg of appropriate plasmid DNA (approximately 2

ItL), and 10 ptL of carrier DNA (10 mg/ml salmon testes DNA stock, which must be boiled for 5

min before each use and flash cooled on ice before addition to the cells). 500 p.L of PEG-LiTE

solution was added to the mixtures and suspended by vortexing. The suspension was incubated at

30C for 1 h. This was followed by heat-shock at 420C for 12 min. Cells were then spun down at

4,000 rpm in a microcentrifuge and the supernatant wasaspirated. The pellet was suspended in

200 p.L of LiTE solution. Aliquots of 50 ptL of this suspension were plated onto solid agar

synthetic media plates supplemented with the appropriate carbon source and amino acids. Plates

were incubated at 300C until colonies were visible (usually 3 to 4 days after transformation).









Solutions

Litium Acetate Tris Base Ethylenediamminetetraacetic Acid (LITE) Solution

5 mL, sterile 10X-TE stock (100 mM Tris pH 7.5, 10 mM EDTA, pH 7.5); 5 mL, sterile

10X-litium acetate stock solution (sterile 1.0 M lithium acetate); 40 mL sterile ultrapure water.

Store this solution at room temperature.

Polyethylenglycol Litium Acetate Tris Base Ethylenediamminetetraacetic Acid (PEG-
LiTE) Solution

5 mL, sterile 10X-TE stock; 5 mL, sterile 10X-litium acetate stock; 40 mL, sterile 44%,

w/v PEG-3350 stock. Store this solution at room temperature.

Tris Base Ethylenediamminetetraacetic Acid (10X-TE) Solution

25 mL of 1 M tris stock, pH 7.5; 5 mL 500 mM EDTA stock, pH 7.5; 250 mL ultrapure

water. Sterilize the solution and store it at room temperature.

Carrier DNA

10 mg salmon testes DNA and 10 mL ultrapure water. Shear by drawing up into a 10 mL

syringe with 18 g during 15 times. Boild, and restore volume to 10 mL. Make 10x-(lmL)

aliquots and store them at -200C.











APPENDIX C
GAS-CHROMATOGRAMS AND MASS SPECTRA OF TOTAL STEROLS

F-DSO Dam. sa yomrs M7 E1I_3
Sbardrd Lc.A.3: sWess '
RT: 6 -1100 SM 0SG I
7.93 N:
1 62H1R 7
TIC F: MS
596 El 36


Tune MiD


B 100 129

so 73 "5 329 AMB

S 69 131 3e3
|| 119 14 33


1004_ i
U2 0
,SE ikoo L TK_


1 291
100 200


393
L39


Hiz


NL 3A.4
5976 El 3W14-1 5 RIF: 7.83-2 AV
I 11 SB: 116 7.58-7.73 8.1.22T: +c
ru ins -I oi.n, .0] +

NL 23E5
5976 E 3m181-193 RI: 49-872 A:
13 SB:E 1 8a22-43 891T:+c
ir n I B .,N0.00.]

NL 1711
5976 El 310-219 RI: 9 05-9 22
B DSH: 11 819897 ,92 -932T:+c
5W40 R f-m [ i0700 00]

IL 2.69
5976 El 3Wr233-2 RI: 9.59.75 At:
W 14 SB: 1 930942 .99-996T:+c
HRFui .m5s ISoD0.0


Figure C-1. Gas chromatography-electron ionization in tandem with mass spectrometric analysis
(GC-EI-MS). TIC of internal standards. Panel A, GC-chromatograms, show that the
TMS derivative of cholesterol (I) eluted first, followed by those for ergosterol (II),
dihydrolanosterol (III) and lanosterol (IV). B, mass spectra of I, II and IV were
similar to those found in the NIST El spectral library.


, ,
















329


129



368


73



250
S281 363
107 121 / 458

69 145
159 247 3
Si443
Jil 173 1 9 I 2 I 23 275 301 313 34


40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40D 420 440 460
(mainlib) Choleterol tnmethylslyl ether
69
100-




363




337


81

119 131 143 1 5

157 253

991 II 378
105 169 185 468


main I I III I I2,1 6 1 21 I 61 267 279 293 453

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
(mainib) Slane, (ergosta-5,7,22-trien-3a-yloxy)tnmethyl-
69
100 69


393







73
50- 109

56 95

81
483
19 135 / \ 498
I 15 16117 1 215227 241 2 2
I 1111 1 1 255
G 11' 201

50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370 390 410 430 450 470 490 510
(mainlib) lane, [[(3a)-lanosta-8,24-d en-3-yl]oxy]trm ethyl-



Figure C-2. The NIST El mass spectra for the TMS derivatives of cholesterol (top), ergosterol

(middle) and lanosterol (bottom).














F'SQ
100 uL
A RT. 5
10

5


10

5


"10




1
5


Data .\L-ynsBOO6O O06 El 10
Wid-type (unsplked,contrml, splitles 1"
00- 10.50 SM. 5G
0 W
I'- -

A


317R2006 12 07.52 PM


799
0



0I
a0 86-
0





0 IVI
:1 ,v A ..


0'i '''u' ''u'''u''''' '''' ''r'' ''*' r'


5 6


7 8
Time (mim)


9 10


FlDSQDatal \Lw~0siOOO606 El 11
100 uL z3delta (unsplked,acntmol splitless 1
RT 500-1050 SM 5G
100

50


101
51


101

5


3117/2006123007 PM


&603
0



0

0 A95
0 sv I


868


IV


n .. . 1. . . 1. .1 . .. Ii. .. .


5 6


7 8
Time (min)


ML1-32ES
rI- MS
TIC F: US
006 El 11

HL1-20ES

40841080 F:
US 6006 El 11
HLS-20ES

457f,84580 F:
MS 6=06 El 11
ML2-49ES

467.846880 F:
MS 600 El 11
ML7-69E4
m&--
49780-41 0 F:
US 06 El 11


9 10


Figure C-3. Total ion chromatograms (TICs) and electron ionization chromatograms (EICs) of

sylilated sterols. A represents wild type and B, is izh3A.


IL1.19E8
ICF: MS
606 El 10

H-111ES

4098-41080 F:
iMS El E10
IL4987ES

isnaDsa so Fr
S 600BEl 1i
417.8A4680 F:
MS 600 El 10
N1112_17ES


ES 5W EI 10
I-6-27E4

1978-4".80 F:
MS 06 El 10


Z100
50















APPENDIX D

CHROMATOGRAMS AND MASS SPECTRA OF ERGOSTEROL ANALYZED BY HPLC-

APCI-MS


SBase Peak Chrom of+TOF MS from 5uM-E+L_LC-APCI-01 wiff Ma 81 e4cps
II
100%- 442

80% 4

eo% 4 IV I
5.40
S40%- 660

20% \


10 20 30 40 50 60 70 80 90 100 11.0 120
Time, mmn


SXIC of+TOF MS 378 8 to 379 8 amu from 5uM-E+LLC-APCI-01 wlff
II
100%- 442
100"Tme m

80% -

60%-

40%-
r
20%.

0% -
10 20 30 40 50 60
Time, mmin

ir lf+TnF hAq' 4nF F n o fQAmll frnm mIImh-F+I i A- pI-nh icitf


0%- 5.40


10%

0%-

,, W I .- , M y -..-,,.,,,,..^- ,. ,,^ i.- .^ ^ -- ^y r~A ^ ^ ^ ^ ^ -i ,. ii.^. -.. ,^.,, ,^ ,,,


10 20 30 40 50 60
Time, mm

l XIC of+TOF MS 4108to411 8 amu from 5uM-E+L LC-APCI-01 liff


Max 2 2e5 cps


7'0 '0 '0 100 11.0 120


Max 9 Oe4 ps


70 80 90 100 11.0 120


Max 8 0e4 ops


0%- 16 59

:0% -

0% -



20% -
nOt ... .. -j


10 20 30 40 50 60
Time, mm


70 80 90 100 11.0 120


Figure D-1. High perfomace liquid chromatography-atmospheric pressure chemical ionization in

tandem, with mass spectrometric analysis (HPLC-APCI-MS) of standards. The

HPLC-APCI-MS BPC (topmost) shows that ergosterol (II) eluted first, followed by

lanosterol (IV) and dihydrolanosterol (III).













155















U ~TOF MSi4370 ho 4~619 miTU~*W SwmcE+CL32 ACI.1 c..ff glierd wulb~atad ~.173c 4.321 'mis ad'S... Mt694sa


265 2*1 2W,
[ I I


0%". 4


1020 1 10 10 00 220 240 00 200 0 2 3 W 3 330 O 3 0 40 420
ViPiA M.


44 5 4M 4000


* +TOF MS:5334to 5.4?0 inlwl Su E+L.ELJ.C-APCO rtf Aqiletr, ubtractd S II 6S.240 mi' mds... Ma 2.7*4 ounts.

1100%. a
IV
40%-







0 1q IY 148 T 1 1 2 Z _23 ,1 1 2 311 4
120 14 100 1W 2 2M0 24 200 280 3 M00 320 3W s s104 4W0 4 40 410 W00
r, amu


* +1Ci1 MS:. SLYL ho 6.71 mlrbftm SwM-E+LJ.C-APCF 5M54111r4.C NIIaBOJO4 p21. 6434 cci, adS...


100%


123 1g a 191 20 217


207 2


uaW 2.S4 wunb.


120 1q0 1 1'0 xO 20 2i0 2d0 2M 20 Wi 0 9 3 0 40 420 4 0 t8 00
'i& j-W


U I MS~ 51~.71 157 SI !mlhrin 241)nZ~M-TJL-AFCWS kiRl~l bg.1f~UhIfltdl1 ~4U73S 514 Ilta ik Ia.


MUi r.odd 0tuck


147? Ie


120 14D 16 19iD 200 220 243 250 25 :0B 320 4 3 :00 203 430 420 440 40 4C80 S D
m& jrtu


Figure D-2. Atmospheric pressure chemical ionization chromatogram (APCI)-mass spectra for

the sterol standards. The figure shows the [(M+H)-H20]+ ion as the base peak for

ergosterol (II), lanosterol (IV), dihydrolanosterol (III) and cholesterol (I).


1m0%


iN~'9


UM.K G.Q4 CoonK.
















SBase Peak Chrom of+TOF MS from wildtype-free3_LC-APCI-01 lff Max 3.3e5 cps


100%- 2.32 281
2 03440 42



50%


10 20 30 40 50 60 70 80 90 100 110 120
Time, min

XIC of +TOF MS 378 8 to 379 9 amu from wiidtype-free3_LC-APC1-01 wiff Max 5.0e5 ops
I
440
100% 440



50% -




10 20 30 40 50 60 70 80 90 100 110 120
Time, mmin

XIC of+TOF MS 388 8 to 369 8 amu from wildtype-free3_LC-APCI-01 wiff Max 1.5e4 ops


100%- 278




58 1
0 58 18 12 382 415 439 4 .-5 6 48 78g 7865 8086.830 g2932 1007 10 60
S 80% 88 70 80 0
10 20 30 40 50 60 70 80 90 100 110 120
Time, mmn

SXIC of+TOF MS 408 to 409 8 amu from wildtype-free3_LC-APCI-01 wiff Max 1.7e4 ops


100% 538

1 53j 1 78
S 01
50%-
2 481
18418 2 13 3.35 7


10 20 30 40 50 60 70 80 90 100 110 120
Time, min

XIC of +TOF MS 410 9 to 411 9 amu from wlldtype-free3_LC-APCl-01 wlff Max 1.9e4 ops

100%- 35
00%



59" 1 10 6 7 III



10 20 30 40 50 60 70 80 90 100 110 120
Time, min




Figure D-3. Base peak chromatogram (BPC) and electron ionization chromatograms (EICs) for

wild type. The chromatograms indicate that ergosterol (II) is present in higher

concentration than lanosterol (IV).














SBase Peak Chrom of+TOF MS from izh3delta-free3_LC-APCI-01 wlff Max 3 Be5 ps.

100% 231 280






0 10
50% I I j

131.. : \ 4 I6.40

10 20 30 40 50 60 70 80 90 100 110 120
Time, mm

,.1 ro T rF I -. 4.00...0. .Or ,.ii.fo ... iLe -fee [ A I .n Ma. 17 p













IV
100.% 438


50%














1 10 2 20 40 5 0 Tie m1
10 20 30 40 50 60 70 80 90 100 110 120
Time, min










































Figure D-4. Base peak chromatogram (BPC) and electron ionization chromatograms (EJCs) for

izh3A. Chormatograms show that ergosterol (II) was also present in higher
concentration than anostero (IV).
50% 5 232

050 124 -1 45 1 4.11425 37 -15 39 7 ,703 775 943982 1 O.521102

10 20 30 40 50 60 70 80 90 100 110 120
Time, min

IC of+TOF MS 408.8 to 409.8 au from izh3elta-free3 -APCI-0 .Wlff Max. 1.7e4 V
IV
538



50%I



1O 20 30 40 50 60 70 80 90 100 110 120
Time, rin

r IC 1 TOF M- 410 B to 411 B aiu from izh3delta-f-ee3 LC-APCI-01 Wlff Max 5e4p

100%- 9 3 m 939



7, 1.77 957
'- 15 101 -254322 420, .53 5 54 _5925650..77740 7.82 839 glO

10 20 30 40 50 60 70 80 90 iiO0 110 120
Time, min



Figure D-4. Base peak chromatogram (BPC) and electron ionization chromatograms (EICs) for

Oh3A. Chormatograms show that ergosterol (II) was also present in higher

concentration than lanosterol (IV).









LIST OF REFERENCES


Arthinggton-Skaggs BA, Crowell DN, Yang H, Sturley SL, and Bard M ( 1996) Positive and
negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast
ergosterol pathway. FEBSLett 392: 161-165

Arthington-Skaggs BA, Jradi H, Desai T, Morrison, CJ (1999) Quantitation of ergosterol
content: Novel method for determination of fluconazole susceptibility of Candida albicans. J
Clin Microbiol 37: 3332-3337

Bagnat M, Keranen S, Shevchenko A, Shevchenko A, and Simons K (2000) Lipid rafts function
in biosynthetic delivery of proteins to the cell surface in yeast. Proc NatlAcad Sci USA 97:
3254-3259

Bagnat M, Chang A, and Simons K (2001) Plasma membrane proton ATPase Pmalp requires
raft association for surface delivery in yeast. Mol Biol Cell 12: 4129-4138

Baida GE, Kuzmin NP (1995) Cloning and primary structure of a new hemolysin gene from
Bacillus cereus. Biochim Biophys Acta 1264: 151-154

Baida GE, KUzmin NP (1996) Mechanism of action of hemolysin 111 from Bacillus cereus.
Biochim Biophys Acta 1284: 122-124

Bailey RB, and Parks LW (1975) Yeast sterol esters and their relationship to the growth of yeast.
JBacteriol 124: 606-612

Baker RT, and Board PG (1989) Unequal crossover generated variation in ubiquitin coding unit
number at the hyman Ubc polyubiquitin. Am JHum Genet 44: 534-542

Baudry K, Swain E, Rahier A, Germann M, Batta A, Ronde S, Mandal S, Henry K, Tint GS,
Edlind T, Kurtz M, and Nickels Jr JT (2001) The effect of the erg26A-1 mutation on the
regulation of lipid metabolism in Saccharomyces cersevisiae. JBiol Chem 276: 12702-12711

Baumgartner U, Hamilton B Piskacek M, Ruis H, Rottensteiner, H. (1999) Functional analysis of
the Zn2CyS6 transcription factors Oaflp and Pip2p. JBiol Chem 274: 22208-22216

Bhuiyan MSA, Ito Y, Nakamura A, Tanaka N, Fujita K, Fukui H, and Takegawa K (1999)
Nystatin effects on vacuolar function in Saccharomyces cerevisiae. Biosc Biotechnol Biochem
63: 1075-1082

Bielawski J, Szulc ZM, Hannun YA, Bielawska A (2006) Simultaneous quantitative analysis of
bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry.
Methods 39: 82-91

Black PN, Fargeman NJ, and DiRusso CC (2000) Long-chain acyl-CoA-dependent regulation of
gene expression in bacteria, yeast and mammals. JNutr 130: 305S-309S









Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 37: 911-917

Bolard J (1986) How do the polyene macrolide antibiotics affect the cellular membrane
properties?. Biochim Biophys Acta 864: 257-304

Breivik ON, Owades JL (1957) Spectrophotometric semi-microdetermination of ergosterol in
yeast. Agric Food Chem 5: 360-363

Carrillo-Mufioz AJ, Giusiano G, Ezkurra PA, Quind6s G (2006) Antifungal agents: mode of
action in yeast cells. Rev Esp Quimioter 19: 130- 139

Cherry JM, Alder C, Ball C, Chervitz SA, Dwight, SS, Hester, ET, Jia Y, Juvik G, Roe T,
Schroeder M, Weng SA, Botstein D (1998) Sacchraromyces genome database. Nucleic Acids
Res 26: 73-79

Choi JY, Stukey J, Hwang SY, and Martin CE (1996) Regulatory elements that control
transcription activation and unsaturated fatty acid-mediated repression of the Saccharomyces
cerevisiae OLE] gene. JBiol Chem 27: 3581-3589

Chung CY, and Obeid LA, (1999) Use of yeast as a model system for studies of sphingolipid
metabolism and signaling. Methods Enzymol 311: 319-331

Cowart LA, Obeid L (2007) Yeast sphingolipids, recent developments in understanding
biosynthesis, regulation, and function. Biochim Biophys Acta 1771: 421-431

Daum G, Tuller G, Nemec T, Hrastnik C, Ballianao G, Cattel L, Milla P, Rocco F, Conzelmann
A, Vionnet C, Kelly DE, Kelly S, Schweizer E, Schuller HJ, Hojad U, Greiner E, Finger K
(1999) Systematic analysis of yeast strains with possible defects in lipid metabolism. Yeast 15:
601-614

DeRusso CC, Black PN, and Weimar JD (1999) Molecular inroads into the regulation and
metabolism of fatty acids, lessons from bacteria. ProgLipidRes 38: 129-197

Dickson R (1998) Sphingolipid functions in Saccharomyces cerevisiae: Comparison to
mammals. Annu Rev Biochem 67: 27-48

Dickson RC, Lester RL, (2002) Sphingolipid functions in Saccharomyces cerevisiae. Biochim
Biophys Acta 1583: 13-25

Dupont D (2006) Use of topical antifungal agents. Therapie 61: 251-254

Dupre S, Haguenauer-Tsapis R (2003) Raft partitioning of the yeast uracil permease during
trafficking alonf the endocytic pathway. Traffic 4: 83-96

Edsall LC, Pirinanow GG, Spiguel S (1997) Involvement of Sphingosine 1-phosphate in nerve
growth factor-mediated neuronal survival and differentiation. JNeurosci 17: 6952-6960









Eide D, Broderious M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from
plants identified by functional expression in yeast. Proc Natl AcadSci USA 93: 5624-5628

Eide D, Zhao H, Butler E, and Rodgers J (1999) Regulation of zinc homeostasis in yeast by the
ZAP1 transcriptional activator. Metals and Genetics 24: 325-338

Einerhand AWC, Kos WT, Distel B and Tabl HF (1993) Characterization of a transcriptional
control element involved in proliferation of peroxisomes in yeast in response to oleate. Eur J
Biochem 314: 323-331

Eisenkolb M, Zenzmaier C, Leitner E, and Schneiter R (2002) A specific structural requirement
for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains
in yeast. MolBiol Cell 13: 4414-4428

Fujita T, Inoue K, Yamamoto S, Ikumoto T, Sasaki S, Toyama R, Chiba K, Hoshino Y,
Okumoto T (1994) Fungal metabolites. Part 11. A potent immunosuppressive activity found in
Isaria sinclairii metabolite. JAntibiot 47: 208-215

Gable K, Slife H, Bacikova D, Monaghan E, and Dunn TM (2000) Tsc3p is a 80-amino acid
protein associated with serine palmitolyltransferase and required for optimal enzyme activity. J
Biol Chem 276: 7597-7603

Gachotte D, Pierson CA, Lees ND, Barbuch R, Koegel C, and Bard M (1997) A yeast sterol
auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme. Proc
NatlAcadSci USA 94: 11173-11178

Gaigg B, Toulmary A, and Schneiter R (2006) Very long-chain fatty acid-containing lipids rather
than sphingolipids per se are required for raft association and stable surface transport of newly
synthesized plasma membrane ATPase in yeast. JBiol Chem 281: 34135-34145

Gaither LA, and Eide D (2001) Eukaryotic zinc transporters and their regulation. Biometals 14:
251-270

Gietz RD, and Woods RA (1994) High efficiency transformation with lithium acetate. In JR
Johnson (ed), Molecular Genetics of Yeast: A Practical Approach, Oxford University Press, NY,
121-134

Gerst N, Ruan B, Pang J, Wilson WK, and Schroepfer GJ Jr (1997) An updated look at the
analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry. JLipidRes
38:1685-1701

Ghannowm MA, and Rice LB (1999) Antifungal agents: Mode of action, mechanisms of
resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12:
501-517

Gitan RS, and Eide D (2000) Zinc-regulated ubiquitin conjugation signals endocytosis of the
yeast ZRT1 zinc transporter. Biochem J346: 329-336









Goldstein AL, Pan X, & McCusker JH (1999) Heterologous URA3MX cassettes for gene
replacement in Saccharomyces cerevisiae. Yeast 15: 507-511

Goldstein AL, and McCusker JH (1999) Three new dominant drug resistance cassettes for gene
disruption in Saccharomyces cerevisiae. Yeast 15: 1541-1553

G6mez-Mount6n C, Lacalle RA, Mira E, Jimenez-Barada S, Barber DF, Carrera AC, Martinez-A
C, and Mafies S (2004) Dynamic redistribution of raft domains as an organizing platform for
signaling during cell chemotaxis. JChem Biol 164: 759-768

Gong P, Hu B, Stewart D, Ellerbe M, Figueroa YG, Blank V, Beckman BS, and Alam J (2001)
Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent
mechanism in chinese hamster ovary cells. JBiol Chem 276: 27018-27025

Grossmann G, Opekarova M, Novakoca L, Stolz J, and Tanner W (2006) Lipid raft-based
membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae.
Erukatyot Cell 5: 945-953

Grossmann G, Opekarova M, Malinsky J, Weig-Meckl I, and Tanner W (2007) Membrane
potential governs lateral segregation of plasma membrane proteins and lipids in yeast. EMBO J
26: 1-8

Guarente L (1983) Yeast promoters and lacZ fusion designed to study expression of cloned
genes in yeast. Methods Enzymol 101: 181-191

Han, G, Gable K, Kohlwein SD, Beaudoin F, Napier JA, and Dunn TM (2002) The
Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty
acid elongase. JBiol Chem 277: 35440-35449

Hannun YA, and Bell RM (1989) Functions of sphingolipids and sphingolipid breakdown
products in cellular regulation. Science 243: 500-507

Hannun YA, and Obeid L (2002) The ceramide centric universe of lipid-mediated cell
regulation: stress encounters of the lipid kind. JBiol Chem 277: 25847-25850

Hapala I, Klobucnikova V, Mazaiiova K, and Kohut P (2005) Two mutants selectively resistant
to polyenes revceal distinct mechanisms of antifungal activity by nystatin and amphotericin B.
Transations 33: 1206-1209

Hauwaerts D, Alexandre G, Das SK, Vanderleyden J, Zhulin IB (2002) A major chemotaxis
gene cluster in Azospirillum brasilense and relationships between chemotaxis operon in alpha-
proteobacteria. FEMS Microbiol Lett 208: 61-67

Hertz GZ, and GD Stormo (1999) Identifying DNA and protein patterns with statistically
significant alignments of multiple sequences. Bioinformatics 15: 563-577









Hiltunen JK, Mursula AM, Rottensteiner H, Wierenga PK, Kastaniotis AJ, Gurvitz A (2003) The
biochemistry of peroxisomal P-oxidation in the yeast Saccharomyces cerevisiae. FEMS
Miocrobiol Rev 27: 35-64

Ho E (2004) Zinc deficiency, DNA damage and cancer risk. JNutr Biochem 15: 572-578

Hsieh M-H, and Goodman HM (2005) A novel gene family in encoding putative heptahelical
transmembrane proteins homologous to human adiponectin receptors and progestin receptors. J
Exp Bot 56: 3137-3147

Huang LE, Arany Z, Livingston DM, and Bunn F (1996) Activation of hypoxia-inducible
transcription factor depends primarily upon redox-sensitive stabilization of its ac subunit. JBiol
Chem 271: 32253-32259

Jiang Y, Vasconcelles MJ, Wretzel S, Light A, Gilooly L, McDaid K, Oh C, Martin CE, and
Goldberg, MA (2002) Mga2p processing by hypoxia and unsaturated fatty acids in
Saccharomyces cerevisiae: Impact on LORE-dependent gene expression. Eukaryot Cell 1: 481-
490

Jiang Y, Vasconvelles MJ, Wretzel S Light A, Martin CE, and Goldberg MA (2001) MGA2 is
involved in the low-oxygen response element-dependent hypoxic induction of genes in
Saccharomyces cerevisiae. Mol Cell Biol 12: 6161-6179

Kandasamy P, Vemula M, Oh C, CHellappa R, and Martin CE (2004) Regulation of unsaturated
fatty acid biosynthesis in Saccharomyces. JBiol Chem 279: 36586-36592

Karpichev IV, Luo Y, Marians RC, and Small GM (1997) A complex containing two
transcription factors regulates peroxisome proliferation and the coordinate induction of 3-
oxidation enzymes in Saccharomyces cerevisiae. Mol Cell Biol 17: 69-80

Karpichev IV, and Small GM (1998) Global regulatory functions of Oafl2p and Pip2p (Oaf2p)
transcription factors that regulate genes encoding peroxisomal proteins in Saccharomyces
cerevisiae. Mol Cell Biol 18: 6560-6570

Karpichev IV, Cornivelli L, and Small GM (2002) Multiple regulatory roles of a novel
Saccharomyces cerevisiae protein, encoded by YOL002c, in lipid and phosphate metabolism. J
Biol Chem 277: 19609-1961

Kastaniotis AJ, and Zitomer RS (2000) Roxl mediated repression: Oxygen dependent repression
in yeast. AdvExpMedBiol475: 185-195

Keen CL, Peters JM, Hurley LS (1989) The effect of valproic acid on 65Zn distribution in the
pregnant rat. JNutr 119: 607-611

Kerridge D (1986) Mode of action of clinically important antifungal drugs. Adv Microb Physiol
27: 1-72









Kerridge D, Koh TY, Marriott MS, and Gale EF (1976) Microbiology and plant protoplasts. In J.
F. Peberdy, A. H. Rose, H. J. Rodger, and E. C. Cocking (ed.), Microbiology andplant
protoplasts. Churchill Livingston, London, England 23-38

Kim, H, Melen, K. & von Heijne G (2003) Topology models for 37 Saccharomyces cerevisiae
membrane proteins based on C-terminal reporter fusions and predictions. JBiol Chem 278:
10208-10213

Koh JY, Suh SW, Gwang BJ, He YY, Hsu CY, Choi DW (1996) The role of zinc in selective
neuronal death after transient global cerebral ischemia. Science 272: 1013-1016

Kontoyiannis DP, Lewis RE (2002) Antifungal drug resistance of pathogenic fungi. Lancet 359:
1135-1144

Kos W, Kal, AJ, van Wilpe S, and Tabak HF (1995) Expression of genes encoding peroxisomal
proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control.
Biochim Biophys Acta 1264: 79-86

Kubler E, Dohlman HG, and Lisanti, MP (1996) Identifiaction of triton X-100 insoluble
membrane domains in the yeast Saccharomyces cerevisiae. JBiol Chem 271: 332975-32980

Kumar A, Xia YP, Laipis PJ, Fletcher BS, and Frost SC (2004) Glucose deprivation enhances
targeting of GLUT1 to lipid rafts in 3T3-L1 adypocytes. Am JPhysiolEndocrinolMetab 286:
568-576

Kupchak BR, Garitaonandia I, Villa NY, Mullen MB, Weaver MG, Regalla LM, Kendall EA,
Lyons TJ (2007) Probing the mechanism of FET3 repression by Izh2p overexpression. Biochim
Biophys Acta 1773: 1124-1132

Kwast KE, Burke PV, Staahl BT, and Poyton RO (1999) Oxygen sensing in yeast: evidence for
the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic
genes. Proc Natl Acad Sci USA 96: 5446-5451

Lampen JO, Arnow PM, and Safferman RS (1960) Mechanism of protection by sterol against
polyene antibiotics. JBacteriol 80: 200-206

Lauwers E, and Andre B (2006) Association of yeast transporters with detergent-resistant
membranes correlates with their cell-surface. Traffic 7: 1045-1059

Leber A, Fischer P, Schneiter R, Kohlwein SD, Daum G (1997) The yeast mic2 mutant is
defective in the formation of mannosyl-diinositolphosphorylceramide FEBS Lett 411: 211-214

Lederberg J (1950) The P-galactosidase of Escherichia coli strain K-12. JBacteriol 60: 381-392

Lee MCS, Hamamoto S, and Schekman R (2002) Ceramide biosynthesis is required for the
formation of the oligomeric H -ATPase Pmalp in the yeast endoplasmic reticulum. JBol Chem
277: 22395-22401









Lester RL, and Dickson RC (1993) Sphingolipids and inositol containing headgroups. AdLipid
Res 26: 253-274.

Luo Y, Karpichev IV, Kohanski RA, and Small GM (1996) Purification, identification, and
properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding
protein that is involved in the activation of POX]. JBiol Chem 271: 12068-12075

Lyons TJ, Gasch AP, Gaither LA, Botstein D, Brown PO, and Eide DJ (2000) Genome-wide
characterization of the Zap p zinc-responsive regulon in yeast. Proc Nat Acad Sci USA 97:
7957-7962

Lyons TJ, Villa NY, Regalla LM, Kupchak BR, Vagstad A, and Eide DJ (2004)
Metalloregulation of yeast membrane steroid receptor homologs. Proc Nat Acad Sci USA 101:
5506-5511

MacDiarmid CW, Gaither LA, and Eide D (2000) Zinc transporters that regulate vacuolar zinc
storage in Saccharomyces cerevisiae. 19: 2845-2855

MacDiarmid CW, Milanick MA, and Eide DJ (2002) Biochemical properties of vacuolar zinc
transport systems of Saccharomyces cerevisiae. JBiol Chem 277: 39187-39194

Mandala SM, Thornton RA, Frommer BR, Curotto JE, Rozdilsky W, Kurtz MB, Giacobbe R A,
Billis GF, Cabello MA, Martin I, Pelaez, F, Harris, GH (1995) The discovery of australifungin, a
novel inhibitor of sphinganine N-acyltransferase from Sporormiella australis. Producing
organism, fermentation, isolation, and biological activity. JAntibiot 48: 349-356

Mao C, Xu R, Bielawska A, Szulc ZM, Obeid LM (2000a) Cloning and characterization of a
Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide. JBiol
Chem 275: 31369-31378

Mao C, Xu R, Bielawska A, Obeid LM (2000b) Cloning of an alkaline ceramidase from
Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase
activity. JBiol Chem 275: 6876-6884

Markwell MAK, Haas SM, Tolbert NE, and Bierber LL (1981) Protein determination in membrane
and lipoprotein samples: manual and automated procedures. Methods Enzymol 72: 296-303

Marini F, Arnow P, and Lampen JO (1960) The effect of monovalent cations on the inhibition of
yeast metabolism by nystatin. J Gen Microbiol 24: 51-62

McDounough VM, Stukey JE, and Martin CE (1992) Specificity of unsaturated fatty acid-
regulated expression of the Saccharomyces cerevisiae OLE1 gene. JBiol Chem 267: 5931-5936

Megumi S, Shin-ichiro M, Mariko A, Tsutoma F, Kazuhiro I, and Haruyki I (2005) Effects of
culture conditions on Ergosterol biosynthesis by Saccharomyces cerevisiae. Biosci Biotechnol
Biochem 69: 2381-2388.









Merrill AH Jr, Wang E, Mullins RE, Jamison WC, Nimkar S, and Liotta DC (1988) Quantitation
of free sphingosine in liver by high-performance liquid chromatography. Anal Biochem 171:
373-381

Merrill AH Jr, Caligan TB, Wang E, Peters K, and Ou J (2000) Analysis of sphingoid bases and
sphingoid base-l-phosphates by high performance liquid chromatography. Methods Enzymol
312: 3-9

Merrill AH Jr, Schmelz EM, Dillehay DL, Spigel S, Shayman JA, Schroeder JJ, Riley RT, Voss
KA, and Wang E (1997) Sphingolipids-The Enigmatic lipid class: biochemistry, physiology, and
pathophysiology. ToxicolApplPharmacol 142: 208-225

Merrill AH Jr, Van-Echten G, Wang E, and Sandhoff K (1993) Fumonisin B1 inhibits
sphingosine (sphinganine) N-acyltransferase and de novo sphingoilipid biosynthesis in cultured
neurons in situ. JBiol Chem 268: 27299-27306

Menaldino DS, Bushnev A, Sun A, Liotta DC, Symolon H, Desai K, Dillehay DL, Peng Q,
Wang E, Allegood J, Trotman-Pruett S, Sullards MC, Merrill AH Jr (2003) Sphingoid bases and
de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action.
Pharmacol Res 47: 373-381

Mo C, Valachovic M, Randall SK, Nickels JT, and Bard M (2002) Protein-protein interactions
among C-4 demethylation enzymes involved in yeast sterol biosynthesis. Proc NatlAcad Sci
USA 99: 9739-9744

Moffett S, Brown DA, Linder ME (2000) Lipid-dependent targeting of G proteins into rafts. J
Biol Chem 275: 2191-2198

Mukhopadhyay K, Kohli A, and Prasad R (2002) Drug susceptibilities of yeast cells are affected
by membrane lipid composition. Antimicrob Agents Chemother 46: 3695-3705

Miullner H, Deutsch G, Leitner E, Ingolic E, and Daum G (2005) YEH2/YLR020c encodes a
novel steryl ester hydrolase of the yeast Saccharomyces cerevisiae. JBiol Chem 280: 13321-
13328

Nakagawa Y, Sugioka S, Kaneko Y, and Harashima S (2001) 02R, a novel regulatory element
mediating Roxlp-independent 02 and unsaturated fatty acid repression of OLE1 in
Saccharomyces cerevisiae. JBacteriol 183: 745-751

Narasimhan ML, Damsz B, Coca MA, Ibeas JI, Yun D-J, Pardo JM, Hasegawa PM, and Bressan
RA (2001) A plant defense protein induces microbial apoptosis. Mol Cell 8: 921-930

Narasimhan ML, Coca MA, Jin J, Yamauchi T, Ito Y, Kadowaski T, Kin KK, Pardo JM, Damsz
B, Hasegawa PM, Yun DJ, and Bressan RA (2005) Osmotin is a homolog of mammalian
adiponectin and controls apoptosis in yeast though a homolog of mammalian adiponectin
receptor. Mol Cell 17: 171-180









Nothwehr SF, Conibear E, and Stevens TH (1995) Golgi and vacuolar membrane proteins reach
the vacuole in vpsl mutant yeast cells via the plasma membrane. JCellBiol 129: 35-46

Oteiza PI, Olin KL, Fraga CG, and Keen CL (1995) Zinc deficiency causes oxidative damage to
proteins, lipids and DNA in rat testes. JNutri 125: 823-829

Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G, Roelofs J, Finley D, and
Gygi S (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol
21: 921-926

Perry DK (2002) Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and
other stress responses. Biochim Biophys Acta 1585: 145-152

Pike LJ (2003) Lipid rafts: bringing order to chaos. JlipidRes 44: 655-665

Pizzirusso M, and Chang A (2004) Ubiquitin-mediated targeting of a mutant plasma membrane
ATPase, Pmal-7, to the endosomal/vacuolar system in yeast. Mol Biol Cell 15: 2401-2409

Rehli M, Krause SW, Schwarzfischer L, Kreutz M, Andreesen R (1995) Molecular cloning of a
novel macrophage maturation-associated transcript encoding a protein with several potential
transmembrane domains. Biochem Biophys Res Commun 217: 661-667

Reiner S, Micolod D, and Schneider R (2005) Saccharomyces cerevisiae, a model to study sterol
uptake and transport in eukaryotes. Biochem Soc Trans 33: 1186-1188

Reiner S, Micolod D, Zellnig G, and Schneiter R (2006) A genomewide screen reveals a role of
mitochondria in anaerobic uptake of sterols in yeast. MolBiol Cell 17: 90-103

Rodriguez RJ, Low C, Bottema CD, and Parks LW (1985) Multiple functions for sterols in
Saccharomyces cerevisiae. Biochim Biophys Acta 837: 336-343

Rutherford JC, Jaron S, and Winge DR. (2003) Aftl and Aft2 mediate iron-responsive gene
expression in yeast through related promoter elements. JBiol Chem 278: 27636-27643

Rutherford JC, and Bird AJ (2004) Metal-responsive transcription factors that regulate iron, zinc
and copper homeostasis in eukaryotic cells. Eukaryot Cell 3: 1-13

Sambrook J, and Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd. edition,
Cold Spring Harbor Lab Press, Cold Spring Harbor, NY

Schnabl M, Daum G, Pichler H (2004) Multiple lipid transport pathways to the plasma
membrane in yeast. Biochim Biophys Acta 1687: 130-140

Serrano R (1988) H -ATPase from plasma membranes of Saccharomyces cerevisiae and Avena
sativa roots: Purification and reconstitution. Methods Enzymol 157: 533-544

Shapiro L, Scherer PE (1998) The crystal structure of a complement-lq family suggests an
evolutionary link to tumor necrosis factor. Curr Biol 8: 335-338









Shimada K, Miyazaki T, Daida H (2004) Adiponectin and atherosclerotic disease. Clin Chim
Acta 344: 1-12

Simons K, and Ikonen E (1997) Functional rafts in cell membranes. Nature 387: 569-572

Sims KJ Spassieva SD, Voit EO, and Obeid LM (2004) Yeast sphingolipid metabolism: clues
and connection. Biochem Cell Biol 82: 45-61

Smith SW, and Lester, RL (1974) Inositol phosphorylceramide, a novel substance and chief
member of a major group of yeast sphingolipids containing a single inositol phosphate. JBiol
Chem 249: 3395-3405

Streit F, Niedmann P-D, Shipkova M, ArmstrongVW and Oellerich M (2001) Rapid and
Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of
Monoethylglycinexylidide. Clin Chem 47: 1853-1856

Swain E, Baundry K, Strukey J, McDonough V, Germann M, and Nickels JT Jr (2002) Sterol-
dependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae. JBiol Chem 277:
26177-26184

Taylor KM, Nicholson RI (2003) The LZT proteins, the LIV-1 subfamily of zinc transporters.
Biochim Biophys Acta 78452: 1-15

Toulmay A, Schneiter R (2007) Lipid-dependent surface transport of the proton pumping
ATPase: A model to study plasma membrane biogenesis in yeast. Biochimie 80: 249-254

Umebayaski K (2003) The roles of ubiquitin and lipids in protein sorting along the endocytic
pathway. Cell Struct Function 28: 443-453

Umebayashi K, and Nakano A (2003) Ergosterol is required for targeting of tryptophan permease
to the yeast plasma membrane. JCellBiol 161: 1117-1130

Valachovi6 M, Hronska L, Hapala I (2001) Anaerobiosis induces complex changes in sterol
esterification pattern in the yeast Saccharomyces cerevisiae. FEMSMicrobiol Lett 197: 41-45

Vallee B, and Riezman H (2005) Liplp: a novel subunit of acyl-CoA ceramide synthase. EMBO
J24: 730-741

Vasconcelles MJ, Jiang Y, McDaid K, Gilooly L, Wretzel S, Porter DL, Martin CE, and
Goldberg MA (2001) Identification and characterization of a low oxygen response element
involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. JBiol Chem
276: 14374-14384

Veen M, Stahl U, Lang C (2003) Combined overexpression of genes of the ergosterol
biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast
Res 4: 87-95









Veen M, and Lang C (2005) Interactions of the ergosterol biosynthetic pathway with other lipid
pathways. Biochem Soc Trans 33: 1178-1181

Veronese P, Ruiz M, Coca MA, Hernandez-Lopez A, Lee H, Ibeas JI, Damsz B, Pardo JM,
Hasegawa PM, Bressan RA (2003) In defense against pathogens. Both plant sentinels and foot
soldiers need to know the enemy. Plant Physiol 131: 1580-1590

Wach A, Brachat A, Pohlmann R, and Phillippsen P (1994) New heterologous modules for
classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808

Watson PF, Rose ME, and Kelly SL (1998) Isolation and analysis of ketoconazole resistant
mutants of Saccharomyces cerevisiae. JMed Vet Mycol 3: 153-162

Woods RA (1971) Nystatin-resistant mutants of yeast: Alterations in sterol content. JBacteriol
108: 69-73

Wu WI, McDonough VM, Nickels JT Jr, Jo J, Fischl AS, Vales TR, Merrill AH Jr, and Carman
GM (1995) Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1. JBiol
Chem 270: 13171-13178

Wu CY, Bird AJ, Winge DR, and Eide DJ (2006) Regulation of the yeast TSA1 peroxiredoxin
by ZAP1 is an adaptive response to the oxidative stress of zinc deficiency. JBiol Chem 282:
2184-2195

Yamaguchi-Iwai Y, Stearman R, Dancis A, and Klausnerl RD (1996) Iron-regulated DNA
binding by the AFT1 protein controls the iron regulation in yeast. EMBO J 15: 3377-3384

Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita, S, Sugiyama T, Miyagishi M,
Hara K, Tsunoda M, Murakami, K, Otheki T, Uchida S, Takekawa S, Waki H, Tsuno NH,
Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R,
Kadowaki T (2003a) Cloning of adiponectin receptors that mediate antidiabetic metabolic
effects. Nature 423: 762-769

Yamuchi T, Hara K, Kubota N, Terauchi Y, Tobe K, Froguel P, Nagai R, Kadowaki T (2003b)
Dual role of adiponectin/Acrp30 in vivo as an anti-diabetic and anti-atherogenic adipokine. Curr
Drug Targets Immune Endocr Metabol Disord 3: 243-254

Yang HM, Bark DA, Bruner A, Gleeson RJ, Deckelbaum G, Aljinovic TM, Pohl R, Rothstein R,
and Sturley SL (1996) Sterol sterification in yeast: a two-gene process. Science 272: 1353-1356

Yun D-J, Zhao Y, Pardo JM, Narasimhan ML, Damsz B, Lee H, Abad LR, Paino D'Urzo M,
Hasegawa PM, and Bressan RA (1997) Stress proteins on the yeast cell surface determine
resistance to osmotin, a plant antifungal protein. Proc Natl Acad Sci USA 94: 7082-7087

Zhang S, Burkett TJ, Yamashita I, and Garfinkel, DJ (1997) Genetic redundancy between SPT23
and MGA2: Regulators of Ty-induced mutations and Tyl transcription in Saccharomyces
cerevisiae. Mol Cell Biol 17: 4718-4729









Zhang S, Skalsky Y, and Garfinkel DJ (1999)MGA2 or SPT23 is required for transcription of the
A9 fatty acid desaturase gene, OLE], and nuclear membrane integrity in Saccharomyces
cerevisiae. Genetics 151: 473-483

Zhao H, Eide D (1996a) The yeast ZRT1 gene encodes the zinc transporter protein of a high-
affinity uptake system induced by zinc limitation. Proc Natl Acad Sci USA 93: 2454-2458

Zhao H, Eide D (1996b) The ZRT2 gene encodes the low affinity zinc transporter in
Saccharomyces cerevisiae. JBiol Chem 271: 23203-23210

Zhao H, and Eide D (1997) Zaplp, a metalloregulatory protein involved in zinc-responsive
transcriptional regulation in Saccharomyces cerevisiae. Mol Cell Biol 17: 5044-5052

Zhao H, Butler E, Rodgers J, Spizzo T, Duesterhoeft S (1998) Regulation of zinc homeostasis in
yeast by binding of the ZAP transcriptional activator to zinc-responsive promoter elements. J
Biol Chem 273: 28713-28720

Zhu Y, Bond J, Thomas P (2003a) Identification, classification, and partial characterization of
genes in humans and other vertebrates homologous to a fish membrane progestin receptor Proc
NatlAcad Sci USA 100: 2237-2242

Zhu Y, Rice CD, Pang Y, Pace M, Thomas P (2003b) Cloning, expression, and characterization
of a membrane progestin recptor and evidence that it is an intermediary in meiotic maturation of
fish oocytes. Proc Natl Acad Sci USA 100: 2231-2236

Zinser E, Paltauf F, and Dunn G (1993) Sterol composition of yeast organelle membranes and
subcellular distribution of enzymes involved in sterol metabolism. JBacteriol 175: 2853-285









BIOGRAPHICAL SKETCH

Nancy Y. Villa was born in La Union (Valle), a small town of Colombia. After receiving a

B.S. in education of chemistry from the Universidad Santiago de Cali, and a B.S. in chemistry

from the Universidad del Valle, she joined the graduate program in department of chemistry of

the University of Florida where she pursued a doctorate, working under the supervision of Dr.

Thomas Lyons.

One of her major interests before starting her PhD was to investigate about the

development and progression of cancer, at biochemical level. This interest was mainly motivated

by familial reasons that put her close to the development and consequences of such a disease.

After completing her PhD, she will join the research group of Dr Grant McFadden, to work as a

post doctoral fellow in the department of Molecular Genetics and Microbiology in the school of

medicine at the University of Florida. In this group, she will investigate biochemical aspects that

determine the malignance of viruses and their implications in the development of cancer.





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1 CHARACTERIZATION OF NOVEL YEAST RE CEPTORS IMPLICATED IN METAL AND LIPID METABOLIC PATHWAYS By NANCY YANETH VILLA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Nancy Yaneth Villa

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3 For my mom and my grandmom Teresa, whose faith and strength have been my driving force to go on, and to face different challeges in my life.

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4 ACKNOWLEDGMENTS I would like to thank Dr. Thom as Lyons for the opportunity to work in his research projects, for the guidance and the support that gave me during my Ph.D. I also want to thank Drs Yusuf Hannun and Ashley Cowart for their help with some of my research projects, and for their incredible generosity. I am very grateful to th e people in the mass spectrometry laboratory at the University of Florida, and the people from the lip idomic core at the Medical University of South Carolina (MUSC). I want to give special thanks to Charlene Alford, at MUSC, for her technical support. Thanks also to my committee members, for their time, their suggestions and useful comments. I am forever grateful to my family and my friends for their unlimited love, and support. I am deeply grateful to my lovely brother Carl os Alberto, who despite the distance was always close to me, enjoying my triumphs and absorbing each of my tears. Without his help and support, getting to this point would never have been possible. Finally, thanks to my lovely God for helpi ng me to grow during this great academic opportunity.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................13 CHAP TER 1 INTRODUCTION..................................................................................................................15 Zinc Homeostasis............................................................................................................... .....15 The Budding Yeast Saccharomyces cerevisiae as a Model S ystem.......................................17 Identification of IZHs on the Sacchar omyces cerevisiae Genome.........................................17 Regulatory Sequences Surrounding the IZH Genes ...............................................................18 The IZH Gene Fam ily Encodes Membrane Proteins.............................................................. 19 The PAQR Family of Proteins................................................................................................ 20 Izh2 is an Osmotin Receptor Protein...................................................................................... 21 The IZHs and their Connection with Lipid Metabolic Pathways ........................................... 22 Summation..............................................................................................................................24 2 METALLOREGULATION OF IZHS ....................................................................................31 Introduction................................................................................................................... ..........31 Materials and Methods...........................................................................................................32 Yeast Strains and Plasmids.............................................................................................. 32 Yeast Media.....................................................................................................................33 Yeast Transformations and Assays.................................................................................35 Preparation of Microsomes.............................................................................................. 36 Western Blot Analysis of Protein Expression................................................................. 36 Immunoprecipitation of Izh2p a nd Western B lot Analysis............................................. 37 Results.....................................................................................................................................38 IZH2 is a Zap1p Target Gene .......................................................................................... 38 IZH2 and IZH4 Are Part of the Hypoxic Response ......................................................... 39 Regulation of IZH4 by Excess of Several T ransitions Metals is only Mga2pDependent....................................................................................................................40 Iron Deficiency Affects the Expression of IZH2 and IZH4 ............................................41 Ubiquitination of Izh2p is Dependent on Nutritional Conditions................................... 42 Discussion...............................................................................................................................43

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6 3 DUAL REGULATION OF THE IZHS BY METALS AND FATTY ACIDS: THE FIRST LINE OF EVIDENCE IMPLICATING THE IZH FAMILY IN LIPID METABOLISM ......................................................................................................................55 Introduction................................................................................................................... ..........55 Materials and Methods...........................................................................................................57 Yeast Strains and Plasmids.............................................................................................. 57 Biochemical Assays......................................................................................................... 58 Results.....................................................................................................................................58 Fatty Acids Exert a Regulatory Effe ct on the Expression of IZHs ..................................58 The Expression of the Izh Proteins is Regulated by Fatty Acids .................................... 59 Transcriptional Regulation of IZH2 and IZH4 in Presence of Metals and Fatty Acids Occurs via Mga2p..............................................................................................60 Discussion...............................................................................................................................61 4 NYSTATIN-RESISTANCE OF IZH3, IS ASSOCIATED TO ALTERATIONS IN THE ERGOSTEROL CONTENT..........................................................................................70 Introduction................................................................................................................... ..........70 Materials and Methods...........................................................................................................72 Yeast Strains and Reagents.............................................................................................72 Yeast Transformations.....................................................................................................73 Yeast Growth Media and Conditions..............................................................................73 Phenotypic Studies.......................................................................................................... 74 Complementation Studies................................................................................................ 75 Sterol Extractions............................................................................................................ 75 Sterol Analysis by Ultraviolet Spectroscopy...................................................................76 Analysis of Sterols by Gas Chromat ography-Mass Spectrom etry (GC-MS)..................77 Analysis of Total and Free Ergosterol by High Performance Liquid Chrom atography-Atmospheric Pressure Ch emical IonizationMass Spectrometry (HPLC-APCI-MS).......................................................................................................78 Results.....................................................................................................................................78 IZH Genes Affect the T oleran ce to the Antifungal Nystatin........................................... 78 Nystatin Induces Alterations in the Tota l Sterol C omposition of Wild Type and izh3...........................................................................................................................80 Gas Chromatography-Mass Spectrometric (GC-MS) Analysis Revealed not Signif icant Differences in the Basal Levels of Total Sterols for Wild Type and izh3............................................................................................................................81 Alterations in the Free Ergosterol Content Were Observed for the Mutant izh3..........81 Addition of Certain Sphingolipids Ameliorate the Aberrant Nystatin Effects on W ild Type and izh Mutants.......................................................................................83 Discussion...............................................................................................................................83 5 POTENTIAL IMPLICATION OF IZHS IN THE SPHINGOL IPID BIOSYNTHETIC PATHWAY............................................................................................................................97 Introduction................................................................................................................... ..........97

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7 Materials and Methods...........................................................................................................99 Strains Plasmids and Yeast Transformations.................................................................. 99 Yeast Growth Conditions................................................................................................99 In Vitro Ceram idase Assay............................................................................................100 Phenotypic Studies........................................................................................................ 102 Total Lipid Extraction................................................................................................... 102 Phospholipid Determination..........................................................................................103 High-Performance Liquid Chromatography Analysis of O -PhthalaldehydeSphingoid Base Derivatives .......................................................................................104 Analysis of Radiolabeled Sphingolipids by One-Dim ensional Thin Layer Chromatography........................................................................................................105 Analysis of Sphingolipids by Electrosp ray-Ionization Tandem Mass Spectrom etry (ESI-MS/MS).............................................................................................................106 Results...................................................................................................................................108 Overexpression of IZHs Produces Increase in the Levels of Free Sphingoid Bases ..... 108 In Vitro Ceram idase Assays Suggest that Izhs May not be Alkaline Ceramidases....... 109 Thin Layer Chromatography of Radio active Sphingolipids R eveals Similar Sphingolipid Profiles for YPC1 and the IZHs ............................................................111 Fumonisin B1 Induces the Acummulation of Sphingoid Bases in YPC1, IZH2, and IZH3 ...........................................................................................................................111 Myriocin Inhibits the Increase in the Leve ls of Sphingolipid Bi osynthesis Mediated by IZH2 and IZH3 ......................................................................................................112 Discussion.............................................................................................................................113 6 CELLULAR LOCALIZATION OF THE Izh2 AND Izh3 PROTEINS BY MEMBRANE FRACT IONATION...................................................................................... 128 Introduction................................................................................................................... ........128 Materials and Methods.........................................................................................................129 Plasmids and Yeast transformations.............................................................................. 129 Growth Conditions........................................................................................................130 Isolation Purification and Characteri zation of Yeast Plasm a Membranes.................... 130 Isolation of Lipid Rafts from Yeast Plasm a Membrane: Procedure 1........................... 132 Isolation of Lipid Rafts from Total Mem branes: Procedure 2...................................... 133 Results...................................................................................................................................135 Izh2p and Izh3p Were Found Enriched in Plasma Membrane......................................135 Izh2p and Izh3p Are Associated with Lipid Rafts........................................................137 Discussion.............................................................................................................................138 APPENDIX A YEAST GROWTH MEDIA AND PROCEDURE.............................................................. 146 B YEAST TRANSFORMATION............................................................................................ 150 Protocol.................................................................................................................................150 Solutions...............................................................................................................................151

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8 Litium Acetate Tris Base Ethylenediamminetetraacetic Acid (LITE) Solution ........... 151 Polyethylenglycol Litium Acetate Tris Base E thylenediamminetetraacetic Acid (PEG-LiTE) Solution................................................................................................. 151 Tris Base Ethylenediamminetetraacetic Acid (10X-TE) Solution ................................ 151 Carrier DNA..................................................................................................................151 C GAS-CHROMATOGRAMS AND MASS SPECTRA OF TOTAL STEROLS................. 152 D CHROMATOGRAMS AND MASS SPECTRA OF ERGOSTEROL ANALYZED BY HPLC-APCI-MS...................................................................................................................155 LIST OF REFERENCES.............................................................................................................159 BIOGRAPHICAL SKETCH.......................................................................................................171

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9 LIST OF TABLES Table page 2-1 List of strains used in Chapter 2........................................................................................ 472-2 Genes induced > 2-fold by zinc excess.............................................................................. 483-1 List of strains used in Chapter 3........................................................................................ 634-1 Sources and genotypes of the strains used in Chapter 4.................................................... 875-1 Strains and genotypes in Chapter 5.................................................................................. 116

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10 LIST OF FIGURES Figure page 1-1 Zap1p activation is zinc dependent. ................................................................................. 261-2 A common budding yeast Saccharomyces cerevisiae cell, and some of the most important and well characterized zinc transporters........................................................... 261-3 Phylogentic analysis of the Izhp family and its closer and more distant homologues in yeast...............................................................................................................................271-4 Regulatory elements surrounding IZH1, IZH2 and IZH4 genes suggest dual role in metal and lipid metabolism................................................................................................ 281-5 Predicted topology for the Izhp family.............................................................................. 281-6 Sequence alignment of important conser ved regions in PAQRs, hemolysins and ceramidases.................................................................................................................... ....291-7 Phylogentic showing the Izhp family, its closer and more distant homologues in yeast, and other organisms.................................................................................................291-8 The Sphingolipid biosynthetic pathway in the yeast Saccharomyces cerevisiae ..............302-1 Zinc regulation of IZH2 .....................................................................................................492-2 Transcriptional regulation of IZH2 and IZH4 by different metals..................................... 502-3 Transcriptional activation of IZH4-lacZ in cells exposed to excess of different metals is dependent on the presence of MGA2 but independent on SPT23. .................................512-4 Iron regulation of IZH2 and IZH4 ......................................................................................522-5 Post-translational effect of iron de ficiency on the overexpression of Izh2p..................... 532-6 Zinc-dependence on the tran slational response for Izh2p.................................................. 543-1 Zap1p-dependent regulation of IZH2-lacZ. The IZH2-lacZ reporter responds to both zinc and exogenous myristate (C14:0)............................................................................... 643-2 Transcriptional regulation of IZHs by exogenous fatty acids. 653-3 Oaf1p/Pip2p dependence of the IZH2-lacZ activity in presence and in absence of myristate (C14:0) and oleate (C18:1), (panels A and B, respectively).............................. 663-4 Post-translational response of the Izhp family upon addition several fatty acids. and D, the expression of Izh2p is highly induced upon addi tion of myristate (C14:0)............ 67

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11 3-5 Dual regulation of IZH2 and IZH4 by exogenous myristate (C 14:0) and cobalt............... 683-6 Regulation of IZH2 and IZH4 by exogenous oleate (C 18:1) and cobalt........................... 694-1 Chemical structures of the sterols analyzed in this chapter............................................... 884-2 Model proposed for the nystatin ac tion in the yeast plasma membrane............................ 894-3 Nystatin-dependent phenotypes......................................................................................... 904-4 Effect of nystatin on izh3 and the wild type strain BY4742 at different stages of growth................................................................................................................................914-5 Ultraviolet spect rophotometric characterization of total 5-7 sterols in wild type strain and izh3..................................................................................................................924-6 Semi-quantitation of to tal ergosterol and 24( 28)-DHE content in wild type and izh3 by UV-spectrophotometry................................................................................................. 924-7 Quantitation of basal levels of total ergosterol and lanosterol of WT and izh3by GC-MS...............................................................................................................................934-8 Total and free ergoste rol content on WT and izh3..........................................................944-9 Sphingoid bases override the toxic effects of nystatin. 954-10 Effect of ceramides and stearylamine in cells exposed to nystatin.................................... 965-1 Chemical structures of the sphingoid base s, ceramides, and sterylamine a structural sphingoid base homolog..................................................................................................1175-2 Synthesis and hydrolysis of the yeast ceramides............................................................. 1185-3 Overview of de novo biosynthetic pathway in yeast.......................................................1195-4 Overexpression of IZH2 and IZH3 produces an increase in the basal levels of the sphingoid bases C18-PHS and C18-DHS...........................................................................1205-5 Overexpression of YPC1 and IZHs induces the increase in the levels of C18-PHS, C20-PHS, and C18-DHS....................................................................................................1215-6 The fluorescent ceramide substrates and the fatty acid product used during the in vitro ceramidase assays.................................................................................................... 1225-7 TLC analysis of in vitro ceramidase activity at different pHs......................................... 1235-8 TLC-autoradiograph of radiolabeled lipid s shows increased levels of PHS and DHS when IZHs and YPC1 are overexpressed......................................................................... 124

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12 5-9 Effect of fumonisin B1 on the production of sphingoid bases......................................... 1255-10 Effect of myriocin on the growth and the sphingolipid biosynthesis of IZH2 and IZH3. ................................................................................................................................1265-11 Overexpression of IZH2 and IZH3 produces an increase in the levels of C26-PHC and C26-DHC..........................................................................................................................1276-1 Lipid composition of the yeast lipid rafts........................................................................ 1426-2 Localization of Izh2p and Izh3p in plasma membrane.................................................... 1436-3 Izh2p and Izh3p are dissociated from lipid rafts prepared from plasma membranes...... 1446-4 Localization of Izh2p and Izh3p in lipid rafts prepared from total membranes.............. 145C-1 Gas chromatography-electr on ionization in tandem with mass spectrometric analysis (GC-EI-MS).....................................................................................................................152C-2 The NIST EI mass spectra for the TMS derivatives of choleste rol (top), ergosterol (middle) and lanosterol (bottom)..................................................................................... 153C-3 Total ion chromatograms (TICs) and el ectron ionization chromatograms (EICs) of sylilated sterols. A represen ts wild type and B, is izh3.................................................154D-1 High perfomace liquid chromatography-atmospheric pressure chemical ionization in tandem, with mass spectrometric analys is (HPLC-APCI-MS) of standards................... 155D-2 Atmospheric pressure chemical ioniza tion chromatogram (APCI)-mass spectra for the sterol standards........................................................................................................... 156D-3 Base peak chromatogram (BPC) and el ectron ionization chromatograms (EICs) for wild type...........................................................................................................................157D-4 Base peak chromatogram (BPC) and el ectron ionization chromatograms (EICs) for izh3...............................................................................................................................158

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF NOVEL YEAST RE CEPTORS IMPLICATED IN METAL AND LIPID METABOLIC PATHWAYS By Nancy Yaneth Villa December 2007 Chair: Thomas Lyons Major: Chemistry In the yeast Saccharomyces cerevisiae, a family of genes was identified by DNA microarrays. These genes were called IZH1-4 ( I mplicated in Z inc H omeostasis) because their expression was dependent on the zinc concentration in the cell. The IZH genes encode membrane proteins with seven transmembrane spanning domains and three highly conserved motifs. Muliple sequence alignments revealed that the IZH gene products (Izhs) belong to a large and ubiquitous family of proteins that includes the progestin and adiponetin receptors (PAQRs). Eleven members of this protein family have been identified in vertebrates, and homologues can be found in species ranging from bacteria to hum ans. The human PAQRs have started to capture the attention of researchers due to their implicat ion in the development of diseases like obesity and type 2 diabetes. Sequence alignment has also indicated that Izh proteins share distant similarity with the yeast alka line ceramidases Ypc1p and Ydc 1p, two enzymes involved in the sphingolipid metabolic pathway. A first approach to the characterizatio n and elucidation of the role(s) for IZHs is presented. First of all, IZH2 is shown to be induced under zinc de ficiency via the zinc sensor Zap1p. Furthermore, the IZH2 gene and its homolog IZH4 respond to excess of zinc, cobalt, and nickel, as well as to iron deficiency via the hypoxic transcription factor Mga2p. Interestingly, when

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14 expressed in media replete in iron and zinc, th e Izh2 protein (Izh2p) seems to be ubiquitinated. However, in a medium deficient in iron or zinc Izh2p does not appear to be ubiquitinated. On the other hand, different lines of ev idence are presented that indicate that IZHs are also implicated in lipid metabolism. In this regard, we initially show that IZH1 and IZH4 are slightly inducted by the fatty acid palmitate, whereas IZH3 is induced by oleate. Interestingly, IZH2 is highly induced by myristateat the transcri ptional and post-translational levels. Other lines of evidence suggesti ng a potential involment of the IZHs in lipid metabolism come from the observation that mutation of the IZH3 gene produces a nystatin resistant phenotype. Nystatin is an antifungal that interacts with ergosterol in the yeast plasma membrane. Sterol analysis indicates that izh3 mutant presents alterations in the sterol composition. Specifically, we demonstrate that izh3 has lower levels of free er gosterol than a wild type strain, thus suggesting a role for IZH3 in the sterol metabolic pathway. Despite the distant similarity with alkaline cer amidases, we could not demonstrate that Izhs function as ceramidases per se; however when overexpressed, the Izhs induced the synthesis of free sphingoid bases. Furtheremore, we show pr eliminary data indicati ng that the overexpression of Izhs can result in the de novo sphingolipid biosynthesis. Finally, strong evidence is pres ented indicating that Izh2p an d Izh3p are plasma membrane proteins that are associated with microdomain s formed by sterols and sphingolipids called lipid rafts. This result not only supports our hypothesis that Izhs are membrane receptors, but also it opens new and exciting avenues regarding the role of these proteins in lipid metabolism.

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15 CHAPTER 1 INTRODUCTION Zinc Homeostasis Zinc is an essential m etal for growth and metabolism of yeast and higher eukaryotes (Wu et al. 2006). Zinc is also an impor tant catalytic cofactor for more than 300 enzymes and a structural component of many proteins (Taylor et al., 2003; Wu et al., 2006). Because the importance of zinc for cells, a vari ety of disorders have been attri buted to alterations in the zinc content. For instance, zinc deficiency is associated with impaired cell division and differentiation, retarded growt h, dysfunction of the immune system, anemia, and defects in appetite among others (MacDiarmid et al., 2000; Wu et al., 2006). Zinc deficiency is also associated with increased levels of lipid and protein oxidation as well as oxidative DNA damage (Keen et al., 1989; Oteiza et al., 1995). In this regard, zinc de ficiency represents a high risk factor for cancer, and other human diseases (Ho, 2004). Notwithstanding the deleterious effects of zinc deficiency to cells, ex cess of zinc is also toxic (Koh et al., 1996). Therefore, organisms have evolved with intricate mechanisms th at regulate the appropriate acquisition, compartmentalization, and storage of zi nc by the cell (zinc homeostasis). Most of the progress in the understanding of zinc homeostasis at the molecular level, comes from pioneering studies performed by Da vid Eide, and coworkers in the budding yeast Saccharomyces cerevisiae. Their findings have started to uncover the mysteries of subcellular distribution of zinc and have re vealed that zinc homeostasis is remarkably more complex than was originally described. A central component of metal ion homeostasis systems is the regulation of the ion flow across lipid membranes. In this regard, lipids se rve as barriers to the diffusion of charged and hydrophobic particles. Compounds and small peptides that bind tightly to zinc may partially

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16 shield the charge and act to ferry the ion across membranes. In eukaryotes metal ion transport is mediated by different families. One of them is named the ZIP family (so called for being Zrt-, Irtlike proteins) because their members play pr ominent roles in transporting zinc and iron from outside the cell to the cytoplasm (Gaither and Eide, 2001). ZIP transporters have also been found to mobilize stored zinc by transporting the metal from within an intracel lular compartment into the cytoplasm. A second group of transporters, the CDF family (C ation D iffusion F acilitator), transports zinc in the direction opposite to that of the ZIP proteins, pr omoting zinc efflux or compartmentalization, by pumping zinc from the cy toplasm out of the cells, or into the lumen of an organelle. In yeast, several members of the ZIP family have been identified. For instance Zrt1p and Zrt2p are encoded by ZRT1 (Zhao and Eide, 1996a), and ZRT2 genes (Zhao and Eide, 1996b) (Z inc R egulated T ransporter 1 and 2 respectively) and constitute the high, and low affinity zinc uptake systems, respectively. Other characterized members of this family Zip1Zip4, and Irt1 of the plant Arabidopsis thaliana, transport zinc (and in the case of Irt1p, iron and manganese) across the membranes of plant cells (Eide et al., 1996). In S. cerevisiae, zinc uptake is controlled at transcript ional level in respons e to intracellular zinc levels. For exampl e, transcription of ZRT1 and ZRT2 is highly induced in zinc-limited cells. Regulation of these genes in response to zinc is mediated by the transcri ption factor Zap1p (Zhao and Eide, 1997). Zap1p (Zinc activator protein) not only regulates the transcription of ZRT1 and ZRT2 but also, its own transcription (Gaither and Eide, 2001). How does Zap1p activate transcription? Studie s conducted by Eide and co-workers have indicated that under zinc de ficiency, Zap1p binds to a consensus sequence of 11 bp (ACCTTNAAGGT), called ZRE (Z inc R esponsive E lement), in the promoter region of Zap1p-

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17 target genes (Zhao et al., 1998) (Figure 1-1). The transcriptional regulato ry effect of Zap1p is repressed in zinc-rep lete cells (e.g. 10 M ZnCl2). The Budding Yeast Saccharomyces cerevisiae as a Model System Saccharomyces cerevisiae is a unicellular and non-pa thogenic eukaryote whose genome was completely sequenced in 1996. Because it is a close relative of pathogenic fungi such as Candida albicans and Candida glabrata (Figure 1-3), and the fact that it shares many homologous genes with higher eukaryotes, this microorganism is a powerful tool to study biochemical events that can furt her be extrapolated to more s ophisticated and evolved systems. In this regard, yeast combines a well-des cribed biochemistry and ease of genetic experimentation. These, characteristics are very useful for mapping out complex signal transduction pathways (Chung and Obeid, 1999). Fu rthermore, metabolic pathways that are poorly understood in higher eukaryotes, including mammals, are more easily understood as a result of studies performed in S. cerevisiae Identification of IZHs on the Saccharomyces cerevisiae Genome The f act that single and multiple deletions of the zinc transporter genes ZRT1, ZRT2 and ZRT3 were not lethal for the cells, suggets, at firs t glance, that these gene s are not essential for cell viability, and suggests the presence of other genes with overlapping functions. Because the above mentioned genes are Zap1p target genes, the ZAP1 regulon was screened by DNA microarrays in order to identi fy new Zap1p-target genes (Lyons et al., 2000). The results of this study revealed 46 new genes as potent ial Zap1p targets. Two of these genes, YDR492w and YOL002c contain a putative ZRE consensus sequen ce in their promoters and were highly expressed under zinc-limitation via Zap1p (Lyons et al., 2000; Lyons et al., 2004). YOL002c and a third homologous gene YOL101c, were induced by high levels of zinc, (Lyons et al., 2004). A fourth homolog YLR023c discoved by sequence alignments, revealed phenotypic

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18 effects on zinc tolerance (Lyons et al., 2004). Due to their transcrip tional metalloregulation, zinc related phenotypes, and highly conserved motifs c ontaining potential metalbinding residues, this novel family of genes was renamed like the IZH family (I mplicated in Z inc H omeostasis), and its members were named as follows. IZH1 ( YDR492w ), IZH2 ( YOL002c ), IZH3 ( YLR023c ), and IZH4 ( YOL101c ) (Lyons et al., 2004). Regulatory Sequences Surrounding the IZH Genes In the prom oter regions of some of the IZHs a group of regulatory sequences have been identified by using RSA-TOOLS ( http://rsat.ulb.ac.be/rsat) (Hertz et al., 1999). First of all, the prom oter regions of IZH1 and IZH2 have a zinc responsive element sequence (ZRE) (Figure 1-4 A and B, respectively). On the other hand, IZH2 and IZH4 have in their promoter regions a putative low oxygen response element (LORE), (Figure 1-4 B and C, respectively). Under hypoxia (low oxygen) or conditions that mimic hypoxia such as excess of cobalt and nickel, the transcription factor Mga2p regulates the transc ription of target genes via the LO RE. It has been proposed that the mentioned transition metals may disrupt the prod uction of reactive oxygen species which may be important signaling molecules in the oxygen response pathway (Huang et al., 1996). Lyons et al 2004, demonstrated that not only cobalt and nick el, but also excess of zinc can mimic hypoxia and induce the transcription of IZH2 and IZH4 Therefore, these two genes have been proposed to be part of the hypoxic response (Lyons et al., 2004). Another speculative regulatory element known as Oleate Response Element (ORE) has been detected in the promoter region of IZH1 IZH2 and IZH4 (Lyons et al., 2004) (Figure 1-4 A, B, and C, repectively). It has been found that in the presence of fatty acids like oleate, the transcription factors Oaf1p and Pip2p form a he terodimer that binds to the ORE consensus sequence in the promoter of target genes (Einerhand et al., 1993).

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19 The binding of Oaf1p and Pip2p to ORE is the proposed mechanism by which both transcription factors regulate the fattyacid dependent gene activation (Karpichev et al., 1997; Kapichev and Small, 1998). Interes tingly, despite the pres ence of ORE in the promoter region of IZH2 this gene is transcriptiona lly regulated by exogenous myristate (a satura ted fatty acid of a carbon chain of 14 carbons, C14:0) (Karpichev et al., 2002; Lyons et al., 2004). In this regard, the results shown in this disse rtation contribute to expand our knowledge with the finding that IZH1 IZH3 and IZH4 are also regulated by fatty acids at tr anscriptional level. The presence of different regulatory elements in the promoter regions of the IZH suggests, at first glance, a dual role for these genes in metal ion homeo stasis and lipid metabolic pathways. The IZH Gene Family Encodes Membrane Proteins The IZH gene fa mily encodes membrane prot eins with predicted seven transmembrane spanning domains (7TMs), and highly conserved motifs (Figures 1-5 and 1-6), which are summarized as follows: (i) a long motif N-te rminal to TM1 that generally resembles PxnGYRxnNEX2Nx2T/SH; (ii) an Sx2Hx2S motif at the C-terminus of TM2; (iii) a DX9GS motif at the beginning of TM3; (iv) a Px2H motif in TM5 where the H residue is generally only conserved in higher eukaryotes; and (v) the loop between TM6 and TM7 containing PER/KxnPG and Hx2F/WH motifs with a cons erved histidine in the middle of TM7 being most common. Furthermore, IZHs encode proteins that belong to the ubiquitous and recently discovered family of membrane protein receptors and named as PAQRs (Progestin and Adipo Q Receptors). Multiple sequence alignments (Figure 1-6) (Lyons et al., 2004), as well as phylogentic analysis (Figure 1-7) performed for the four yeast prot eins, revealed a high similarity between these proteins and the PAQRs. Like th e Izhs, PAQR genes encode prot eins with seven transmembrane domain proteins.The topology model presented in Figure 1-5 has been confirmed for the yeast Izh2p and Izh4p (Kim et al., 2003), as well as the hu man PAQR1 and PAQR2 (Zhu et al., 2003).

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20 .Because Izhs are membrane proteins with seven transmembrane domains, it has been we have proposed that the Izhs are membrane r eceptors. Interetingly, phylogenetic analysis has revealed that some members of the Izhp family such as Izh1p, Izh2p, and Izh3p have homologous proteins in other f ungi, some of which are very pa thogenic (Figure 1-3). Since S. cerevisiae is a non-pathogenic fungus investigating the role of thes e proteins can shed light into the mode of action of pathogenic fungi. The PAQR Family of Proteins Even though PAQRs are widely dispersed throughout eubacteria and ubiquitous in eukaryotes, very little is known a bout PAQR genes from any of thes e organisms. In fact, the first published characterization of a member of the PAQR protei n family was of a protein cloned from the opportunistic pathogen Bacillus cereus This protein, when expressed in E. coli conferred hemolytic activity onto the host strain, and therefore, it was named hemolysin III, (Hly3) (Baida and Kuzmin, 1995). A subsequent study suggested that Hly3 was capable of inducing large pores in erythroc yte membranes on the order of 3035 in diameter (Baida and Kuzmi, 1996). Rehli et al., 1995 reported that the gene PAQR11 was highly induced during differentiation of human macrophages from monocytes. The gene product, PAQR11, was postulated to function as a receptor based on the f act that, like G-protein c oupled receptors, it has seven predicted transmembrane domains. However, no sequence similarity with known Gprotein coupled receptors was found (Rehli et al., 1995). The next relevant published finding was the discovery of a PAQR-like gene in the -proteobacterium Azospirillum brasilense In this case, the PAQR encodes a chimeric protein that consists of an N-te rminal Hly3-like domain fused to a C-terminal domain that is identical to the CheA hisidine kinase involved in chemotaxis (Hauwaerts et al., 2002).

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21 In humans, the PAQR family of genes en codes membrane protein receptors, which mediate hormonal signaling (Hsieh et al., 2005). For example, PAQR1 and PAQR2 (also called AdipoR1, and AdipoR2, respectively) function as receptors for the insulin-sensitizing hormone adiponectin (AdipoQ), (Yamuchi et al., 2003a). Adiponectin is an adipocyte-derived proteinaceous hormone related to tumor necrosis f actor (Shapiro and Scherer, 1998) that acts as an anti-diabetic and ant-atherogenic adipokine by enhancing insulin sensitivity (Yamauchi et al., 2003b). In fact low circulating levels of AdipoQ ar e associated with type 2 diabetes, obesity and coronary artery disease (Shimada et al., 2004). In a separate study the human PAQR5 (mPR PAQR7 (mPR and PAQR8 (mPR proteins were identified as membrane receptors for the hormone progesterone (Zhu et al., 2003a). These receptors transmit rapid and non-genomic steroid signals (Zhu et al., 2003b). Interstingly, adiponectin and steroid receptors are part of the same family, which remains to be mystery si nce they recognize hormones that are highly different structurally. Izh2 is an Osmotin Receptor Protein Osm otin is a secreted polypeptide found in the tobacco leaves, which is closely related to the natural sweetener thaumatin (Veronese et al., 2003). Both proteins belong to the PR-5 family of plant defensins. Unlike thaumatin, osmotin is a potent antifungal that induces apoptosis in Saccharomyces cerevisiae by signaling suppression of cellular stress responses via RAS2/cAMP (3,5-cyclic Adenyl Mono Phosphate) (Narasimhan et al., 2001). The antifunga l activity of osmotin is dependent on the funga l cell wall composition (Veronesse et al., 2003). For example, while glycoproteins repress the osmotin acti on, phosphomannans enhance its toxicity (Yun et al., 1997). In addition to this, the interation betw een osmotin and specific plasma membranes receptors, is required for the osmo tin antifungal activity (Narasimhan et al., 2005). Recently, it

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22 was reported that the overexpressi on of the yeast Izh2 protein medi ated the apoptotic effects of osmotin via a RAS2 signaling pathway (Narasimhan et al., 2005). A possible mechanism by which Izh2p mediates cell death i nvolves the interaction of osmo tin with Izh2p. This interaction activates RAS2/cAMP pathway with the concom itant suppression of stress responses and the subsequent accumulation of reactive oxyge n species and cell death (Narasimhan et al., 2005). Therefore, Izh2p has been recognized as an osmotin receptor (OsmoR). The IZHs and their Connection w ith Lipid Metabolic Pathways The first insight about the role of the IZHs in a pathway different from metal homeostasis was postulated by Karpichev et al., 2002. In that report, the YOL002c ( IZH2 ) gene was shown to be highly induced in cells grown in the presence of myristate (C14: 0) as the sole carbon source. In contrast, mutations of this gene produced de fects in the growth of cells were exposed to myristate. This observation suggested a regulatory role for IZH2 in lipid metabolism. During those studies, it was also found that the mutation of IZH2 ( izh2) produced a resistant phenotype against the antibiotic nystatin. Because nystatin pr eferentially binds to ergosterol in the yeast plasma membrane (Lees et al., 1995), it was proposed that the izh2 mutant has alterations in sterol composition. Furthermore, Cherry et al., 1998 reported that the IZH3 transcription is induced and the IZH4 transcription is repressed by defects in the ergos terol biosynthetic pathway. These observations, combined with the fact that some Izhp vertebrate orthologs (e.g. membrane progestin receptors) function as a recept ors for structurally related steroids, identify ergosterol metabolism as a likely biochemical pathway in which to place the IZH genes and consequently the Izh proteins. Besides sterols, the yeast membranes cont ain sphingolipids. In the yeast plasma membrane, sterols like ergosterol and sphingolipid s are the main components of the detergent-

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23 resitant microdomains termed lip id rafts (Pike, 2003; Schnabl et al., 2004). Rafts are conceived as platforms that mediate the sorting of proteins, and are implicated in membrane trafficking pathways like endocytosis and exocytosis (Lawrens and Andr, 2006). Interestingly, the sphingolipid pathway is also directly connected with other meta bolic pathways, including fatty acid synthesis, elongation a nd sterol metabolism (Sims et al., 2004). Furthermore, as mentioned before, multiple sequence alignments and phylogenetic analysis have also revealed that Izhs (or yeast PAQRs) have distant similarity to a family of yeast membrane proteins known as alkaline ceramidases (Figures 1-6 and 1-7). Ceramidases are enzymes that catalyze the deacy lation of ceramides to generate sphingoid bases and fatty acids (Figure 1-8). Ceramide and its metabolites act as signaling molecules for apoptosis and proliferation. Theref ore, the ceramide/sphingoid base ratio is considered to be a rheostat that governs these even ts. Three types of ceramidases have been identified and classified as acidic, neutral, and alkaline according to their optimum pH (Mao et al., 2000a; Mao et al., 2000b). Acidic ceramidase is localized in lysosomes and is primarily responsible for catabolism of ceramide. On the other hand, neutral and alkaline ceramidases have been implicated in signal transducti on and cell regulation (Merrill et al., 1997; Dickson, 1998). Thus far, two alkaline ceramidases, Ypc1p and Ydc1p, have been cloned and characterized in S. cerevisiae (Mao et al., 2000a; Mao et al., 2000b). Although Yp c1p catalyzes the deacylation of phytoceramide to phytosphingosin e, Ydc1p catalyzes the deacylation of dyhydroceramide to dyhydrosphingosine. In this disse rtation, we have esta blished a connection between IZHs and the fatty acid, sterol and sphingolipid metabolic pathways. Structurally, sphingolipids are defined and di stinguished by the presence of a sphingoid base backbone. In mammalian cells, this is usually C18-sphingosine, whereas in yeast cells, this

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24 sphingoid backbone is C18-phytosphingosine (Figure1-8). Othe r variations, in yeast include C20phytosphingosine, C18-dihydrosphingosine, C20-dihydrosphingosine, and a variety of other hydroxylated analogs of C18-phytosphingosine. Collectively, sphingosine, dihydrosphingosine, phytosphingosine, and related long-chain amino ba ses are also termed as sphingoid bases. The next building block in the sphi ngolipid structure is ceramide, (or phytoceramide in yeast). Ceramide is derived from sphingosine by the acylat ion at the 2-amino position by a fatty acid of varied carbon chain length; with C26 being the most abundant fatty acid found in yeast ceramides. Ceramide is the backbone of more complex sphingolipids. S. cerevisiae has three complex sphingolipids, inositol phosphocer amide (IPC), manosylinositol phosphoceramide (MIPC) and manosyl di-inositolphosphoceramide (M(IP)2C) (Figure1-8). Ceramide is at the center of the sphingolipid pathway regulating the synthesis of various sp hingolipids. In addition to this, ceramide is considered the point where animal and fungal sphingolipid biosynthesis begins to diverge. Summation This research entails the study of the IZH fa mily of genes in the yeast Saccharomyces cerevisiae. Investigating the role(s) of this family of genes has been one of our main aims. Based on our observations, we propose that IZHs have a dual role in metal and lipid metabolic pathways. Different lines of evidence that support these hypotheses are addressed and discussed herein. In Chapter 2, we show th e transcriptional and post-transla tional responses of some of the IZHs by several metals such as zinc, iron, cobalt and nickel. In Chapter 3 we demonstrate that the expression of some of the IZH genes and proteins are also regulated by exogenous fatty acids at transcriptional and translational levels, respec tively. Genetic and bioche mical studies suggest that IZHs can also be implicated in the sterol metabol ic pathway (Chapter 4) as well as in the sphingoilid pathway (Chapter 5). In Chapter 6, the localization of the proteins Izh2p and Izh3p in

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25 plasma membrane and in the detergent-resistan t microdomains termed lipid rafts supports our hypothesis that some members of the Izh family ar e implicated in metabolic pathways such as sterols and sphingolipids. This investigation is just the starting point to understand more complicated metabolic pathways in higher eukaryotes. In this dissertation, and according to the yeast nomenclature, a gene is reprented by IZH A protein encoded by the IZH gene is called Izhp, or Izh pr otein. A single mutation of the IZH gene is referred to as izh or simply izh, and a wild type strain is in some cases represented like WT.

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26 Figure 1-1. Zap1p activation is zi nc dependent. Under zinc de pletion, Zap1p binds to a zinc response element (ZRE) consensus sequence in the promoter region of target genes inducing their expression. Figure 1-2. A co mmon budding yeast Saccharomyces cerevisiae cell, and some of the most important and well characterized zinc transporters.

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27 Figure 1-3. Phylogentic an alysis of the Izhp family and its closer and more distant homologues in yeast.

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28 5 ZRE ORE IZH1 3 5 3 5 ZRE ORE IZH2 3 5 3 LORE 5 ORE IZH4 3 5 3 LORE A B C Figure 1-4. Regulator y elements surrounding IZH1, IZH2 and IZH4 genes suggest dual role in metal and lipid metabolism. In the figure, ZRE stands for zinc responsive element, LORE is the low oxygen response element, and ORE is oleate response element. Figure 1-5. Predicted topology for the Izhp famil y. Izh are membrane proteins with seven transmembrane spanning domains and f our highly conserved motifs facing the cytoplasm (black circles in the figure). The C-terminus is proposed to be outside whereas the N-terminus is inside the cell, specifically in the cytoplasm.

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29 Figure 1-6. Sequence alignment of important co nserved regions in PAQRs, hemolysins and ceramidases. The alignment was performed using CLUXTALX. Predicted transmembrane segments (TMs) are indica ted by solid bars. Highly conserved regions are highlighted in grey. Figure 1-7. Phylogentic showing the Izhp family, its closer and more distant homologues in yeast, and other organisms.

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30 Figure 1-8. The Sphingolipid bios ynthetic pathway in the yeast Saccharomyces cerevisiae

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31 CHAPTER 2 METALLOREGULATION OF IZHS Introduction Elucidating the role of genes of unknown function is som etimes a difficult task; especially if very few precedents exist that mark the route to follow. This chapter constitutes a first approach to determine the role of a group of genes of unknown function termed IZHs The catalyst for this chapter were pioneer ing studies conducted by David Eide and coworkers, in the yeast S. cerevisiae, which revealed the existence of a complex system, orchestrated by different gene produ cts that regulate the homeostasis of zinc and iron in the cell. Part of this system includes the IZH family of proteins (Lyons et al., 2000). Two members of the IZH family, IZH1 and IZH2 were found to be highly induced under zinc deficiency via the transcription factor Zap1p. Interestingly, the expression of IZH2 and its homologue IZH4 was induced by excess of zinc in a Zap1p-independent manner. Overall, the results presented contribute to our understandings about metalloreuglation in the yeast S. cerevisiae. The fact that IZH2 is positively affected by two opposite effects (zinc deprivation, and excess of zinc), opened the possi bility to explore new avenues in which the IZHs could function. In fact, excess zinc, cobalt a nd nickel, as well as iron chelation, can modulate the expression of genes via the hypoxia sensor Mga2p (Lyons et al., 2004; Vasconcelles et al., 2001). As mentioned in Chapter 1, under hypoxia (low oxygen), or in the presence of toxic metals, the transcription factor Mga2p is believed to bind a low oxygen responsive element (LORE) in the promoter regi ons of Mga2p-target genes. The presence of putative LOREs in the promoter regions of IZH2 and IZH4 led us to speculat e that these two genes can be Mga2p-target genes. With this in mind, evidence supporting the regulatory effect exerted by certain transition metals on the transcriptional response of IZH2 and IZH4 is

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32 presented. Additionally, we show that the response of IZH2 to metals like zinc and iron also occurs at post-translational level, and that su ch a response is dependent on the cellular metal status. We confirm that the tran scriptional response of IZH2 under zinc deficiency is Zap1dependent, and that IZH2 is a Zap1p-target gene (Lyons et al., 2004). Also, we show that IZH2 and IZH4 are Mga1p-target genes (Lyons et al., 2004). Besides the effect exerted by certain meta ls on the transcriptional activation of IZH2 we show that zinc and iron regulate the accumulati on of the Izh2 protein. Indeed, under zinc or iron deficiency, the expression of Izh2p was strongly observed. By contrast, growth in a medium replete in either iron or zinc decreases the expression of th e Izh2 protein. Furthermore, we present evidence suggesting that Izh2p seems to be ubiquitinated in medium that is iron or zincreplete. Taken together, the results presented in this chapter constitute a pivotal piece of evidence implicating two members of the IZH family in metalloregulation. Furthermore, our results match with the idea that in some cases, transcriptional and translational responses are directly related. Materials and Methods Yeast Strains and Plasmids The yeast strains used in this study are liste d in T able 2-1. Those strains were obtained from two different sour ces, (i) EUROSCARF ( http://web.uni-frankfurt.de/fb15/m ikro/euroscarf) and (ii) from Dr. David Eide yeast collection ( eided@missouri.edu).The IZ H promoters were fused to a lacZ reporter gene as follows. PCR-amplifie d genomic fragments from approximately 1,000 bp upstream to ATG were inserted by homologous recombination, into the episomal YEp353 vector, and between the EcoRI and BamHI sites (Lyons et al., 2000). Resultant IZHlacZ promoter reporter fusions were used to perform -galactosidase assays.

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33 For protein expression, the C-terminus of the Izh2p was tagged with the triple hemagglutinin epitope (3xHA). The HA-epitope tag is a peptide from human influenza hemagglutinin protein, which has th e amino acid sequence, YPYDVPDYA (Mo et al., 2001). The tagged construct was genera ted by Dr. Thomas Lyons, according to published procedures (MacDiarmid et al., 2002). Briefly, the ZRC1 promoter and open reading frame in the YCp ZRC1 -3xHA plasmid were exchanged with those of IZH2 This was accomplished by gap repair of Age 1-digested YCp ZRC1 -HA to generate the p IZH2 -3xHA. This construct has the IZH2 gene driven by its native promoter and retains the ZRC1 terminator sequence. To provide a galactose inducible c onstruct, the native IZH2 promoter was exchanged with the GAL1 promoter using gap repair of p IZH2 -3xHA plasmids previously cut with Eco R1. The resultant p IZH2 3xHA plasmid contained the GAL1 promoter to drive protein overproduction. In both cases, the tagged or untagged protein is overexpressed using the galactose i nducible promoter ( GAL1 ). Yeast Media In this chapter, -galactosidase assays and protein expression were perform ed using different types of media, which are brie fly described as follows. While all the -gaslactosidase assays were performed in a medium that uses glucose as a carbon source, protein expression was carried out in either a growth medium supplemeted with glucose or galactose. Synthetic medium supplemented with either glucose (also called dext rose), or galactose, as a carbon source (SD or SGal, respectively) was used as a medium that c ontains all metals. To limit either zinc, or iron availability, or both, chelexed synthetic medium supplemted with either dextrose (CSD) or galactose (CSGal) was used according to Lyons et al., 2004. Finally, to fully limit availability of iron, low iron medium (LIM) was used according to Kupchak et al., 2007. LIM was supplementd with an aprropriated carbon source.

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34 Each growth medium was prepared as follows (Apendix A for more details). One liter (1 L) of synthetic medium was prepared by disso lving 1.7 g Yeast Nitrogen Base (YNB) without amino acids and ammonium sulfate (Fisher). This medium was then supplemented with 5 g of ammonium sulfate (Fisher), 2% alpha (+) gluc ose (99%, anhydrous), or 2% D (+) galactose (Across-Organics), and 0.01% of a ppropriate amino acids (Sigma). For -galactosidase assays under excess of metals like zinc, cobalt, a nd nickel; synthetic medium (SD medium) was supplemented with ZnCl2 to a final concentration of 3 mM (excess), whereas CoCl2 and NiCl2 were added to a final concentration of 400 M. One liter of CSD medium was prepared by di ssolving, 20 g of dextrose, 5.1 g of YNB without divalent cations, amino acids, ammonium sulfate, and phosphates (Qbiogene), and 0.1 g of appropriate amino acids, into sterile nano-pure water. Chelex-100 ion exchange resin (25 g) from Sigma was added, and the cult ure was stirred overnight at 4oC. After removal of the resin, the pH was adjusted to 4.0 with HCl, and th e following were added to recommended final concentration: MnSO4, CuSO4, CaCl2, MgSO4, and KPO4 monobasic (Appendix A, for details), nano-pure water was added to 1 L. This medium is devoid of iron or zinc To generate medium that is replete in these metals either zinc or iron was added back to CSD to a final concentration of 10 M (repletion). The solution was then filter-s terilized into polycarbonate flasks. Before being used, all plastic used for CSD media pr eparation and cell culturing was washed with Acationox detergent (Baxter Scientif ic Products, McGaw Park, IL). One liter of LIM was prepared by dissolvi ng, 1.7 g of YNB without amino acids and ammonium sulfate (Fisher), 20 g of glucose (o r galactose) (Acros Organics), 20 mL 1.0 M sodium citrate (pH 4.2), and 5 g of ammonium sulf ate. This medium was then supplemented with 0.1 g of appropriate amino acids, as well as 1.0 mM EDTA, at pH 8.0. MnCl2 was added back to

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35 LIM to a final concentration of 20 M, and ZnSO4 was added to a final concentration of 0.8 g/mL (or 5 M). Iron deficiency and iron repleti on were generated by adding either, 1 M or 1 mM FeCl3, respectively. The solution was then filter sterilized into polycarbonate flasks. Yeast Transformations and Assays Yeast transform ations were perf ormed to introduce plasmids in to appropriate strains. To do this, single colonies of each strain were grown for 1 overnight (e.g. 12-18 h) in 1X YPD to saturation. Aliquots of the overnights were i noculated in fresh 1X-YPD and grown to OD600 of 1.0 to subsequently being used for yeast transformations. Yeast transformations were performed by using standard procedures and using th e lithium acetate method (Gietz and Woods, 1994) (Appendix B). Promoter reporter activities were measured by using -galactosidase assays, which are spectrophotometric assays. In this type of assays the enzyme -galactosidase, which is encoded by the Escherichia coli lacZ gene hydrolyzes the synthe tic chromogenic substrate o-nitrophenyl-D-galactopyranoside (ONPG), generating o-nitrophenol, which is yellow in aqueous solution. The course of this reaction is followed by m onitoring absorbance at 420 nm (Lederberg, 1950). For yeast, the -galactosidase assay is performed by permeabilizing the cells with 0.1% sodium dodecylsulfate (SDS) and chloroform (1:1, v/ v), and then by suspending the cells in galactosidase assay buffer, pH 7.0. After incubati on with ONPG for a period of time, the reaction is terminated with a solution of 1 M Na2CO3 and the OD420 is measured. Before each assay, cells were inocul ated from overnight cultures (OD600 = 3-4) into the appropriate media to an initial OD600 of 0.1. Cells were grown at 30oC to mid-log phase (OD600 of 0.5). -galactosidase activity was assayed as described by Guarente et al., 1983, and is expressed in Miller un its. The activity was calculated as follows: (A420 x 1000) / min x mL of

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36 culture used x culture A600). Results for experiments are re ported are the product of three independent samples; each experi ment was done in triplicate. Preparation of Microsomes Microsom es were prepared according to the procedures described by Gable et al., 2000; Mo et al., 2002 with some minor modifi cations as follows. Cells in early exponential phase of growth (OD600 of 0.8) were centrifuged for 5 min, at 3,000 rpm and 4oC. Pellets were washed twice with sterile cold water, and re-suspende d at 2 mL/g wet cell weight in mitochondrial isolation buffer (MIB), which is composed of 0.6 M mannitol, 20.0 mM HEPES-KOH, pH 7.4, 1.0 mM EDTA, pH 7.5, and the protease inhibi tors, 1.0 mM phenylmethylsulfonyl fluoride (PMSF), and 2 g/mL of pepstatin A (Sigma) was prep ared according to published procedures (Gitan and Eide, 2000). Glass beads (acid-washed glass beads, from Sigma) were added to just below the meniscus (approximately 250 L), and cells were lysated by six cycles (1 min each time) of vortexing with cooling on ice (1 min) between each cycle. Unbroken cells, beads, and debris were removed by centrifugation at 3,000 X g, 4oC, for 10 min. The low speed supernatant was then ultra-centrifuged at 130,000 X g (45,000 rp m), for 90 min using a Beckman 75 Ti rotor, to provide the microsomal pellet. Microsomal pe llet, which is enriched in membrane proteins, was then resuspended in 200 L MIB buffer supplemented with 15% glycerol. Western Blot Analysis of Protein Expression Protein concentration was determ ined by the BCATM protein assay (Pierce) using bovine serum albumin (BSA) as standard. Samples we re suspended to 1 mg/mL in 1X SDS-PAGE loading buffer, prepared by mixing 0.0625 M Tris -HCl, pH 6.8, 2% SDS, 10% glycerol, 1% mercaptoethanol, 0.001% bromophenol blue, and sterile water. Prot ein suspensions were warmed at 37oC for 30 min followed by centrifugation at 12,000 rpm during 50 sec. Equal concentrations

PAGE 37

37 of protein (22 g per lane) were loaded onto a 10% SD S-PAGE gel followed by Western blot analysis. Western blot was performed following standa rd procedures (Sambrook and Russell, 2001) as follows. After separation in an SDS-PAGE gel, proteins were transf erred to a polyvinylidene fluoride membrane (PVDF) (fro m Millipore) at 80 volts, 4oC, and during 1 h using a tank transfer chamber (Bio Rad). Blots were then wash ed three times, 5 min each time with 1X TrisBuffered Saline solution, (TBS), pH 7.4, (TBS is 20 mM Tris-base, 500 mM NaCl, pH 7.4) supplemented with 0.05% Tween 20 (Fisher), followed by blocking in 1X TBS-Tween 20, supplemented with 5% nonfat milk, for 1 overnight, at 4oC and with constant agitation. For detecting Izh2p-3xHA, blots were probe d for 2 h, with the primary antibody rabbit polyclonal IgG anti-HA at a dilution of 1/500 (HA-probe (Y-11): sc-805, from Santa Cruz). After washing three times (5 min each time) with 1X TBS-Tween 20, blots were probed for 1 hr at room temperature with the secondary antibody horseradish peroxidase-conjugate goat antirabbit IgG obtained from Santa Cruz (at a d ilution of 1/10000, v/v). The bound antibodies were detected by the ECL Western blot ting detection system and usi ng the super signal west pico chemiluminescence kit (Pierce), and exposing a CL-XPsureTM film (Pierce). Immunoprecipitation of Izh2p and Western Blot Analysis The m icrosomes were solubilized at 1 mg /mL with 2 mM sucrose monolaurate, > 97% TLC grade, (Fluka) for 20 min at room temp erature. After centrifugation at 33,500 rpm for 30 min in a 75 Ti rotor (Beckman Coulter), the supe rnatant (containing the solubilized microsomes) was collected. Soluble microsomes (100 L) were incubated with 20 L of mouse monoclonal IgG anti-HA with agarose beads-conjugate [HAprobe (F-7): sc7392 AC, Santa Cruz], as the immunoprecipitated antibody, for one overnight, at 4oC and with constant agitation. Suspension

PAGE 38

38 was spun down at 1,000 rpm for 50 sec, and the resu ltant precipitates were washed three times with 500 L of cold 1X Phosphate Buffered Saline (1X PBS), pH 7.4 (Fisher). Proteins were eluted by re-suspending in 60 L 1X SDS-PAGE loading buffer. SDS-PAGE protein separation and subsequent Western blot analysis were perfor med in identical fashion as described in former section. To investigate the ubiquitination of Izh2p, a bl ot was probed with a ra bbit polyclonal antiubiquitin obtained from Abcam, as primary antib ody (1/8000, v/v dilution), for 2 h and at room temperature followed by incubation with a horse radish peroxidase-conjugated goat anti-rabbit (sc-2004 Santa Cruz) at a dilution of 1/10000, for 1 h at room temperature, as secondary antibody. Blots were developed in iden tical fashion as described before. Results IZH2 is a Zap1p Target Gene IZH2 possesses a putative zinc responsive elem ent (ZRE) in its promoter region, which has the sequence TCCTCTAGGGT. In a wild type strain, the IZH2-lacZ construct yielded 2-fold more activity under zinc deficiency than under zinc repletion (8.1 0.9 vs. 3.6 0.3, Figure 2-1 A). In a zap1 strain, however, the induction of IZH2 -lacZ under zinc deficiency was highly repressed, whereas a sl ight induction of the IZH2-lacZ activity was observed under zinc repletion (2.9 0.0 vs. 5.2 0.2) (Figure 2.1 A). Taking to gether, our data demonstrate that the IZH2-lacZ activity is induced by zinc deficiency via Zap1p. Furthermore, under zinc repletion, the IZH2lacZ activity is also dependent on the presence of the ZAP1 gene. From a different stand point, we also found that zinc deficiency causes a significant accumulation of the Izh2 protein (Figure 2-1 B), suggesting that zinc depr ivation has an post-translational effect on Izh2p. Thus, we can conclude that IZH2 is a bona fide Zap1p-target gene.

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39 IZH2 and IZH4 Are Part of the Hypoxic Response To illus trate with examples those genes that can be expressed under hypoxia or conditions that mimic hypoxia like excess of zinc and ir on deficiency, table 2-2 was generated (Lyons et al., 2004). This table lists genes with an average induction of > 2-fold in cells exposed to 3 mM zinc (excess) (n=2), and includes known targets of the Mga2p hypoxia sensor, OLE1 and Ty1 elements (Vasconcelles et al., 2001; Zhang et al., 1997; Lyons et al., 2004). The remaining genes are known to be induced by either low pO2 (hypoxia) (Cherry et al., 1998; Kastaniotis and Zitomer, 2000), or by iron deficiency via the Aft1p iron-responsive tr anscription factor (Rutherford et al., 2003). A screen for regulatory elemen ts in the promoters of high zincregulated genes by using RSA-TOOLS, generate d probability-based consensus matrices that matched with two groups of genes. One group has promoters containing the LORE (low oxygen element). The promoters of the other group have the FeRE (iron responsive element). With these matrices, 750 bp of the promoters of all genes shown in Table 2-2 were scanned, and found that most of the O2-regulated promoters contained putative LO REs and that all of the Aft1p-target promoters contained putative FeREs The IZH4 promoter contains a potential LORE sequence between -189 and -197 bp, but does not contain a FeRE, suggesting th at it is a target of Mga2p in stead of Aft1p. To address this, we tested the effects of mga2 and aft1 mutations for their effects on IZH4-lacZ activity. Figure 2-2 A, shows that although the induction of IZH4-lacZ in response to zinc was still 2-fold in an aft1 mutant, the basal levels of activity of th e reporter construct was increased 5-fold. Figure 2-2 B confirms that the LORE -lacZ hypoxia reporter is also induced by high zinc in an Mga2p-dependent fashion (data genera ted by Brian Kupchak), as well as by aft1 deletion (recall that LORE is the low oxygen responsive element). Figure 2-2 C shows that basal and

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40 zinc-inducible expression of IZH4-lacZ depends on Mga2p. In addition, other stimuli that are known to induce the hypoxic response in yeast, such as high Co2+ and Ni2+ (Vasconcelles et al., 2001), also induce IZH4-lacZ These responses are not seen in an mga2 strain. The IZH2 promoter also contains a putative LORE c onsensus sequence between -137 and -145 bp. IZH2lacZ is weakly induced by Co2+ and Ni2+ in an Mga2p-dependent manner (Figure 2-2 C), (data generated by Lisa Regalla). As observed for LORE -lacZ (control), the increase of the IZH2-lacZ activity under excess of zinc is not maintained in an mga2 mutant, suggesting that Mga2p is responsible for maintaining elevated expression in high zinc (Figure 22 C). Since Mga2p binds to LORE, the promoter reporter constructs, LORE -lacZ and OLE1-lacZ were used as positive controls to see the effect of c obalt on their transcriptional resp onse. Figure 2-2 D confirms that these reporters are also induced by Co2+ via Mga2p. Regulation of IZH4 by Excess of S everal Transition s Metals is only Mga2p-Dependent MGA2 and its paralog SPT23 show considerable sequen ce homology, with 43% of the amino acids being identical and 60% being similar (Jiang et al., 2001). Therefore, function of Mga2p and Spt23p is in many cases redundant (Zhang et al., 1997). In fact, Zhang et al., 1999 reported that one of the Mga2p-target genes, OLE1 was transcriptiona lly activated by both MGA2 and SPT23 under hypoxic conditions. Because IZH4 was strongly regulated by excess of zinc cobalt, and nickel via Mga2p, we investigated if SPT23 had the same effect as MGA2 on the transcriptional re sponse of this gene. By using -galactosidase assays, we show that although the IZH4-lacZ activity slightly decreases in presence of cobalt in a spt23 strain (39.5 3.4 for WT vs. 20.9 0.2 for spt23), no significant change in the promoter reporter activity was observed under excess of zinc and nickel in the mutant strain (Fig ure 2-3). In contrast, the IZH4-lacZ activity was fully repressed in an

PAGE 41

41 mga2 knockout strain under the same conditions. These results indicate that the transcriptional regulation of IZH4 under hypoxic mimicking-conditions is mainly dependent on Mga2p but not on Spt23p. Iron Deficiency Affects the Expression of IZH2 and IZH4 Besides th e excess of certain tr ansition metals, iron chelation ha s a similar regulatory effect on Mga2p-target genes (Vasconcelles et al., 2001). In order to examine the effect of iron deficiency on the expression of IZH2 and IZH4 -galactosidase assays were performed. To do so, a chelexed synthetic medium (CSD), where th e iron concentration was limited to 50 nM (iron deficiency) or 10 M (iron repletion) was used. Figur e 2-4 A and B shows that under iron deficiency Mga2p is required to induce the IZH2-lacZ IZH4-lacZ, and OLE1-lacZ activities. However, LORE-lacZ activity was just slightly affected under those conditions, suggesting the possibility of a different co-activator for Mga2p in the promoter regions of IZH2 IZH4 and OLE1 Interestingly, although the -galactosidase activities under the two tested conditions are similar for the positive controls, the effect of ir on deficiency is still more prominent than iron repletion. These results constitute another pi ece of evidence that strongly suggests that IZH2 and IZH4 are Mga2p-target genes. Since iron deficiency has a dominant effect on the transcription of IZH2 we were interested in investigating if the accumulation of Izh2 protei n was also affected by iron deficiency. With this in mind, the Izh2p was expressed from the pIZH2 -3xHA plasmid, in which the IZH2 gene is driven by its own promoter. Protei n expression was performed in CSD medium supplemented with glucose as carbon source, deficient of iron, or supplemented with 10 M FeCl3 (iron repletion). As expected, iron defici ency also induces the accumulation of Izh2p, (Figure 2-4 C).

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42 Ubiquitination of Izh2p is Dependent on Nutritional Conditions We tested if Izh2p was post-translationally m odified when overexpressed in low iron medium (LIM). To investigate this, pRS316GAL1-IZH2 -3xHA construct was used, which provides galactose inducible expression. Overe xpression of Izh2p was carried out in replete media supplemented with galactose (SGal). Synt hetic medium supplemented with glucose (SD) was used as control medium to demonstrate specif icity of the band for Izh2p. In addition to this, LIM medium supplemented with galactose or glucose (LIMGal and LIMD, respectively) was also used to test the effect of iron deprivation and iron re pletion, on the expression of Izh2p. When the Izh2p was overexpressed in synthetic co mplete medium, or in LIMGal containing 1 mM FeCl3 (iron repletion), a group of bands at higher molecular weight than the one corresponding to the Izh2p molecu lar weight (36.3 KDa) was seen in the Western blot (Figure 25 A, lanes 4 and 12, respectivel y). Interestingly, when Izh2p wa s overexpressed in LIMGal (1 M FeCl3, iron deficiency), only the band at 36.3 KDa was observed (Figure 2-5 A, lane 8). Initially, we speculated that those bands at hi gher molecular weight could be due to the formation of complexes between Izh2p and othe r proteins. Alternativel y, we envisioned the possiblity that Izh2p is ubiquitina ted in media replete of iron or other metals, and that under iron deprivation (1 M FeCl3) the ubiquitination of Izh2p does not occur (Figure 2-5 A lane 8). To test these possibilities, we fi rst immunoprecipitated Izh2p expresse d in SGal and in LIMGal (1 M FeCl3). The Western blot analysis showed a ba nd at a molecular weight between 75 KDa and 50 KDa besides the band at 36.3 KDa (Figure 2-5 B, lane 1). To determine if the band at higher molecular weight was the result of Izh2p ubiqui tination, another Western blot was carried out and an antibody against the ubiquitin protein was used to investigate ubiquitination. Ubiquitin is a small and highly conserved eukayotic protein with a molecular weight of about 8.5 KDa (Baker

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43 and Baker, 1987; Peng et al., 2003). Interestinlgy, only the band between 75 KDa and 50 KDa was observed (Figure 5-2 C, lane 1), suggesti ng that Izh2p when overexpressed in a synthetic complete medium is undergoing ubiquitination. To explore the effect of zinc deficiency and zinc repleti on on the overexpression of Izh2p, Western blot analysis was carried out. In this case, Izh2p was expressed in a chelexed synthetic medium supplemented with galactose as a carbon source (CSGal), and either no ZnCl2 (zinc deficiency) or10 M ZnCl2 (zinc repletion) was added to the growth medium. Interestingly, similar results than those obtained with synthetic complete medium and LIMGal (1 mM FeCl3) were obtained (Figure 2-6, A and B). After im munoprecipitation, the band between 75 KDa and 50 KDa was observed only when Izh 2p was overexpressed in CSGal (10 M ZnCl2) (Figure 2-6 B). These results suggest that the overexpression of Izh2p in a me dium replete of zinc also induces the protein ubiquitination. To confirm this it is necessary to do a Western blot and use the anti-Ub antibody to see if the band between 75 KDa and 50 KDa is in fact the result of ubiquitination. Discussion The discovery of the IZ H family, along with the discove ry that the expression of IZH1 IZH2 and IZH4 is zinc-dependent, opened the possibility that this family of genes is implicated in zinc homeostasis. This hypothesis wa s then supported with the finding that IZH1 and IZH2 have a putative zinc responsive element (ZRE) in their promoter regions (Lyons et al., 2000) (Figure 1-3, panels A and B for details). The presence of a ZRE is necessary and sufficient to confer Zap1p-regulated expression onto a prom oter (Zhao and Eide, 1996a; Zhao and Eide, 1996b). Robust evidence is presented herein, that demonstrates that IZH2 is a bona fide Zap1p-

PAGE 44

44 target gene, and, therefore, a poten tial role for this gene in the metabolism of zinc is envisioned (Lyons et al., 2004). On the other hand, the fact that the IZH2 and IZH4 are induced by high zinc implies the existence of a transcription apparatus that is i nvolved in the modulation of the transcriptional response of these two genes under excess of me tals. Indeed, in the promoter regions of IZH2 and IZH4 a putative LORE consensus sequence was reported by Lyons et al., 2004, (Figure 1-4 panels B and C, respectively). LORE is a low oxygen response element and the DNA binding site for the hypoxia sensor Mga2p (Jiang et al., 2001). Besides low pO2 (hypoxia), excess of metals like zinc, cobalt, or nickel, have been proven to be inducers of the hypoxic response (Lyons et al., 2004; Vasconcelles et al., 2001; Gong et al., 2001; Rutherford et al., 2003). High metals are believed to induce Mga2p by displacing iron from important sites. Not surprisingly low iron also induces Mga2p. Herei n, we present evidence that suggests that the function of some of the IZHs goes beyond a role in zinc metabolism. For example IZH4 and to a lesser extent, IZH2 are induced under excess of Zn2+, Co2+, Ni2+, as well as by iron deficiency, and deletion of Aft1p, the iron-sensing transc ription factor (Figur e 2-2 A, B, and C). Interestingly, we have found that the metalloregulation of IZH2 and IZH4, under the conditions used, depends on the Mga2p hypoxia-responsive transcription fact or, suggesting that IZH2 and IZH4 are Mga2p-target genes and part of the hypoxic response (results published by Lyons et al., 2004). In addition to Mga2p, Spt23p, an Mga2p-re lated protein, was found to regulate hypoxic genes (Zhang et al., 1997; Nakagawa et al., 2002). In fact, studies performed by these groups indicated that both proteins are required for transcription of OLE1, a well known hypoxic gene. During the course of those studies, it was also found that, when synthesized, Spt23p and Mga2p

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45 are dormant proteins anchored through their C-terminal tails on the endoplasmic reticulum or nuclear envelopes. Under hypoxia, both proteins ar e ubiquitinated with the subsequent release of the N-terminal transcription factor domains into the cytosol, where they transcriptionally activate of OLE1. Although, we tested the effect of the spt23 deletion in the tran scriptional response of IZH4-lacZ under excess of metals, we did not see any significant effect (Figure 2-3), indicating that the transcrip tional response of IZH4 under the tested conditions is exclusively dependent on Mga2p. In Chapter 1, we mentioned that Izhs are homologous with the hemolysin 3 subfamily (Hly3). In the -proteobacterium Azospirillium brasilence Hly3 genes encode a chimeric protein with a Hly3-like N-terminus fused to the CheA chemotaxis histidine kinase (Hauwaerts et al., 2002). The involvement of Hly3-CheA from A. brasilense in hypoxia sensing emphasizes the importance of our finding that IZH2 and IZH4 are hypoxic genes, and suggests a conservation of function across species. Even though our studies regard ing transcriptional regulation provides striking information related to the role exerted by two the IZHs in the metabolism of metals, we were also interested to explore effect of iron and zinc on Izh2p accumu lation. To do this, the expression of Izh2p in metal replete media (CSD + 10 M ZnCl2 or FeCl3) versus media that are deficient in either zinc or iron (CSD ZnCl2 or FeCl3) were investigated. Our data, i ndicate that Izh2p is also induced under deprivation of either meta l (Figure 2-1 B and Figure 2-4 C, respectively), suggesting that the effect of zinc and iron deficiency goes be yond a simple transcriptio nal response. In this scenario, is possible that Izh2p exerts a role di rectly by scavenging either of these metals to supply the cell requirements. Alternatively, Izh2p can signal other molecules (e.g. metal transporters) that will directly uptake the required metal. In this regard, Kupchak et al., 2007

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46 found that overexpression of IZH2 constitutively represses FET3 under iron deficiency. FET3 encodes a high affinity iron transporter (Fet3p), which is implicated in high-affinity iron-uptake (Rutherford and Bird, 2004). Another important finding was that when Izh2p was overexpressed in a medium supplemented with metals and in a medium replete of iron, the protein appears to be ubiquitinated (Figure 2-5 A, lanes 4 and 12, B a nd C, lane 1). Ubiquitination is a cellular mechanism that mediates the sorting of proteins to the ensosomal/vacuolar pathway in response to nutritional signals (Pizzirusso and Chang 2004). In this sense, it is plausible that a high dosage of Izh2p is the driving force for the activation of the ubiquitin pathway, which results in the trafficking of the protein to the vacuole fo r further degradation. Another posiibility is that since Izh2p is a membrane protei n, its ubiquitination occurs to be removed from the membrane to further fulfill other roles within the cell. On the other hand, in an iron lim iting medium (LIM, containing 1 M FeCl3), Izh2p is not ubiquitinated, suggesting and inherent role for Iz h2p under metal deprivation (Figure 2-5 A, lane 8, and B and C, lane 3). Takeng together, the results pres ented in this chapter let us envision that Izh2p is directly involved in the regulation of zinc and iron homeostasis. At transcri ptional level, more studies are required to completely understand the mechanisms by which the responses of IZH2 and IZH4 are mediated by metal concentrations.

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47 Table 2-1. List of strains used in Chapter 2 Strain Mutation Source/Derivation Genotype BY4742 Wild type EUROSCARF MAT ; his3; leu2; ura3; lys2 DY1457 Wild type David Eide MAT ; ade6, his3; trp1; leu2; ura3; can1-100c ZHY6 zap1 David Eide MAT ;ade6; his3; trp1;leu2; ura3; can1-100c Y15968 mga2 EUROSCARF MAT ; his3; leu2;lys2; ura3; YIR033w::KanMX4 Y14869 spt23 EUROSCARF MAT a; his3; leu2;met15 ura3 ; YKL020c::KanMX4 Y04438 aft1 EUROSCARF MAT a; his3; leu2;met15 ura3 ; YGL071w::KanMX4

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48 Table 2-2. Genes induced > 2-fold by zinc excess Group Gene name Fold induction Induced by SSN6 deletion or by low oxygen IZH4 OLE1 HSP26 YGL039w ERG3 PIR3 YOR38w HSP30 COS10 IZH2 NCE103* AHP1 YGR161c HSP104* YOL106w 6.6 3.4 3.4 2.9 2.8 2.6 2.3 2.3 2.2 2.1 2.0 2.0 2.0 2.0 2.8 Miscellaneous PDR3 MGA2 UBS1 HSP150 2.6 2.3 2.1 2.0 Iron metabolism FIT3 FIT2 TAF1 TIS11 ENB1 ARN1 FTR1* 7.6 7.2 6.2 3.6 3.4 3.0 2.4 Iron metabolism FRE1 SIT1 FET3 HMX1 2.3 2.2 2.0 2.0 Ty retrotansposons YBL005w-A (YBLWTy1-1) YER138c (YERCTy1-1) YER160c (YERCTy1-2) YHR214c-B (YHRCTy1-1) YML045w (YMLWTy1-2) YBR012w-A/-B (YBRWTy1-2) YCL019w/20w (YCLWTy2-1) YJR026w/27w (YJRWTy1-1)* YJR028w/29w (YJRWTy1-2) YML039w/40w (YMLWTy1-1) YMR045c/46c (YMRCTy1-3)* YMR050c/51c (YMRCTy1-4) 2.1 2.2 2.3 2.2 2.6 2.2/2.2 2.1/2.4 2.2/2.5 2.2/2.2 2.2/2.6 2.0/2.6 2.3/2.6 *Genes in bold have promoters containing putative regulatory elements scoring > 7.0 when using the LORE or FeRE matrices generated by RSA-T OOLS. *LORE-containing, FeRE-containing Tables 2-1 and 2-2 were reproduced, with permissi on, from Lyons TJ, Villa NY, Regalla, LM, Kupchak BR, Vagstad A, and Eide DJ (2004) Metalloregulati on of yeast steroid receptors homologs. Proc Natl Acad Sci USA 101: 5506-5511 (Table 2, supplemet ary information, and Table 1, page 5508).

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49 Figure 2-1. Zinc regulation of IZH2 Panel A shows that the activity of the IZH2-lacZ promoter reporter is increased by zinc deficiency in a Zap1p-depe ndent manner (black bars). On the other hand, the IZH2-lacZ activity is repressed under zinc repletion (10 M ZnCl2) (grey bars). Panel B is a Western bl ot showing that the expression of Izh2p under zinc deficiency is also induced.

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50 Figure 2-2. Transcriptional regulation of IZH2 and IZH4 by different metals. Panel A shows the effect of aft1 mutation on IZH4-lacZ activity. B, effect of zinc excess, Mga2p, and Aft1p on LORE-lacZ activity. In panels A and B, grey bars show activity in + zinc (10 M ZnCl2), white bars show activity in + + zinc (3 mM ZnCl2). C shows the Mga2p dependence of IZH4-lacZ and IZH2-lacZ activities in cells exposed to 10 M ZnCl2 (grey bars), 3 mM ZnCl2 (white bars), 400 M Co2+ (hatched bars), or 400 M Ni2+ (black bars). D, shows the effect of Mga2p + zinc (10 M ZnCl2, grey bars), and 400 M Co2+ (hatched bars) on LORE -lacZ and OLE1-lacZ activities. Panels A, B, and C were reproduced, with permission, from Lyons TJ, Villa NY, Regalla, LM, Kupchak BR, Vagstad A, and Eide DJ ( 2004) Metalloregulation of yeast steroid receptors homologs. Proc Natl Acad Sci USA 101: 5506-5511 (Figure 3, page 5508).

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51 WT galactosidase activity (Miller Units) 0 10 20 30 40 50 60 No metals 3 mM Zn 2+ 400 M Co 2+ 400 M Ni 2+ spt23mga2IZH4-lacZ Figure 2-3. Transcriptional activation of IZH4-lacZ in cells exposed to excess of different metals is dependent on the presence of MGA2 but independent on SPT23.

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52 Figure 2-4. Iron regulation of IZH2 and IZH4 A and B show the effect of iron deficiency, and the presence of Mga2p, on the transcriptional regulation of IZH2-lacZ IZH4-lacZ, LORE -lacZ, and OLE1-lacZ activities. C is a West ern blot showing that the expression of Izh2p is positively modulated by iron deficiency. In the panels corresponding to -galactosidase activities, gray bars show activity in + iron (10 M FeCl3, iron repletion), and black bars show activity in iron (no FeCl3, iron deficiency).

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53 Figure 2-5. Post-translational effect of iron deficiency on th e overexpression of Izh2p. Figure shows Western blot analysis of Izh2p under different nutritional conditions. A shows the Izh2-3xHA protein expres sion in different growth media (e.g. SD, SGal, LIMD, and LIMgal). B is aWestern blot of immunoprecipitated samples. C shows the ubiquitination of Izh2p. In panels A and B, an antibody against the HA epitope tag was used to identify Izh2-3xHAp. In panel C, an antibody against ubiquitin was used to determine protein ubiquitination. In the figure, SD and SGal stands for synthetic medium, supplemented with glucose and gala ctose, respectively. LIMD and LIMGal represent low iron media, supplemented with glucose and galactose, respectively. Iron deficiency is 1 M FeCl3 and 1 mM FeCl3 is iron repletion.

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54 Figure 2-6. Zinc-dependence on the translational response for Izh2p. A is a Western blot showing the overexpression of Izh2p in cells exposed to zinc deficiency (no ZnCl2 added to the growth medium) and + zinc repletion (10 M ZnCl2). B shows a Western blot for the Izh2-3xHA immunopr ecipitates. Izh2p was overexpressed in chelexed synthetic medium supplemented w ith galactose (CSGal). In the figure + HA and HA represent HA-tagged a nd untagged Izh2protein, respectively.

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55 CHAPTER 3 DUAL REGULATION OF THE IZHS BY METALS AND FATTY ACIDS: THE FIRST LINE OF EVIDENCE IMPLICATING THE IZH FAMILY IN LIPID METABOLISM Introduction Saccharomyces cer evisiae is able to survive on a wide ra nge of growth media due to its ability to activate pathways that enable the utilization of ferm entable and non-fermentable carbon sources. Fatty acids are one of those nutrients that tightly regulate gene expression when used as a sole carbon source (Choi et al., 1996; DeRusso et al., 1999; Black et al., 2000; Kandasamy et al., 2004). In the S. cerevisiae genome, a myriad of genes of known and unknown function have been found to be activated or represse d, at transcriptional and transl ational levels, when cells are grown in media supplemented with fatty acids. Perh aps one of the most interesting examples that illustrate the regulatory effect exerted by fatty aci ds is the oleate-dependent activation of genes encoding proteins implicated in the fatty acid -oxidation pathway and peroxisomal proliferation (Hiltunen et al., 2003). In S. cerevisisae peroxisomal -oxidation is the only means to catabolize long chain fatty acids (Luo et al., 1996). Oleate, a very abundant fatty acid in yeast cells, is a cis -9 monounsaturated fatty acid cont aining an acyl chain of 18 carbons in length, (Vasconcelles et al., 2001). Despite the positive effect exerted by fatty acids in the transcription of specific genes, other genes are transcriptionally repressed in response to chan ges in a carbon source. One of those genes that illustrate such an opposite effect is OLE1 This gene encodes -9 fatty acid desaturase, an enzyme involved in the formation of unsaturated fatty acids. In presence of oleate, the expression of OLE1 is repressed. Conversely, transcription of this ge ne is induced when cells are grown in the presence of saturated fatty acids (McDonough et al., 1992). Interestingly, the

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56 hypoxia sensors MGA2 and SPT23 have been implicated in the transcription of OLE1 (Kandasamy et al., 2004). Jiang et al., 2002, reported that in S. cerevisiae the presence of the LORE (low oxygen response element) is important for the transcriptional regulation of target genes via Mga2p, and that certain lipids like unsaturated fatty acids can repress the LOREdependent induction of Mag2p targes like OLE1 Thus, hypoxia and unsaturated fatty acids work in opposing manners (Kwast et al., 1999; McDonough et al., 1992; Nakagawa et al., 2001; Vasconcelles et al., 2001). In the promoter regions of genes regulated by certain fatty acids, a consensus sequence termed as an Oleate Response Element (ORE) has been identified (Kos et al., 1995). This sequence is the binding site for the transcri ption factors Oaf1p and Pip2p. Upon addition of oleate, these two proteins bind to ORE as a heterodimeric complex, and mediate oleatedependent transcriptional activation (Karpichev and Small, 1998; Baumgartner et al., 1999). Interestingly, three of the four IZH genes, ( IZH1, IZH2, and IZH4 ), contain putative ORE sequences in their promoter regions (Figur e 1-4, panels A, B, and C for details). In this chapter, we present some evidences that suggest other roles for the IZHs besides their implication in the metabolism of metals. First of all, we show that IZH2-lacZ promoter reporter fusion responds independently to both zi nc and the addition of myristate. Second, we demonstrate a dual regulatory eff ect exerted by metals and fatty acids. For example, we found that certain fatty acids and metals like cobalt when added to the same growth medium produce an additive effect on the induction of IZH2-lacZ and IZH4-lacZ activities. Even more interesting is the finding that these regulatory effects occur at transcriptional leve l via the hypoxic sensor Mga2p. This last result illuminat es the idea that diverse metabo lic pathways like metals and lipids can converge to the poi nt where genes like the IZHs, are up-regulated.

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57 Also, we show that supplementation of growth medium with fatty acids as a sole carbon source, elicits the transcriptional and post-translational response of IZHs In this regard, one of the most promising results is the strong transc riptional and post-trans lational induction of IZH2 by myristate (a saturated fatty acid with a chai n of 14 carbons in length). Surprisingly, the transcriptional induction of IZH2-lacZ occurs via Oaf1p/Pip2p. This finding was unexpected, since these two transcription f actors are known to be activated by oleate but not by myristate. Furtheremore, we also show that Oaf1p and Pi p2p are required to maintain the weak but still measurable IZH2-lacZ activity in presence of oleate. Overall, the regulatory effect s of different fatty acids on the transcription of the IZHs, is investigated. Furtheremore, a dual regulatory effect of metals and fatty acids on the trnacription of IZH2 and IZH4 is established during this study. Taken together the results presented herein, suggest a dual implication of IZHs in metals and lipid metabolism. Materials and Methods Yeast Strains and Plasmids Yeast strain s used in this chapter are liste d in table 3-1. As men tioned elsewhere, the IZHlacZ fusions were generated by gap repair of the plasmid vector YEp353 (Lyons et al., 2000). Briefly, PCR products were gene rated from genomic DNA that contained 1,000 bp of the target promoter sequence flanked by regions of vector homology. These fragme nts were gel-purified (Promega) and co-transformed into the BY4742 wild type strain with Eco RIBam HI-digested YEp353; transformants were selected for Ura+ prototrophy. Plasmids were then transferred to Escherichia coli and confirmed by sequencing. Although the IZH2 O pen R eading F rame (ORF) has two ATG codons, only the fusion of the second in-frame ATG of in the IZH2 (ORF) to lacZ resulted in a functional promoter-reporter construct ( IZH2-lacZ).

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58 Biochemical Assays Yeast transform ations were performed accordin g to standard procedures, in identical fashion as described in Chapter 2. Promoter reporter activities were measured by -galactosidase assays (Chapter 2). Before each -galactosidase assay, cells were grown aerobically during one overnight in synthetic medium containing dextrose (SD medium). When required, and unless otherwise indicated, the SD medium was supplemented with 1 mM of the respective fatty acid, and 0.5% Tergitol-NP40 (Sigma). To limit zinc availability, chelex ed-synthetic medium (CSD) was used. CSD was prepared in identical fashion as described in Chapter 2 (Ape ndix A for more details). When myristate was used along with zinc, a 37.5% (w/v) stock solution of the fa tty acid was dissolved in 50% EtOH, 25% Tween-40 and then added to the CSD growth medium to a final concentration of 0.375% (Lyons et al., 2004). Protein expression, immunoprecipitation, and Western blot anal ysis were performed as described in Chapter 2. Results Fatty Acids Exert a Regulatory Effect on the Expression of IZHs Previously Karpichev et al., 2002 reported that the expression of IZH2 was induced by exogenous myristate. In that pub lication, they also reported th at addition of m onounsaturated fatty acids like oleate induced the Oaf1p/Pip2p-depe ndent regulatory effect of target genes. As mentioned before, putative ORE sequences are present in the IZH2 (-159 to -167 bp), IZH1 (-302 to -328 bp), and IZH4 (-204 to -263 bp) promoters (Karpichev et al., 2002). Herein, we have confirmed that IZH2-lacZ responds independently to both, zi nc and the addition of exogenous myristate (Figure 3-1) (Lyons et al., 2004).

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59 We have also found that while IZH1-lacZ and IZH3-lacZ respond to exogenous oleate (C18:1), stearate (C18:0), and palmitate (C16:0) (Figures 3-2 A and C, respectively), the IZH4lacZ reporter responds to the addi tion of palmitate (Figure 3-2 D), and the transcriptional activation of the IZH2-lacZ reporter was exclusively observed in presence of myristate (Figure 32 B). Interestingly, we also found that the transcripti onal regulation of IZH2 by myristate and oleate is Oaf1p/Pip2p-dependent (Figures 3-3 A and B, respectively). These results suggest that the Oaf1p/Pip2p complex is not ex clusively activated by oleate, and that myristate can also mediate the activation of this transcription factor complex. The Expression of the Izh Proteins is Regulated by Fatty Acids Due to the e ffect exerted by several fatty acids in the transcriptional response of the IZH gene family, we decided to investigate if this e ffect also exists at leve l of protein accumulation. To do this, 3xHA epitope-tagged IZH constructs under the control of thei r own promoter or a galactose inducible promoter (for protein overexpression) were used. A polyclonal antibody against the HA tag, probe Y-11 was used to reco gnize each of the Izh1-3xHA fusion proteins in the Western blots. Cells expr essiong untagged Izh proteins or the empty plasmid vector (pRS316-GAL1 ), were used as negative controls. Interestingly, we did not s ee any change in the expression profile of Izh1p when the IZH1 contained its own promoter (Figure 3-4 A). However, when Izh1p was overexpressed in galactose, a slightly induction of Izh1p expression, upon addition of plamitate, was observed (Figure 34 B). Likewise, Izh2p was induced by myristate (Figures 3-4 C and D). Also, the expression of Izh3p was induced only when the cells were spiked with oleate for 2 h (Figure 3-4 F). In c ontrast, the expression of Izh3p was fully repressed upon incubation with oleate for 12 h (Figure 3-4 E). This could be at tributed to toxic effects of oleate during long periods of incubation. To de tect Izh3-3xHAp, the protein had to be concentrated by immunopreciopitation. Immunoprecipitati on was done in identical fashion as

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60 explained in Chapter 2 and using a mouse monocl onal anti-HA conjugated w ith agarose beads to pull down the 3xHA-tagged Izh3p. Immunoprecipitates were then detected by Western blot. For Izh4p, addition of plamitate did not produce any si gnificant effect on the levels of accumulation of Izh4p (Figure 3-4 G). In general, the results obtained with the We stern blots indicate th at the effect of the exogenous fatty acids goes beyond a mere transcrip tional response, and that certain fatty acids constitute a sufficient stimulus to induce the expression of the majority of the IZH gene products. Taken together, these results represent additi onal evidence suggesting a strong implication of IZHs in lipid metabolism. Transcriptional Regulation of IZH2 and IZH4 in Presence of Meta ls and Fatty Acids Occurs via Mga2p. In the former chapter, the Mga2p-dependent regulation of IZH2-lacZ and IZH4-lacZ activities by excess of metals was reported. Nakagawa et al., 2001; Vasconcelles et al., 2001, reported that OLE1 has an LORE in its promoter region and that its expression was induced in response to excess of cobalt and saturated fatty acids via Mga2p. In that study, the expression of OLE1 was also found to be repressed by unsaturated fatty acids like oleate (C18:1) and palmitoleate (C16:1). Like OLEI, IZH2 and IZH4 contain in their promoter regions a low oxygen responsive element (LORE) and an oleate responsive element (ORE) (Karpichev et al., 2002; Lyons et al., 2004). Therefore, it is plausibl e that under similar stimuli, IZH2 and IZH4 respond like OLE1 To investigate if the expression of IZH2 and IZH4 upon the simultaneous addition of Co2+and myristate, as well as Co2+ and oleate were Mga2p-dependent, we performed reporter gene assays. Surprisingly, we found that the simultaneous addition of Co2+ and myristate produced an additive effect on the IZH2-lacZ activity (Figure 3-5 A) On the other hand, the inducible effect of Co2+

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61 on the IZH4-lacZ reporter activity was partially repressed upon addi tion of myrsistate (Figure 35 B). The transcriptional response of the promote r-reporter fusions analyzed in this study, were Mga2p-dependent (Figure 3-5 A and B). In addition to this, we also investigated the effect of adding oleate and cobalt to the same growth medium. IZH2 and IZH4 respond similarly to OLE1 under these external stimuli and this response is Mga2p dependent. In effect, oleate repressed the Co2+-dependent induction of the IZH2-lacZ reporter (Figure 3-6 A). Moreover, these effects were even more stringent when the MGA2 gene was knocked out. Similarl y, the inducible effect of Co2+ in the IZH4-lacZ reporter activity was also fully repressed by ol eate (Figure 3-6 B). These tran scriptional responses were also confirmed for the positive controls LORE -lacZ and OLE1-lacZ (Figure 3-6 C and D, respectively). Overall, our results suggest a c onnection between the hypoxia and the fatty acid metabolic pathways. In this scen ario, it is totally feasible that the hypoxia pathway controls the levels of unsaturated fatty acids in S. cerevisiae, by regulating the expression of three genes OLE1 IZH2, and IZH4. Discussion Saccharomyces cer evisiae cells can grow on a variety of carbon sources, including fatty acids. When used as a sole carbon and energy so urce, fatty acids elicit the transcriptional and translational up-regulation of different genes. Herein, we report that IZHs respond to exogenous fatty acids at the transcriptional and post-tr anslational levels, suggesting a role for the IZHs in lipid metabolism. Also, we demonstrate that zinc deficiency and the addition of myristate acid have an additive effect on the induction of the IZH2-lacZ activity and that res ponse to each stimulus is independent of each other. The transcriptional response of IZH2 under zinc deficiency is Zap1dependent, whereas the induction of the gene by myristate appears to be dependent on the

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62 presence of the transcription factors Oaf1p/Pip2p. Although oleate did strongly induce IZH2lacZ activity, per se, single mutations of OAF1 and PIP2 produce repression on the IZH2-lacZ activity, indicating that both transcrip tion factors are required for activation. Another interesting finding was that oleate overrides the cobalt-induction of IZH2 lacZ and IZH4-lacZ activities, and that such a repressing e ffect is Mga2p dependent. Furthermore, myristate and cobalt have a strong additive effect on the IZH2-lacZ activity. These results suggest a critical role for IZH2 and IZH4 under diverse stimuli such as hypoxia and exogenous fatty acids. The results presented in this chapter, consti tute a first piece of ev idence that reveal the promiscuity of this family of yeast proteins Their dual regulation by metals and fatty acids makes of these proteins an attractive target of research. Understanding how such divergent metabolic pathways can transcriptio nally and post-translationally a ffect a family of proteins is vital because it can contribute to the elucidati on of how these metabolic pathways are tightly regulated. Undoubtedly, alternative hypotheses need to be addresse d to decipher this metabolic puzzle. For future work, it is imperative to inve stigate the mechanism by which some of the IZH genes can respond to different nu tritional stimuli. A first experi ment that can illuminate the results presented in this chapter is a DNA micr oarray experiment using the nutritional conditions reported herein. With this type of experiment we can have a global idea about which genes and which metabolic pathways are affected under hypoxi c conditions and the use of fatty acids as a sole carbon source. Microarray data can then be compared to the -galactosidase data reported in this chapter. Furthermor e, Western blots can be used to expl ore the post-transla tional response of the IZH and other genes found by DNA microarrays.

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63 Table 3-1. List of strains used in Chapter 3 Strain Mutation Source/Derivation Genotype BY4742 Wild type EUROSCARF MAT ; his3; leu2; ura3; lys2 Y10355 oaf1 ( yaf1 ) EUROSCARF MAT ; his3; leu2; lys2; ura3 ; YAL051w::KanMX4 Y11660 pip2 ( oaf2 ) EUROSCARF MAT ; his3; leu2; lys2 ura3;; YOR363c::KanMX4 Y15968 mga2 EUROSCARF MAT ; his3; leu2;lys2; ura3; YIR033w::KanMX4

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64 Figure 3-1. Zap1p-depe ndent regulation of IZH2-lacZ. The IZH2-lacZ reporter responds to both zinc and exogenous myristate (C14:0). Black bars show reporter activity in zinc, and gray bars show reporte r activity in + zinc (10 M ZnCl2). In this, and all other figures showing lacZ data, a representative experime nt performed in triplicate is shown and the error bars re present standard deviati on. Figure reproduced, with permission from Lyons TJ, Villa NY, Re galla, LM, Kupchak BR, Vagstad A, and Eide DJ (2004) Metalloregulation of yeast steroid receptors homologs. Proc Natl Acad Sci USA 101: 5506-5511 (Figure2 D, page 5507).

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65 Figure 3-2. Transcriptional regulation of IZHs by exogenous fatty acids. A, ZH1-lacZ and IZH3-lacZ activities are slightly induced by addition of palmitate (C16:0), stearate (C18:0) and oleate (C18:1). B, shows that while the IZH2-lacZ activity is highly induced by myristate (C14:0) (panel B-left), the IZH4-lacZ activity is induced by palmitate (C16:0), (panel B-right).

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66 Figure 3-3. Oaf1p/Pi p2p dependence of the IZH2-lacZ activity in presence and in absence of myristate (C14:0) and oleate (C18:1) (panels A and B, respectively).

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67 Figure 3-4. Post-translational re sponse of the Izhp family upon addition several fatty acids. Panels A and B show the effect of palmita te (C16:0) in the expression of Izh1p. The effect exerted by C16:0 is remarkable when Izh1p was overexpressed, panel B. C and D, the expression of Izh2p is highly induced upon additi on of myristate (C14:0). E and F, time-dependence of Izh3p expression upon addition of oleate (C18:1). G, no effect of C16:0 on the e xpression Izh4p was observed.

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68 Figure 3-5. Dual regulation of IZH2 and IZH4 by exogenous myristate (C14:0) and cobalt. A and B show the Mga2p dependence of IZH2-lacZ and IZH4-lacZ activities in cells exposed to 400 M Co2+, 1 mM myristate, and the addition of both 400 M CoCl2, and 1 mM C14:0. A shows that exogenous C14:0 has an additive effect on the Co2+dependent induction of IZH2-lacZ activity. B, the addition of C14:0 partially represses the inductive effect of Co2+on the IZH4-lacZ activity.

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69 Figure 3-6. Regulation of IZH2 and IZH4 by exogenous oleate (C18:1) and cobalt. A, Mga2p dependence of IZH2-lacZ activity in cells exposed to 1 mM C18:1. Panels A, B, C, and D, show the repressive effect of 1 mM C18:1 in the Co2+-dependent induction of the promoter -reporter activities Mga2p-independence of the IZH4-lacZ LORElacZ, and OLE1-lacZ activities upon addition of C18:1 is shown in panels B, C, and D, respectively.

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70 CHAPTER 4 NYSTATIN-RESISTANCE OF IZH3, IS ASSOCIATED TO ALTERATIONS IN THE ERGOSTEROL CONTENT Introduction Sterols are essential structur al and regulatory components of eukaryotic cell m embranes (Figure 4-1) (Veen et al., 2003; Mllner et al., 2005). Although cholesterol is the main sterol in mammals, ergosterol is the most abundant sterol in fungal membranes (Reiner et al., 2006). In yeast, sterols are found in two main forms: (i) free sterols and (ii) steryl esters. Free sterols accumulate in the plasma membranes wher e their main role is to maintain membrane integrity and fluidity (Veen et al., 2003). Steryl esters, on the other hand, are found in lipid particles, where they serve as storage reservoirs or as intermediates in intracellular transport (Zinser et al., 1993, Yang et al., 1996; Valachovi et al., 2001). When free sterols are required, esterified sterols are hydrolyzed producing free st erols and fatty acids, which are then mobilized to plasma membranes to fulfill the membrane requirements (Kffel et al., 2006). Despite its structural role, ergos terol has been recently implicat ed in regulation of cell cycle progression, cell polarization and membrane fu nction during mating, endoc ytosis and vacuolar fusion as well as protein sorti ng along the secretory pathway and signal transduction (Reiner et al., 2006). Because sterols are required to maintain the fl uidity and integrity of the fungal membranes, the sterol pathway has emerged as a potential target for the development of antifungal drugs (Carrillo-Muoz et al., 2006). The reason for this is the spec ificity of some antifungals toward distinct sterols. For example, so me antifungals like azole s inhibit enzymes at sp ecific steps of the sterol pathway (Gachotte et al., 1997), and others like nystatin and amphotericin B exert their action by interacting directly with ster ols like ergosterol (Sharma, 2006).

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71 Currently fungal infection is one of the major causes of mortality in immunocompromised patients such as those with AIDS. In addition, treatments of chemotherapy, organ transplantation, and environmental stresses like UV radiation ar e also potential causes of fungal infection (Kontoyiannis et al., 2002). Members of the Candida spp family, in particular, are the major cause of virulence (Mukhopadhyay et al., 2002). Besides azoles, pol yene antifungals like amphotericin B and nystatin have been widely used in the treatment of fungal infections (Dupont, 2006). Although S. cerevisiae is a non-pathogenic fungus, its genetic sim ilarity to Candida spp makes it a safe model system to investigate th e mechanisms that govern polyene effects. Nystatin, a polyene antifungal (Figure 4-2 C), isolated from Streptomyces noursei (Marini et al., 1960), selectively target s ergosterol in the fungal plasma membrane (Lees et al., 1995) generating pores in the plasma membrane through which the leakage of nutrients occurs and the concomitant cell death (Lampen et al., 1962) (Figure 4-2 panels A a nd B). Despite the variety of studies performed, the mechanism by which nystati n exerts its fungicidal effect still remains unclear. Karpichev et al., 2002 reported that mutation of the fungal gene IZH2 ( izh2) caused resistance to nystatin (or a normal growth in pres ence of nystatin). Nystatin resistance has been associated with mutations that lead to changes in the sterol composition or the sterol content in the cell membranes (Lampen et al., 1960; Hapala, et al., 2005). Although we could not confirm the results reported by Karpichev et al., 2002, we found that the mutation of the IZH3 gene ( izh3) was consistently resistant to nystatin. In this chapter, we report for the first time th e identification and initial characterization of izh3 as a novel nystatin resistant mutant. As a first approach to this discovery, phenotypic

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72 studies revealed that izh3 grows better than wild type and the single mutations izh1, izh2, and izh4in a medium supplemented with nystatin Second, complementary studies where the IZH3 gene was re-introduced into the izh3 mutant, rescued the nys tatin-dependent growth defects. Finally, by using different analyt ical techniques, we demonstrate that izh3 shows alterations in the sterol composition; particularl y, we have discovered that this mutant has less free ergosterol than wild type, suggesting that the IZH3 gene could be involved in the sterol biosynthetic pathway. Even though nystatin requires ergosterol for toxicity (Bhuiyan et al., 1999), recent studies have revealed that sphingolipids can also be targets for the ny statin action (Leber et al., 1997). Phenotypic studies conducted to see the dual effect of nystatin and certain sphingoid bases in the growth pattern of the wild type st rain BY4742 and the single and multiple izh mutants suggest that sphingoid bases can ameliora te the toxic effects of nystatin. This observation also opens the possibility that the IZH3 gene could be implicated in relate d sterol metabolic pathways such as the sphingolipid biosynthetic pathway. Materials and Methods Yeast Strains and Reagents The stra ins used in this chap ter are described in Table 4-1. Deletions were generated by Dr. Thomas Lyons (during his post-doctoral fellow) using PCR-based gene disruption using short flanking homology, according to Wach et al., 1994. Multiple, mutations were generated from heterozygous quadruple knockout strain engin eering by successive rounds of mating and sporulation. The kanMX4 (kanamycin) markers in the izh2, izh3 and izh4 strains were replaced with the hphMX4 (hygromycin), natMX4 (nourseothricin), and ura3MX4(URA+) cassettes, respectively (Goldstein and McCusker, 1999).

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73 Nystain (Nys), sterylamine, and ergosterol, la nosterol, and cholestero l were obtained from Sigma. The sphingolipids C18-phytosphingosine (C18-PHS) and C18-dihydrosphingosine (C18DHS), C2-phytoceramide (C2-PHC), C16-phytoceramide (C16-PHC), and C18-pytoceramide (C18PHC) were obtained from Avanti Polar Lipids. The solvents for GC-MS or HPLC-MS analysis were from Burdick & Johnson. Yeast Transformations Yeast transform ations were performed as described before. In this case, the centromeric plasmid vector pRS315CEN (empty vector), or the plasmid vector expressing IZH3 via its own promoter were introduced into the izh3 mutant. Likewise, the pRS316GAL1 (empty vector) or the plasmid vector expressing IZH3, were transformed into izh3. Finally, the pRS316GAL1 and the plasmid expressing each of the IZHs were also transformed into the wild type strain BY4742. Yeast Growth Media and Conditions Cells were grown aerobically, during one overn ight in liquid synthetic m edia, prepared with 0.67% Yeast Nitrogen Base without ami no acids and ammonium sulfate (Fisher), and supplemented with 0.5% ammonium sulfate, 2% gl ucose and appropriate amino acids at a final concentration of 0.01% (SD medium). For gene ov erexpression, glucose was replaced with 2% galactose as carbon source (SGal medium). For gr owth in solid media, the SDor the SGalmedium was supplemented with 1X bacto agar (Fisher). Cell growth in liquid media was monitored using a UV-VIS 170-2525 SmartSp ec Plus spectrophotometer (Bio-Rad). For ultraviolet spectrometric, or gas chromatography mass spectrometric analysis (GCMS) of total sterols, cells (50 mL liquid culture) containing th e WT strain, or the izh3 mutant were grown to stationary phase (OD600 of 3.0-4.0), followed by centrifugation at 3000 rpm, 4oC

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74 for 5 min. Pellets were washed twice with cold sterile nano-pure water, a nd their net wet weight was determined. When used, 5 U/mL of nystatin wa s added to the growth media at a very early OD600 (e.g. OD600 of 0.1). Once again, cells were grown to stationary phase (OD600 of 3.0-4.0), followed by harvesting in identical fa shion as described just before. For high performance liquid chromatography-ma ss spectrometric analysis (HPLC-MS) of total and free sterols, cells (5 mL) where the izh3 strain was transformed with the empty vector pRS315CEN (same than izh3 strain), or the pRS315CEN expressing IZH3 (same than WT), were grown for one overnight, to saturation. Fres h liquid media (50 mL) were inoculated with aliquots of the overnights and grown to logarithmic phase (OD600 of 1.0). Cells were then harvested by centrifugation at 3,000 rpm, 4oC and for 5 min. Resultant pellets were washed twice with cold sterile water, and freeze dried by using a lyophilizer. Dried cells were further used for sterol extractions. Phenotypic Studies Cells were grown overnight to saturation (OD600 of 3.0) at 30oC, in synthetic medium supplemented with glucose as a carbon source. Cells were then se rially diluted at OD600 = 1.0, 0.1, and 0.01, and aliquots of 5 L of these dilutions were plat ed on synthetic medium agar plates supplemented with 5 U/mL, or 10 U/mL nystatin dissolved in DMSO. Nystatin solutions were always prepared and immediat ely used to avoid degradation. For the phenotypic studies in pr esence of sphingolipids, +/12.5 M C18-PHS, and C18DHS sphingoid base-like stearylamine (d issolved in 100% ethanol), +/0.5 M C2-PHC, C16PHC and C18-PHC (dissolved in a mixture of cholorform : methanol at a ratio 1:1, v/v) were used to plate 5 L of each serial dilution. Plates were incubated in the dark, at 30oC for 3 days; and observed for growth.

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75 Complementation Studies The izh3 mutant strain, containi ng the kanMX4 cassette was transformed with the centromeric plasmid vector pRS315 (pRS315CEN) or with the pRS315 -CEN plasmid, expressing the IZH3 via its native promoter. To s ee the effect of overexpressing IZH3 in presence of nystatin, the mutant izh3 was also transformed with both the pRS316 plasmid containing the strongly inducible GAL1 promoter (pRS316GAL1 ), or with the pRS316 -GAL1 plasmid expressing IZH3 in a medium supplemented with galactose. Sterol Extractions For UV and GC-MS analysis, sterols were extr acted accord ing to the protocol described by Arthington-Skaggs et al., 1999. Briefly, pellets were suspen ded in 3 mL of 25% alcoholic potassium hydroxide solution (25 g of KOH and 35 mL of sterile nano-pure water, and brought to 100 mL with 100% ethanol), and mixed by vortexing for 1 min. Cells suspensions were transferred to 13 X 100 mm borosilicate glass tubes with screw-caps, and in cubated in the dark in 85oC for 2 h. Following incubation, tubes were allo wed to cool at room temperature. Sterols were then extracted with 4 mL of a mixture of sterile nano-pure water and nheptane (1:3, v/v) followed by vigorous vortex mixing during 3 min. The heptane layer was transferred to new sterile 13 X 100 mm borosilicate glass test tubes with screw-cap. The obtained sterol extracts were used for UV spectrophotometric analysis. Extractions of total and free ergosterol were performed adapting the protocols described by Bailey and Parks, 1975; Megumi et al., 2005. For semi-quantitative ster ol analysis of total and free ergosterol, 0.026 moles of the internal standard cholesterol (e.g. 3 l 8.7 mM solution of cholesterol prepared in ethyl a cetate) were added to dried cells before extraction. Samples were briefly dried under a stream of N2, and followed by sterol extraction. On the other hand, for

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76 quantitation, 0.625 M cholesterol was added to the samples after extraction and just before injection into the HPLC-MS instrument in order to compensate for variations in ionization efficiency and stability of the samples. Total sterol extraction, was perf ormed as follows. Cells were suspended in 1 mL absolute ethanol followed by addition of 6 mL of methanol and 0.4 g of KOH. Saponification of the mixtures was carried out by heating at 75oC for 40 min. After the mixture was cooled down at room temperature, 2 mL of sterile water was ad ded, and the sterols were extracted twice with 2 mL of petroleum ether, each time. The fractions of petroleum ether were collected and evaporated to dryness under a stream of N2, and then weighed. For free ergosterol extraction, dr ied cells were suspended in 400 L of DMSO, and heated for 1 h at 100oC. After cooling at room temperature, the mixture was mixed with 3 mL of sterile H2O and then extracted three times with 2 mL of petroleum ether. After each extraction, suspensions were briefly centrifuged to help layer separation. Extracts containing the free ergosterol, (enriched in the petroleum ether layer) were combined and evaporated to dryness under a stream of N2. Sterol Analysis by Ultraviolet Spectroscopy For qualitative and sem i-quantitat ive analysis of ergosterol and 5, 7, 22, 24(28)dehydroergosterol [4(28)-DHE], total sterol extracts were scanned spectrophotometrically between 190 and 310 nm using a UV-visible CARY Varian spectrophotometer. Quantitation of the ergosterol and 24(28)-DHE contents was pe rformed according to the method described by Breivik and Owades, 1957, applying the equations: % ergosterol + % 24(28)-DHE = (A281 / 290) / pellet weight. % 24(28)-DHE = (A230 / 518) / pellet weight. % er gosterol = (% ergosterol + % 24(28)-DHE) % 24(28)-DHE, where 290 and 518 are the E values (in percentages per

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77 centimeter) determined for crystalline ergoste rol and 24(28)-DHE, resp ectively (Breivik and Owades, 1957). Results are reported as the percenta ge of each sterol per wet weight of cells Analysis of Sterols by Gas Chroma tography-Mass Spectrometry (GC-MS) Before GCMS analysis, sterol extracts as we ll as ergosterol, lanosterol and cholesterol were converted into their more volatile trimet hylsilyl (TMS) ether count erparts following the procedure described by Gerst et al., 1997 with some modifications Briefly, non-saponifiable lipid extracts (100 L) were transferred into glass scre w-cap GC vials, and spiked with 5 L of a 2.58 nM solution of cholesterol. After vortexi ng thoroughly, suspensions were dried under a stream of N2. Dried samples, and standa rds were treated with 200 L of a N,O bis(trimethylsilyl)-trifluoroacetamine (BSTFA) w ith 1% trimetylchlorosilane (TMCS), (Fluka). The silylation reactions were carried out at 45oC, in the dark, for 1 h. Silylated sterols were identified by GC-MS, by comparing their retentio n times with the silylated standards. This was performed by Dr. Cristina Dancel at the Mass Spectrometry Laboratory, department of Chemistry, University of Florida) Tandem GC-MS was done on a Thermo Scientif ic Trace DSQ instrument, equipped with an Rtx-5MS column (15 m x 0.25 mm i.d. x 0.25 m d.f.). 2 l of each solution was introduced into the capillary column at 1-min splitless mode, with helium carri er gas flowing at a rate of 0.7 mL/min. The injector was kept at 225oC while the transfer line and ion source were at 200oC. The GC column was maintained at 200oC for 2 min and then heated to 280oC at 20oC/min. Mass spectra were generated via elect ron ionization (EI) and compared against those in the NIST EI Spectral Library (Appendix C). The silylated deri vatives of standard ergosterol and lanosterol were analyzed to validate retention times and to generate a calibration cu rve with cholesterol as the internal standard.

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78 The calibration curve was gene rated by plotting the peak ar ea ratio of the standards ergosterol and lanosterol to the p eak area of cholesterol against the concentration in picomolar (pM) of each standard. The concentrations of ergosterol and lanosterol in the samples were determined from the calibration curve, and expressed in picomolar (pM). Analysis of Total and Free Ergosterol by High Performance Liquid ChromatographyAtmospheric Pressure Chemical I onizati onMass Spectrometry (HPLC-APCI-MS) HPLC-APCI-MS analysis of sterols was done (i n conjunction with Dr. Cristina Dancel at the Mass Spectrometry laboratory, department of Chemistry, University of Florida), using an Agilent 1200 Series HPLC System e quipped with a Phenomenex Luna C18 column (150 x 2.0 mm, 5 ). Sterol extracts were dissolv ed in 9:1 isopropanol-hexane and 5 L of each solution was injected. Methanol was used as the mobile phase in isocratic mode and at a flow rate of 0.4 mL/min. Ions were obtained by atmospheric chemical ionization (APCI) and detected with an Agilent 6210 time-of-flight (TOF) mass spectrom eter. For semi-quantitation, and quantitation analyses, a stock solution of 25 M ergosterol was prepared in 9: 1, v/v isopropanol : hexane, and diluted with methanol before injection. Semi-qua ntifitation of total and fr ee ergosterol was based on comparing the peak areas of the sterol chromatograms as follows. The ratio of peak area for ergosterol to peak area for cholesterol was calcula ted, and normalizing to OD600 of 1.0. Free ergosterol quantitation was based on the response factor method and using c holesterol as internal standard. Results IZH Genes Affect the Tolerance to the Antifun gal Nystatin We have observed that single and multiple deletions of the IZH genes grow with wild type characteristics (Figure 4-3 A) indicating that none of these genes is required for viability. However, under exposure to the antifungal drug nysta tin, defects in the grow th were observed for

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79 the wild type BY4742 and for all the izh mutant strains except for izh3which displayed a remarkably resistant phenotype under these conditions (Figure 4-3). In order to investig ate if the resistant phenotype observed for izh3 was not due to side mutations during the period of incubation with nystatin, complementation studies were performed. If izh3 is in fact resistant to nystati n, we would expect that when the IZH3 gene is re-introduced into the izh3 mutant strain, the sensitivity to ny statin must rescue. In order to investigate this, the centr omeric plasmid pRS315CEN (empty vector), and the pRS315CEN plasmid vector containing the IZH3 gene driven by its own promoter were introduced into the izh3 strain by yeast transformation. Like wise, the effect of overexpressing IZH3 gene in presence of nystatin was also investigated by introducing the pRS316GAL1 plasmid vector or the pRS316 -GAL1 containing the IZH3 gene into the izh3 by the same procedure. In both cases, IZH3 restored the wild type nystatin -growth defect in the resistant strain (Figures 4-3 B and 4-3 C). In addition, when the all the IZH1, IZH2, and IZH4 genes and the vector control (pRS316GAL1) were overexpressed in a medium supplemen ted with 2.5 and 5 U/mL nystatin, the cells grew better. Interesti ngly under these conditions IZH3 showed defects in its growth. At a higher concentration of nystatin (e.g.10 U/ mL) cells did not survive (Figure 4-3 D). These latter results confirm that the presence of the IZH3 gene produces sensitivity to nystatin and that the presence of IZH1 IZH2 and IZH4 has the opposite effect conf erring resistence to nystatin. Another important aspect was inve stigating if the resistance of izh3 to nystatin occurred at a particular stage of growth, or if it was independent on the stat e of growth. In yeast, a growth curve has different stages, which can be mon itored by measuring the op tical density of the growth cultures (OD600) (Figure 4-4 A). To investigate if th e effect of nystatin was dependent of the cell growth stage, phe notypic studies of WT and izh3 were analyzed in presence of nystatin,

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80 at different OD600 [e.g. 0.25 and 0.5 (logarithmic phase) and 3.4 (stationary phase)]. Interestingly, izh3 always showed resistance to nystatin independently of the stage of growth (Figure 4-4 B). Nystatin Induces Alterations in the Total Sterol Composition of Wild Type and izh3 Because nystatin preferentially targets sterols in the plasma membrane, we envisioned the possibility that ce lls harboring the izh3 mutation shows alterations in the sterol composition or the sterol content, and therefore that the IZH3 gene is required to main tain the appropriate levels of sterols in the membranes. We explored this possibility by determining the sterol content in both wild type and izh3. To do this, ultraviolet spectrophotometry was used as a first approach. This technique takes advantage of the unique spec tral absorption patterns of ergosterol and its precursor 24(28)-DHE. In fact, both sterols have a characteris tic UV spectrum with three peaks at 271, 281, and 293 nm, with a maximum at 281 nm (Figure 4-5 A). In addition, 24(28)-DHE has a side-chain diene that re sults in absorptions at 230, a nd 235 nm (Figure 4-5 B). The UV spectra of the sterol extr acts of wild type and the izh3 indicate that these strains have the same sterol profile. In addition a p eak at 205 nm was also identifie d, which corresponds to squalene (Figures 4-5 A and B). In absence of nystatin, the c ontent of ergosterol and 24(28)-DHE was the same (Figure 4-6 A), although, the levels of 24(28)-DHE were ve ry low (< 0.2%). Interestingly, upon addition of of nystatin, 24(28)-DHE seems to be accumula ted (Figure 4-6 B). On the other hand no significant difference was observed for the levels of ergosterol (Figure 4-6 B). These results suggest that nystatin induces the production of precursors of ergosterol. Unfortunately, UV-VIS did not allow us to identify sterols other rath er than squalene, ergosterol and 24(28)-DHE.

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81 Gas Chromatography-Mass Spectrometric (GCMS) Analysis Revealed not Significant Differences in the Basal Levels of T otal Sterols for Wild Type and izh3 Even though, UV spectrophotometry has been widely used for the analysis of sterols that contain the 5,7diene systems, identification of other sterols is outside the capability of this technique (Woods, 1971). Therefore, GC-MS was used as an alternative method not only to validate the UV-spectrophotometric re sults but also to characterize other sterols and to quantify them by using standards. Before analysis, samples had to be derivatiz ed by silylation with BSTFA containing 1.0% TMCS. The resultant silyla ted ethers were then suitable for electron ionization and mass spectrometric analysis. A ca libration curves using er gosterol and lanosterol allowed to quantitfy the content of these ster ols in the samples analyzed (Figure 4-7 A) (Appendix C). Interestingly, the data generated with this technique suggest that both wild type and izh3 have the same basal levels of ergosterol and lanosterol (Figur e 4-7 B). Since 24(28)DHE is not commertially availa ble, we could not quantify th is sterol in our samples. Alterations in the Free Ergosterol Co ntent Were Observ ed for the Mutant izh3 Ergosterol can be present in tw o forms, forming esters with fatty acids (steryl ergosterol) and as free ergosterol. As mentioned elsewhere in this chapter, free ergos terol is primarily found in plasma membrane where it play s a structural role, contributing to maintain the integrity of the plasma membrane (Veen et al., 2003). To investigate the total and free ergosterol content in wild type and the mutant izh3, HPLC-APCI-MS was used. One advantage of using HPLC-APCI-MS over GC-MS is that the first one does not require pre-treatment of the samples (or derivatization), which is time consuming, and can affect the result s if the dreivatixation is incomplete. The use of HLPC-MS let us establish significant conclusions regarding the er gosterol content of izh3versus that one of wild type. First, we c onfirmed that the total ergosterol content was

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82 similar for both samples (Figure 4-8 A).This obs ervation was in agreetment with the results obtained with UV-spectrophotometry and GC-MS analysis. Because ergosterol is the most abundant sterol in yeast, and a well known-target for nystatin, we hypothesize that the izh3s plasma membrane has alterations in the ergosterol content. Plasma membrane has been proposed to be the place where nystatin makes a first contact (Sharma, 2006). Since fr ee ergosterol is predominantly found in plasma membrane, we reasoned that izh3 could have less free ergosterol than wi ld type; and consequently nystatin would have less chance to be deleterous for the mutant yeast membrane. To test this hypothesis, we first semi-quantifed the free ergosterol content of izh3and wild type, by using HPLC-APCIMS. As we expected, the content of free ergosterol in the mutant was dramatically diminished (Figure 4-8 B). In an effort to quantify free ergosterol in both WT (pRS315CEN-IZH3 ) and the mutant izh3(pRS315CEN transformed with a izh3 strain) single standard quantitation method was used. To do this, the peak area for ergosterols m/z 379.3, was normalized to the peak area corresponding to cholesterol at m/z 369.3 (internal standard). Concen tration of ergosterol in each sample was calculated using a 3.75M standard solution, and normalized to OD600 (For each experiment OD600 was 1.0, see materials and methods for more details). Free ergosterol content is reported in M. This method allowed us to quantify free ergosterol and demonstrate that izh3 has altered sterol composition compared to wild type, (Figure 4-8 C) (Appendix D). Our results not only support our hypothesis, but also, suggest an implication of IZH3 in the sterol biosynthetic pathway.

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83 Addition of Certain Sphingolipid s Ameliorate the Aberrant Nyst atin Effects on Wild Type and izh Mutants In addition to sterols, sphingolipids are othe r important components of plasma membranes (Eisenkolb et al., 2002). Kerridge, 1986 and Leber et al., 1997 suggested that besides sterols, certain sphingolipids like the mannosyl-diionos ytolphosphorylceramide, the most abundant sphingolipid if S. cerevisiae, are also be targets for nystatin. With this in mind, we investigated the effect on the growth of wild type and the single, and multiple izh mutations upon the simultaneous addition of nystatin and sphingolipids to the growth culture. To do this, phenotypic studies in solid agar-synther ic medium were conducted. Interestingly, the sphingoid bases C18PHS and C18-DHS were able to alleviate the toxic eff ects of nystatin (Figure 4-9). In fact, in a medium supplemented with any of the sphingoid bases (e.g. C18-PHS or C18-DHS) and nystatin, the growth of wild ype and the single and multiple izh mutations was normal (Figure 4-9). On the other hand, ceramides and stearylamine (a s phingolipid-like compound) fa iled to alleviate the toxic effects of nystatin (Figur e 4-10 A and B, respectively). Taken together, these results confirm our idea that sphingolipid s and sterols like ergosterol c ould be potential ligands for nystatin. Discussion In this chapter, we have identified izh 3 as a novel nystatin-resist ant mutant. Even more interesting, we have discovered this phenotype is associated with al terations in the sterol content for the mutant strain. This finding suggests a potentia l implication of IZH3 gene in sterol metabolism. We first, have shown that the growth of izh3, upon addition of nystatin, was normal compared to the defects in the growth observed for the wild type strain and the other single and multiple mutation strains (Figure 4-3 A). The discovery that IZH3 rescues the sensitivity of the

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84 cell to nystatin, suggests that the obser ved nystatin resistant phenotype for izh3 is not due to side-mutations and that is in fact, the mutation of IZH3 per se, which produces such a phenotype (Figure 4-3 B, C, and D). Additionall y, we found that the overexpression of IZH1 IZH2 and IZH4 but not IZH3 ameliorate the toxic effect of nys tatin (in a concentration-dependent manner), with the concomitant resistant phenotype (Figure 4-3 D). Another important discovery was that the resistant of izh3 to nystain is independent on th e growth stage (Figure 4-4 B). Based on this result we could speculate that the izh3 plasma membrane has a lipidic composition that is perhaps different from the other izh mutants and the wild type. Thus, experiments that elucidate th e lipidic composition of the izh3 plasma membrane can generate valuable information that contributes to understand the izh3 resistant phenotype. Because nystatin preferentially interacts with ergosterol (Ghannoum and Rice, 1999; Sharma, 2006), this lipid became the first candidate to investigate. Also, we contemplated the possibility that IZH3 could have a role in the sterol bios ynthetic pathway. It is known that upon mutation, genes that are implicated in the sterol pathway have alterations in the sterol composition or sterol co ntent (Arthinggton-Skaggs et al., 1996; Baudry et al., 2001). Therefore, as a first approach to justify the observed phenot ypes, we used different analytical techniques like UV-spectrophotometry, and gas-chromatogra phy/mass spectrometry (GC-MS), to analyze total sterol composition and content in wild type and izh3 differences in the total sterol profile and content of both samples were de tected. However, upon addition of nystatin, alterations in the sterol composition of both strains were observed by UV-spectrophotometry. For instance, while an increase in the levels of the intermediate 24(28)-DHE was detected no significant changes in the ergostero l content were observed with al l of the techniques used during the analysis. This result confirms the specificity between nystatin and ergosterol.

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85 Free ergosterol, and esterified ergosterol a ccounts for total ergoste rol in yeast. Free ergosterol is accumulated at high concen tration in plasma membrane (Zinser et al., 1993), being a suitable target for nysta tin. We hypothesized that izh3 is resistant to nystatin due to alterations in the free ergosterol content. We explored th is possibility using HP LC-MS analysis of free ergosterol versus total ergosterol. The advantage of using HPLC-M S is that it does not require derivatization like the GC-MS technique. Interestingly, we found that izh3 is more deficient in free ergosterol than its wild type counterpart. This result truly explains the resistance phenotypes observed by this mutant. On the other hand, the leve ls of total ergosterol a ppear almost identical for both the mutant and wild t ype. It is quite possible that izh3 has more esterified ergosterol than wild type. Theref ore, the presence of IZH3 is necessary to maintain the levels of free ergosterol in the plasma membranes. Besides sterols, the effects of an tifungals like azoles are also associated with defects in the sphingolipid content. For example, genes involved on the synthesis of phytosphingosine ( SUR2 or LCB1 ), are down-regulated by azole drugs (V een and Lang, 2005). Our observations that sphingoid bases reconstitute the normal growth of wild type and mutants seem reasonable. We do not discard the the possibility that once adde d to the growth media, the sphingoid bases are embedded in the plasma membrane protecting it agai nst the toxic effects of nystatin. It is also possible that when added simultaneously to the cells, nystatin competes for a place in plasma membrane with the sphingoid bases. Therefore, experiments conducted to test these possibilities are necessary. Based on our results, it is important to inve stigate the transcriptional response of the IZH3 gene in order to have a better id ea about the role, if any, that this gene plays in the sterol pathway. Overall, the results presented in this chapter open an avenue regarding the role of IZH3

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86 that demands further investigation. For exampl e, the steryl ester composition of WT and izh3 must be investigated. It is plausible, that izh3 has more steryl esters than the WT strain, which could explain why the mutant strain has less fr ee ergosterol available for the nystatin action.

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87 Table 4-1. Sources and genotypes of the strains used in Chapter 4 Strain Mutation Source/Derivation Genotype BY4742 Wild type EUROSCARF MAT ; his3; leu2; ura3; lys2 TLY9 izh1 Thomas Lyons MAT ; his3;leu2; ura3;lys2; izh1::kanMX4 TLY50 izh2 Thomas Lyons MAT a ; his3; leu2; ura3; lys2; izh2::hphMX4 Y01578 izh3 Thomas Lyons MAT a; his3; leu2; ura3; met15; lys2; izh3::KanMX4 TLY46 izh4 Thomas Lyons MAT ; his3; leu2; ura3; lys2; izh4::ura3MAX4 TLY20 izh1izh2 Thomas Lyons MAT ; his3; leu2; ura3; lys2; izh1:: kanMX4; izh2:: hphMX4 TLY41 izh2izh4 Thomas Lyons MAT a; his3; leu2; ura3;lys2; izh2:: hphMX4; izh4:: ura3MAX4 TLY45 izh1izh2izh4 Thomas Lyons MAT a; his3; leu2; ura3;lys2; izh1:: KanMX4; izh2:: hphMX4; izh4 ::ura3MX4 TLY23 izh1izh2izh3izh4 Thomas Lyons MAT ; his3; leu2; ura3; lys2; izh1:: KanMX4; izh2:: hphMX4; izh3:: natMX4; izh4::ura3MX4

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88 HO CH3H3C H3C CH3 H3C H3C CH3CH3H3C H3C H3C CH3CH3CH3H3C H3C HO CH3CH3H3C CH2H3C CH3CH3H3C CH3H3C HO HO SqualeneLanosterol24(28)-Dehydroergosterol Ergosterol Choleste r ol Figure 4-1. Chemical structures of th e sterols analyzed in this chapter.

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89 CH3O O H3C HO OH OHOH O O OH HO NH2O OH OH COOH H3C OH OHNystatinCH3C Figure 4-2. Model proposed for the nystatin acti on in the yeast plasma membrane. A shows an intact yeast plasma membrane. B the in teraction between nystatin and ergosterol produces the formation of holes in the plas ma membrane with the consequent leakage of nutrients. C shows the chem ical structure of nystatin.

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90 Figure 4-3. Nystatin-dependent phenotypes. A shows the growth of wild type and the izh mutant strains on synthetic medium agar pl ates supplemented with +/nystatin. B and C illustrate the complementation of the nystatin-dependent phenotype by the pRS315-CEN plasmid and pRS316GAL1 repectively. In B, the single copy plasmid (pRS315), or pRS315 expressing IZH3 was transformed into the izh3strain. In panel C, the plasmid containing the pRS316GAL1 or expressing IZH3, was transformed into the izh3strain. D shows the effect of different concentration of nystatin onto the growth of cells overexpressing IZHs In this case the pRS316GAL1 containing each IZH was tested. When CEN plasmid was used plates were supplemented with 2% glucose. For gene overexpression, 2% galactose was used.

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91 Figure 4-4. Effect of nystatin on izh3 and the wild type strain BY4742 at different stages of growth. Panel A shows a typic growth curv e generated to illustrate the different growth stages of both WT and izh3. B shows that izh3 is remarkable resistant to nystatin regardless its growth stage.

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92 Figure 4-5. Ultraviolet spectrophotometric characterization of total 5-7 sterols in wild type strain and izh3. A represents the UV spectra for WT and izh3 obtained in absence of nystatin. B shows the UV spectra for WT and izh3, when the cells are exposed to 5 U/mL of nystatin. Figure 4-6. Semi-quantitation of total ergosterol a nd 24(28)-DHE content in wild type and izh3 by UV-spectrophotometry. A shows the basal levels of ergosterol and 24(28)-DHE. B shows the levels of both st erols in cells exposed to 5 U/mL of nystatin. In the figure, the content of each sterol is expresse d as the percentage of total ergosterol per wet weight of cells.

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93 Figure 4-7. Quantitation of basal levels of total ergosterol and lanosterol of WT and izh3by GC-MS. In the figure, A shows a calibra tion curve generated with the standards ergosterol, lanosterol, and c holesterol (internal standard). B shows the concentration (pM) of total ergosterol and la noterol derived form the calib ration curve. Data are the mean standard deviation of three experiments.

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94 A Peak area (Total Erg / Chol) / OD 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 B 0 2 4 6 8 10 Peak area (Free Erg / Chol) / OD 600WT izh3WT izh3 Figure 4-8. Total and free ergosterol content on WT and izh3. The figure shows the semiquantitative analysis of total ergosterol, A and free ergosterol B, using HPLC-APCIMS. C shows the quantitation analys is of free ergosterol for WT and izh3 by using HPLC-APCI-MS. Data shown in each panel represent the mean standard deviation of two independent experiments.

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95 Figure 4-9. Sphingoid bases overrid e the toxic effects of nystatin. To generate the figure, 5 l of 10-fold OD600 serial dilutions were spotted on ag ar-simthetic medium (e.g. from the left to the right OD600 of 1.0, 0.1, and 0.01, respectively) and incubated for 3 days at 30oC.

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96 Figure 4-10. Effect of ceramides and stearylamine in cells exposed to nystatin. The figure shows that contrary to the effect of sphingoid bases, cer amides and the sphingoid base homolog stearylamine, are unable to amelio rate the toxic effect of nystatin.

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97 CHAPTER 5 POTENTIAL IMPLICATION OF IZHS IN THE SPHINGOL IPID BIOSYNTHETIC PATHWAY Introduction Sphingolipids are a group of ubi quitous lipids f ound in all e ukaryotic cells where they comprise 10% to 20% of the total lip id species found in membranes (Smith et al., 1974). Structurally, sphingolipids are formed by three elements; the sphingoid base backbone (or long chain base), a very long chain fa tty acid, and a head group. In yeas t, fatty acids with a chain of 26 carbons in length, is common (Les ter and Dickson, 1993). The yeast Saccharomyces cerevisiae, has only three complex sphingolipids named inositol phosphoryl ceramide (IPC), mannosyl inositol phosphorylceramide (MIPC), and mannosyl diinositolphosphoryl ceramide, (M(IP)2C) (Dickson, 1998). The yeast S. cerevisiae has served as unique model to dissect and understand metabolic pathways and has emerged as a scaffold upon which sphingolipid metabolism and function can be elucidated. Originally, sphingolipids were considered only as structural components whose main role was to protect the cell surface against potential harsh and hostile environments (Hannun and Bell, 1989; Meer et al., 2002). In fact, in plasma membranes, sphingolipids are found tightly packed with sterols forming microdoma ins termed lipid rafts (Toulmay et al., 2007). Recent studies have demonstrated that the role of the sphingolipids goes beyond a si mple structural role, and that they can also play roles as second messe ngers implicated in signa l transduction. In this sense, sphingolipids can mediate cellular differentiation and apoptosis (Merrill et al., 1997; Edsall et al., 1997; Hannun and Obeid, 2002; Di ckson and Lester, 2002). Multiple sequence alignments as well as phylogene tic tree analysis have revealed that the Izhp family shares distant homology with a grou p of enzymes known as alkaline ceramidases. Ceramidases hydrolyze the amide linkage of ceram ide to generate a free sphingoid base and the

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98 corresponding fatty acid (Figure 5-1). In yeast, two alkaline ceramidas es (Ypc1p and Ydc1p) have been characterized (Mao et al., 2000a; Mao et al., 200b). Interestingly, Ypc1p and Ydc1p also catalyze the reverse ceram idase reaction with the concomitant formation of phytoceramide and dihydroceramide from free sphingoid bases and fatty acids (Mao et al., 2000a; Mao et al., 2000b; Valle et al., 2005), (Figure 5-2). Th is latter reaction is us ually catalyzed by two ceramide synthases called Lag1p and Lac1p (Valle et al., 2005). Different observations have highlig hted the possibility that the IZHs could be implicated in the sphingolipid biosynthetic pathway. Herein, we present evidence that indicates that the overexpression of the IZHs results in an increas e in the levels of s phingoid bases and certain ceramides. However, in vitro ceramidase assays suggest, that Izhs are not alkaline ceramidases. Alternatively, we investigate the possibility that the IZHs may modulate the levels of sphingolipids by affecting the de novo sphingolipid biosynthesis. As is well known, sphingoid bases can be synthesized de novo as follows. In this case, serine is condensed with palmitoylCoA via serine palmitoyl transferase (SPT), to form 3-keto-dehydrosphingosine, which by successive reactions is converted to the sphingoid bases dehydrosphingosine and phytosphingosine (Perry, 2002; Menalindo et al., 2003; Cowart and Obeid, 2007) (Figure 5-3 A). In an effort to investigate if the IZHs could be implicated in de novo sphingolipid biosynthesis, three different experi mental approaches are explored. First, qualitative analysis of radioactive sphingolipids indi cates that the levels of sphingoid bases produced under IZH overexpression are bigger than thos e produced by the vector contro l, but similar to the levels produced when YPC1 is overexpressed. Second, the use of my riocin, an inhibitor that blocks de novo biosynthesis of sphingoid base s via serine palmitoyl transfer ase, significantly reduces the levels of sphingoid bases produced when the Iz hs are overexpressed. Howe ver, the effects of

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99 myriocin on the levels of sphingoid bases found when YPC1 was overexpressed and those in the vector control are barely affected. Finally, the effect of fumonisin B1 (FB1), an inhibitor that inhibits ceramide synthase, was investigated. Our results indicate that after treatment with this drug, an accumulation of sphingoid bases in all th e samples analyzed is produced, suggeting that the role of Izhs is more related to the de novo sphingolid biosynthesis. Although, still preliminary, our results suggest a connection between the levels of sphingolipids (specifically sphingoid bases and ceramides) and the high dosage of IZHs in the cell. Materials and Methods Strains Plasmids and Yeast Transformations For the stud ies described in this chapter, the strains and their respective genotypes are described in table 5-1. The YPC1 and IZH1-4 open reading frames were placed under the control of galactose inducible GAL1 promoter, by using the plasmid v ector pRS316, which contained the URA selection marker gene. The pRS31 6-GAL1 (empty vector) and th e vector containing the YPC1 and each of the IZHs (pRS316-GAL1-YPC1 and, pRS316-GAL1IZH1-4, respectively) were transformed with the wild type strain BY4742, or with the double mutant ypc1ydc1 (Gietz and Woods, 1994). The double mutant strain ypc1ydc1was kindly provided by Dr. Howard Riezman (howard.riezman@biochem.unige.ch). Yeast Growth Conditions Unless otherwise stated, the com pounds used fo r these studies were dissolved in either 100% ethanol, or DMSO. When mentioned, the dete rgent Tergitol NP-40 was used to facilitate the uptake of certain sphingolipids by the cells. At low percentage, this de tergent does not have any significant effect on the growth of cells. The inhibito rs, myriocin and fumonisin B1 (FB1) were obtained from Sigma.

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100 For gene overexpre ssion, cells lacking YPC1 and YDC1 or wild type cells were transformed in identical fashion as mentioned elsewhere in this diss ertation. When used, Nacetylphytosphingosine (or C2-PHC), (Avanti Polar Lipids), wa s prepared in 100% ethanol. Cells in early logarithmic phase, (e.g. OD600 of 0.2) were spiked with C2-PHC to a final concentration of 278 nM, and grown to OD600 was 0.8 (usually for 1 h). When fumonisin B1 (FB1) was used, cells were prepared according to the procedure described by Wu et al., 1995 with some minor modificat ions. Cells were grown at 30oC in 30 mL of uridine-limited synthetic medium (SGalUri), supplemented with 0.005% Tergitol NP-40 (Sigma); in the absence or in presence FB1 to 100 M. Cells were grown to OD600 of 0.8 and then harvested to collect pellets. Likewise, when myriocin was used, cells we re grown in 30 mL of synthetic medium (SGal-Uri), in the absence and in presence of 1.0 M myriocin were grown to OD600 of 0.8. Cells were harvested and resultant pellets were washed twice with cool sterile water and used for total lipid extraction. In Vitro Ceramidase A ssay Microsomes were isolated from the yeast cells according to Mao et al., 2000a; Mao et al., 2000b with some minor modifications as follows. Ce lls were suspended in 0.5 mL of lysis buffer A (20 mM Tris-HCl, pH 7.4, 1.0 mM EDTA, and the protease inhibitor mix form Sigma). Acidwashed glass beads were added to just below the meniscus (1/3 beads per every 1/3 of cell pellets). Cells were homogenized thre e times (3 min for 30 sec), and at 4oC using a Mini-beadbeater-8 (Biospec Products) set at the maximum speed. The cells were chilled on ice for 1 min between homogenizations. The re sultant supernatant was transf erred to a new tube after centrifugation at 2,000 rpm for 10 min. Unbroken cells and cell debris were removed by

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101 centrifugation at 4,000 rpm for 10 min. To pellet the membrane fraction, the supernatant was centrifuged at 40,000 rpm for 40 min at 4oC. The membrane fraction was rinsed gently with the lysis buffer A and suspended in 100 L of the same buffer A. Protein concentration was determined by the BCA kit (Pierce), and using BSA as standard. Ceramidase activity was determined by the re lease of NBD-fatty acid from fluorescent substrates, NBD-C12-ceramide, NBD-C12-dihydroceramide (Matreya) and NBD-C12phytoceramide (kindly provi ded by Dr Yusuf A. Hannun, hannun@musc.edu) described by Mao et al., 2003 with som e modifications. Briefly, 20 L of microsomes (containing 10 g of protein) in buffer B (25 mM Tris-HCl, 0.5 mM CaCl2at three different pHs, 8.6, 9.4, and 7.0) was mixed with 20 L of each substrate in buffer B with 0.4% Nonidet P-40 in a 1.5 mL microfuge tube. After incubation at 30oC for 60 min, the reactions were stoppe d by boiling for 5 min, and dried in a heating block at 80oC during 20 min in the dark. The C12-NBD-dodecanoic acid, (generously provided by Dr. Yusuf. A. Hannun) was used as marker for the completion of the ceramidase reaction. Additionally, this fluorescent fatty acid in one of the products released from the catalytic breakdown of NBD-C12-sphingolipids by the ceramidase s. Reaction mixtures were dissolved in 30 L of chloroform : methanol (2:1, v/v). 25 L of each sample, the substrates and the NBD-dodecanoic acid was applie d onto a silica gel 60 TLC pl ate (Whatman) and resolved by the solvent system composed by chloro form : methanol: 4.2 N ammonium hydroxide (90:30:0.5, v/v/v). Fluorescent lip ids were detected by scanning the TLC plate in a StormTM 860 chromatoscanner (Molecular Dinamics), operate d in the fluorescent mode, with an excitation wavelength of 450 nm and an emission of wavelength of 525 nm.

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102 Phenotypic Studies Yeast cells where the YP C1 and YDC1 genes were mutated ( ypc1ydc1), and containing the pRS316GAL1 (empty vector) or overexpressing each IZH gene or YPC1 were first grown to stationary phase. After spinning down, pellets were suspended in ur idine-limiting synthetic medium supplemented with galactose, a nd appropriated amino acids. Aliquots (5 L) of the overnight were plated at OD600 of 1.0, 0.1, and 0.01, onto solid-agar uridine-limiting synthetic medium supplemented galactose, and appropriate amino acids, with or without myriocin. When used, myriocin was prepared in DMSO (Sigma), and added fresh to solid-agar medium to concentrations of 0.1 M and 1.0 M. Plates were incubated at 30oC during 3 days, and followed by growth analysis. Total Lipid Extraction Unless otherwise stated, all reag ents used during this proced ure were of high quality and the solv ents used were HPLC grade. Total lipi d extraction was performe d according to the BlighDyer method (Bligh and Dyer, 1959), with some modifications. Briefly, pe llets were suspended in 0.8 mL of water, and 3.0 mL of a mixture of methanol : chloro form (2:1, v/v). After vortexing thoroughly, suspensions were le ft during 1 overnight, at 4oC to permeate the cell wall and the yeast plasma membrane. Cell debris was discar ded by centrifugation at 3,000 rpm for10 min, and the supernatant was transferred to a sterile 13 X 10 mm glass sc rew capped borosilicated tubes (Fisher). Supernatant was then mixed with 1.0 mL of chloroform and 1.0 mL of water and vortexed vigorously for 1 min. The resultant su spension was settled down during 30 min at room temperature, followed by centrifugation at 3,000 rp m for 10 min to help the separation of two phases. The aqueous phase was aspirated, and th e organic one was transferred to a new 13 X 10 mm glass screw capped borosilicated t ube, and dried under a stream of N2. Dried fraction was

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103 suspended in 300 L chloroform. One-third of this suspension (100 L) was dried and used to determine phospholipids (this procedure is descri bed in the next section). The remaining twothird fraction, (200 L) were suspended in 800 L of a 0.125 M methanolic KOH solution and incubated during 75 min in a 37oC water bath to hydrolyze the acy l glycerolipids. After cooling at room temperature, 1.4 mL of chloroform and 200 L 0.3 M HCl were added to the mixture, followed by the addition of 400 L of a solution of 1.0 M NaCl in 5% glycerol. Lipids were extracted by vortexing vigorously during 3 min. Centrifugation at 3,000 rpm, during 5 min allowed the formation of two pha ses. Again, the aqueous phase was aspirated and the organic one was washed with 1 mL of neutralized water (prepared by mixing 1.0 M ammonium hydroxide and water at a ratio of 1:300, v/v). After aspirating the aqueous phase, the organic phase, which is enriched of sphingol ipids was dried under a stream of N2, and stored at -20C until HLPC analysis. Phospholipid Determination Phospholipid determ ination from the lip id extracts was performed, with some modifications according to Merrill et al., 1988. Briefly, standards ranging from 0-80 nmol of N2HPO4 (e.g. 0, 5, 10, 20, 40, 60, 80 L 1.0 mM NaHPO4) and one-third of the dried lipid fraction (as described in former section) were mixed in 600 L of ashing buffer prepared by mixing 10 N H2SO4, 70% perchloric acid, and water (1 :9:40, v/v/v), and heated at 160oC during 1 overnight (18 h). After cooling at room temperature, the lipid fraction was mixed, by vortexing, with 900 L of sterile water, 500 L of 0.9% (w/v) ammonium molybdate (Fisher) and 200 L of a fresh solution of 9%, w/v L-ascorbic acid (Sigma). Standards and samples were incubated during 30 min in a 45oCwater bath. Absorbances were measured at 820 nm using a Safire Xluor4D microplate read er (version 4.50). A standard ca libration curve was generated by

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104 plotting the nmol of the standa rds against their ab sorbance at 820 nm. The obtained curve was used to calculate the nmol of phos pholipids present in each sample. High-Performance Liquid Chromatography Analysis of O -Phthalaldehyde-Sphingoid Base Derivativ es Standards used in this section were obtained from Avanti Polar Lipids. Extracts containing sphingolipids and the standards Deryhtro -C18-DHS, Dribo-C18-PHS, C20-PHS, and L -threo C18-DHS, were derived according to Merrill et al., 2000 with some minor modifications, and by using the fluorescent reagent O -phthaladehyde (OPA), (Sigma). The following protocol was performed by Charlene Alford, (Medical Univers ity of South Carolina, MUSC) who also ran the samples in the HPLC instrument. The two-third dried lipid fractions were first dissolved in 100 L of methanol. FiftyL (50 L) of the standards and sample s dissolved in methanol were mixed with 50 L of the OPA reagent for derivatizati on. After thoroughly mixing, the mixtures were incubated for 20 min under darkness, and at room temperature. The samples and the standards were then centrifuged briefly to clarify and kept at 4oC until HPLC analysis. The OPA reagent was prepared by mi xing 5 mg of OPA in 100 L 100% ethanol, with 5 L of 2mercaptoethanol and 9.9 mL of a solution of 3%, w/v boric acid (H3BO3), which was prepared in water, and adjusting its pH to 10.5 with KOH. Before HPLC analysis samples and standards were spiked with 25 pmol of the internal standard Lthreo -C18-DHS prepared in methanol (Lthreo -C18-DHS is a non natural sphingoid base). Fluor escent derivatives were analyzed using a C18 analytical Ultrasphere column with an intern al diameter of 46 mm, and a length of 250 mm. The HPLC (Breeze system Waters binary HPLC model 1525) was coupled with a Shimadzu RF-551 spectrofluoremetric detect or (Beckman Coulter). The solv ent system was methanol : 5 mM potassium phosphate, pH 7.0, (90:10, v/v). The OPA derivatives were detected using the spectrofluoremetric detector, with an exci tation wavelength of 345 nm and an emission

PAGE 105

105 wavelength of 455 nm. The sphingoid bases were identified by comparing their elution profile with those of the standards. Analysis of Radiolabeled Sphingolipids by On e-Dimensional Thin Layer Chromatography As describe before, yeast cells where the YPC1 and YDC1 genes were m utated ( ypc1ydc1), and containing the pRS316-GAL1 (empty vector) or the pRS316-GAL1 expressing each IZH gene or YPC1 were first grown to statio nary phase. After spinning down, pellets were suspended in uridine-limiting synthe tic medium supplemented with galactose. Fresh synthetic medium supplemented with 2% galactos e and appropriate amino acids was inoculated with aliquots of the overnights to an OD600 of 0.1. Cells were to an OD600 of 0.5. Aliquots of 3.0 mL of each culture was transferred to 50 mL capped Falcon tubes and spiked with 150 L 1.0 mCi/mL [3H]serine (American Radiolabelled Ch emicals, ART-246) during 40 min at 30oC. Subsequently, 1.0 mL of each culture was tr ansferred to 13 X 10 mm glass screw capped borosilicate tubes and spun down at 3,000 rpm, 4oC for 5 min. After aspirating the supernatant, the pellet was washed with cold sterile water, and immediately used for lipid extraction. Lipid extraction was performed according to the Mandala procedure (Mandala et al., 1995). Briefly, pellets were suspended by vortexing in 1 .0 mL of the Mandala re agent [95% ethanol : water : diethylether : Pyridine : NH4OH, (15:15:5:1:0.018, v/v/v/v/v)]. Suspensions were then incubated in a 60oCshaking water bath for 15 min. After centrifugation at 3,000 rpm, 10oC for 5 min, the supernatant was pipeted off and save d. Pellets were re-suspended again in 500 L of the Mandala reagent, and extraction was repeated one more time. The tw o supernatants were combined and dried under a stream of N2. Dried samples were then suspended in 50 L of a mixture of chloroform : methanol : water, (1 :2:0.1, v/v/v) and vortexed hard for 15-20 sec.

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106 Sphingolipids were analyzed by one-dimensional chromatography (TLC), on Whatman Partisil LK6D silica gel 60 20 X 20 cm size thin layer thickness 250 m plates, (Whatman). To do this, aliquots of 25 L of each suspension and 10 L of the standards phytoceramide (PHC), dihydroceramide (DHC), phytosphingosin e (PHS), dihydrosphingosine (DHS), phosphatidylserine (PS), phosphatidyli nositol (PI), (type of standard s) were applied to the TLC pate, which was previously treated with 150 mL acetone for 45 min and equilibrated for 3 h in 100 mL of the solvent mixture choloroform : methanol : 4.2 N ammonium hydroxide, (9:7:2, v/v/v). Lipids were resolved by using the same mixture of solvents. When the level of solvent mixture reached 1 cm form the t op edge, the plate was taken out the chamber and allowed to dry at room temperature. The non-radioactive standard s were visualized in iodine vapor. Radioactive bands on the TLC plate were visualized by autoradiography after treatment with EN 3HANCE (NEN Life Science) and exposure to a tritium sc reen. Radiolabelled lipids were compared with the standards. Analysis of Sphingolipids by ElectrosprayIonization Tandem Mass Spectrometry (ESIMS/MS) All the experim ents described in this section were performed using the strain ypc1ydc1. Internal standards were either prepared by th e Lipidomics Core, at MUSC, or obtained from Avanti Polar Lipids or Matreya. All solv ents used were obtained from Fisher. Sphingolipid analysis was performed by HPLC-Tandem mass spectrometry (HPLC/MS/MS). High performance liquid chro matography (HPLC) was performed in a Surveyor equipped with a quaternary HPLC pump, Surveyor an autosampler, and a BDS Hypersil column (C8 150 x 3.2 mm; 3 m particle size), (Phenomenex). The HPLC system was coupled with a triple quadrupole mass spectrome ter equipped with a Thermo Finigan, PE Sciex

PAGE 107

107 Electrospray Ion Source (EIS), a syringe pump, and syringes (5 L-1 L) and a nitrogen generator (Parker Hannifin Corp). Cells containing the plasmid vector (pRS316GAL1 ) or the vector containing each of the genes YPC1 IZH2 and IZH3 (pRS316GAL1 YPC1 pRS316GAL1 IZH2 pRS316-GAL1IZH3 ) were grown in synthetic media supplemented with galactose to OD600 = 0.8. Sample and standards preparations were carr ied out in the lipid analysis we re prepared by the Lipidomics Core, at the Medical University of South Carolina (MUSC). Total lipid extractions were performed according to Bielawski et al., 2006 with some modifications. A mixture of the following internal standards was used, 1.0 M solution containing C17-sphingosine, C17sphingosine-1-P, 17C16-Ceramide, and 18C17-Ceramide and C6-phytoceramide in methanol were used. Pellets were first spiked with 50 L of the internal standards solutions, and then mixed by vortexing with 2.0 mL of the extraction mixture iso-propanol : water : etha nol : pyridine : 25% ammonia, (5.0:1.5:1.0:0.2:0.04, v/v/v/v/v). Extracts were incubated at 60oC for 15 min and then vortexed thoroughly. Suspensions were centrifuged for 5 min at 3000 rpm, and the supernatant was collected. Pellets were suspended in another 2 mL of the extr action mixture and the procedure was repeated. The two supernatants we re combines and dried under a stream of N2. Before analysis, extracts were suspended in 150 L of mobile phase (1.0 mM ammonium formate in methanol containing 0.2% formic aci d), and vortexed. Extracts were centrifuges at 4,000 rpm for 5 min, and the supernatant was transf erred to an autosampler HPLC vial with 200 L insert. 20 L of this supernatant was in jected on the HPLC system. For quantitation, calibration curves for each of the internal standard s were prepared as follows. Cells were substituted with bovine serum albumin (BSA). This artificial matrix was spiked with a known amount of the target analyt e (sphingolipid of intere st), and a constant

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108 amount of the corresponding internal standard. The above described extraction procedure was followed. Standard extracts were analyzed by HPLC/MS/MS system in positive MRM mode (Multiple Reaction Monitoring). Calibration curv es were generated by plotting the peak area ratio of analyte (sphingolipid of interest) / internal standard, against the concentration in pM of each sphingolipid of interest. For quatitation, the peak areas were determined using extracted ion chromatograms. The amount of each sphingol ipid detected was normalized to the OD600 of each sample; and the results are reported in pmol lipid/OD600. Results Overexpression of IZHs Produces Increase in the Levels of Free Sphingoid Bases Ypc1p and Ydc1p degrade ceram ides to pr oduce sphingoid bases a nd fatty acids (Mao et al., 2000a; Mao et al., 2000b). Because the Izhs have distant similarity with the yeast alkaline ceramidases, we questioned whether the IZHs could be implicated in the sphingolipid metabolic pathway. With this in mind, we first characterized and quantified the levels of sphingoid bases in cells overexpressing the IZH genes. To do this, HPLC analysis of fluorescent sphingolipid derivatives was performed. In a first trial, the pRS316GAL1 (empty vector) or the vector expressing IZH2 IZH3 and YPC1 were transformed with a BY4742 wild type strain. C18-PHS and C18-DHS (the two major sphingoid bases found in yeast) were detected by HPLC. Not surprisingly, an increase in the levels of these sphingoid bases was observed when IZH2 and IZH3 were overexpressed. But even more prominent was the fact that the overexpression of IZH3 produces more sphingoid bases than YPC1 (Figure 5-4). Although at first glance, we could envision the possibility that IZHs encode proteins that play sim ilar functions to ceramidases, we wanted to make sure that the results obtained we re not because we were using a wild type strain, which contain the ceramidase genes YPC1 and YDC1 Therefore, we decided to investigate the

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109 levels of sphingoid bases when the IZHs and YPC1 were overexpressed in the double mutant strain ypc1ydc1. With this in mind, two different experi ments were performed. First, the basal levels of sphingoid bases were investigated by HPLC. The results obtained indicated that the levels of the sphingoid bases C18-PHS, C20-PHS, and C18-DHS were increased, (Figure 5-5 A, B, C). To investigate if an external stimulus like the addition of a ceramide could activate the production of sphingoid bases in the an alyzed samples, the effect of C2-PHC was monitored by HPLC. Although C2-PHC is a non natural ceramide, it ha s the advantage that is less hydrophobic that the natural C26-PHC. Therefore, it can easily be deliv ered into the cells. When used, C2-PHC was added to a final concentration (278 nM) for a short period of time (e.g. 1 h) to avoid possible artifacts. An increase in the levels of sphingoid bases was observed for the IZHs and YPC1 (Figure 5-5 D, E, and F). Taken together, these results truly represent a first approach, and the first piece of evidence suggesting a possible implication of IZHs in the sphingolipid metabolic pathway. In Vitro Ceramidase A ssays Suggest that Izhs May not be Alkaline Ceramidases There are two ways to synthesize sphingoid bases. First, it can proceed by de novo sphingolipid biosynthesis, a process that starts with the condensation of serine and a fatty acid which is usually palmitic acid through the action of serine palmitoyl transferase (SPT). Second, sphingoid bases can be synthesi zed from hydrolysis of ceramide by the action of ceramidases (Figure 5-6 A). To investigate if the IZHs encode ceramidases, the vector pRS316 containing the GAL1 inducible promoter (pRS316GAL1 ) and the vector expressing the YPC1 and the IZHs (e.g. pRS316GAL1-YPC and pRS316GAL1-IZHs respectively) were transformed with the yeast double mutant strain ypc1ydc1. Protein expression was induced in 2% galactose. To do this, microsomes were prepared, as described under materials and methods, and assayed for

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110 ceramidase activity at different pHs (pH 8.6, pH 9.4, and pH 7.0). Flurorescent ceramides, NC12:0-NBD-phytoceramide, and N-C12:0-NBD-dihydr oceramide, were used as substrates for the ceramidases, and the N-C12:0-NBD-dodecanoic acid was used as a maker for the ceramidase reaction product (Figure 5-6). TLC analysis was performed to identify the florescent products. We did not observe any significant difference between the empty vector and the Izhs in terms of the intensity of the band corresponding to the fluo rescent fatty acid (Figure 5-7 A, B, C, and D). This suggests that Izhs are not ceramidases; howev er, it is possible that the substrates used for this assay were not appropriate. The f act that this band, only appears when YPC1 was overexpressed at alkaline pH (e .g. pH 9.4), indicates that under the conditions used only Ypc1p is acting as a ceramidase. Each experiment was carried out in duplicat e. Although, it is well known that the optimal pH for the alkaline ceramidase activity is in a range of 8 to 9, we wa nted to investigate if the pH had any effect on the enzymatic reaction. In this assay, the cata lytic action of the ceramidase produces the release of the fluorescent fatty acid. It is important to remember that C26-PHC or C26-DHC, are the natural substrates for alka line ceramidases. However, fluorescent C26ceramides are not commercially available. Thus, to do these experiments, we had to use artificial florescent substrates. This could be one of the reasons by which we did not obtain the expected results. The preliminary data presented in this section represent a va luable piece of information that suggests that Izhs may not be ceramidases. However, additional studies need to be conducted to rule out this idea.

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111 Thin Layer Chromatography of Radioactive Sphingolipids Reveals Si milar Sphingolipid Profiles for YPC 1 and the IZHs The altered levels of sphi ngoid bases observed when the IZHs were overexpressed may be due to a role for IZH genes in de novo sphingolipid biosynthesis. To test this, cells were labeled with [3H]serine during 40 min. In yeast, serine and palmitic acid are the two precursors of de novo sphingolipid biosynthesis (Cowart and Obeid, 2007) (Figure 5-3 A). After addition of [3H]serine to the growth medium, we expected its rapid incorpor ation into the cells followed by the efficient biosynthesis of labeled sphingolipids. Interestingly, when we analyzed the sphingolipid profiles by TLC, an increase on th e levels of PHS, DHS and other complex sphingolipids were observed when the IZHs and YPC1 were overexpressed compared to the vector control (Figure 5-8). These results suggest that de novo biosynthesis of sphingoid bases is induced under IZH and YPC1 overexpression, supporting our hypothe sis about the implication of the IZHs in this part of the biosynthetic pathway. Fumonisin B1 Induces the Acummulation of Sphingoid Bases in YPC1, IZH2, and IZH3 Fumonisin B1 is a mycotoxin that bears structural similarities to sphingoid bases (Wu et al., 1995), (Figure 5-3 B). It has been shown that in mammalian cells, as well as in yeast, FB1 promotes the accumulation of sphingoid bases by inhibiting the activity of the ceramide synthases Lac1p/Lag1p (Merrill et al., 1993; Wu et al., 1995) (Figure 5-3 A). To continue investigating the implication of Izhs in de novo biosynthesis of sphingoid bases, we decided to use FB1. When used, FB1 was added to a final concentration of 100 M to cells where the genes YPC1 and YDC1 were mutated and containing pRS316GAL1 (empty vector), or the vector containing YPC1 IZH2 or IZH3 Once exposed to FB1, cells were harvested and total lipids were extracted, and analyzed by HPLC as described under materials and methods. Some observations that are worth mentioning are the following: (i ) the addition of FB1 resu lted in the accumulation

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112 of sphingoid bases such as C18-PHS, C20-PHS, and C18-DHS (Figure 5-9 A, B, C), (ii) The vector control accumulated more C18-PHS and C20-PHS than YPC1 IZH2 and IZH3 (Figure 5-9 A and B, respectively). This result suggests that in YPC1 IZH2 and IZH3 the synthesis of such as sphingoid bases is somehow blocked under treatment with FB1. Myriocin Inhibits the Increase in the Levels of Sphingolipid Biosynthesis Mediated by IZH2 and IZH3 Myiocin is another po tent inhibitor of de novo sphingolipid biosynthetic pathway, which inhibits serine palm itoyl-CoA transferase (SPT), the rate-limiting enzyme of the sphingolipid pathway (Figure 5-3 A and B) (Fujita et al., 1994). We reasoned that if the IZHs are implicated in the synthesis of sphingolipids de novo the inhibition of SPT would suppress the increase of sphingoid bases produced when the IZHs are overexpressed. To test this, we first performed phenotypic studies to determine the minimal inhib itory concentration of myriocin. These studies indicated that 1 M myriocin had an inhibitory effect in the growth but, at that concentration, it was not lethal for the cells (Figure 5-10 A). Theref ore, cells grown in liqui d medium were treated with 1 M myriocin for 30 min. HPLC analysis only allowed the identification of C18-PHS, which is one of the most abundant sphingoid ba ses in yeast. Interes tingly, upon addition of myriocin, the levels of C18-PHS were noticeably reduced when IZH2 and IZH3 were overexpressed, compared to those detected under overespression of YPC1 and the empty vector (Figure 5-10 B). Therefore, our results buttress our hypothesis that IZHs contribute to modulate de novo sphingolipids biosynthesis. Finally, to analyze the effect of overexpr ession in the production of ceramide, HPLCTandem mass spectrometric analysis was performed. Our results indicate th at the overexpression of IZHs also has an effect in the synthesis of C26-PHC and C26-DHC, which are the most abundant ceramides in yeast (Figure 5-11, A and B). These latter results not only support our

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113 hypothesis about the im plication of the IZH genes in de novo sphingolipid biosynthesis, but also suggest that the IZH genes can be implicated in the s ynthesis of complex sphingolipids like ceramides. Taken together, the reults presented in this ch apter indicate that an in crease in the dosage of the IZH gene s results in consistent with increases in the levels of sphingoid bases and ceramides. In this sense, we envisioned the possibility that IZHs can be implicated in the biosynthesis of this sphingolipids de novo Discussion The prim ary objective of this chapter was to stablish a connection between the the IZHs and the sphingolipid biosynthetic pathway. Par ticularly, we set out to determine if the IZH family plays role(s) in the sphingolipid pathway. To th is end, biochemical studies were performed. One first observation that sugg ests an implication of IZHs in the sphingolipid pathway was increased levels of certain sphingolipids detected when the IZHs were overexpressed. To explain this observation, two different possibilities were explored. First of all, the IZH genes encode alkaline ceramidases. As mentioned before, ceramidases are enzymes that catalyze the deacylation of ceramide to produce a sphingoid base and a fatty aci d. Our hypothesis regardi ng the role of Izhs as possible ceramidases was genera ted based on the fact that the IZHs might encode membrane proteins that have distant similarities with this group of enzyme s. To test that hypothesis, HPLC analysis was first performed to measure the leve ls of sphingoid bases. Data generated by this means indicate that under overexpressing conditions, IZHs increase the levels of sphingoid bases. As a second approach, in vitro ceramidase assays were perfor med. However, our data suggest that under our experimental conditions, Izhs do no t appear to be alkaline ceramidases, per se. However, it is imperative to do more studies that help test this hypothesis.

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114 Although our ceramidase assays did not support our original hypothesis regarding the role of Izhs as ceramidases, they illuminated the possibility that Izhs can modulate the de novo synthesis of sphingolipids. Sphingoid bases can be synthesized by two different mechanisms. First, complex sphingolipids can be converted to free sphingoid bases and fatty acids through the action of alkaline ceramidases (Mao et al., 2000a; Mao et al., 2000b). Second, free sphingoid bases can be synthesized de novo starting with the condensation of serine and palmitoyl-CoA (Cowart and Obeid, 2007) (Figure 1-8 for details). To investigate a possible role of IZHs in the de novo sphingolipid biosynthesis we generated radi olabeled sphingolipids by treating the cells with [3H]serine. The data generated with this type of experiments revealed a moderate but still measurable increase in the levels of PHS and DHS when the IZHs were overexpressed (Figure 58). As a second approach, we used myriocin and fumonisin B1 two drugs that inhibit different satges of de novo biosynthesis of sphingolipids (Figur e 5-3 A). When myriocin was used, a significant decrease in the levels of sphingoid bases wa s observed in cells overexpressing IZH2 and IZH3 (Figure 5-10 B). This result suggests the possibility that IZHs encode proteins directly implicated in de novo sphingoid base biosynthesis. On the other hand, an accumulation of sphingoid bases was observed upon treatment with fumonisin B1, suggesting that IZHs can also mediate the synthesis of more complex sphingolipids, (Figure 5-9 A and B). Our current data support but do not ab solutely prove the implication of IZHs in sphingolipid biosynthesis. However, different pos sibilities are envision ed. First of all, the IZH genes could modulate the synthesi s of sphingoid bases and complex sphingolipids. From this standpoint, it is possible that these genes exert a regulatory role, modulating other genes that are directly implicated in the de novo production of sphingoid bases. On the other hand, is plausible that IZHs are directly implicated in the synthesis of sphingolipids, perhaps forming complexes

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115 with other proteins. It is also entirely possible that IZHs exert a regulatory role, modulating other genes that are directly implicated in the de novo production of sphingoid bases. Future studies are required to clarify the mechanism by which IZHs modulate the production of sphingoid bases and ceramides. Among the myriad of possibilities, the following experiments are imperative. To really prove if IZHs encode alkaline ceramidases more a sensitive assay is required. In this regard the use of radioactive ceramide substrates like [3H]phytoceramide and [3H]dehydroceramide could be used. Following a similar procedure than that one described in the in vitro ceramidase can be followed. Besides qualitative analysis, quantitation of the levels of radiolabeled ce ramides and sphingoid bases can be performed by using a scintillation counter. To cont inue investigating the implication of IZHs in the de novo biosynthesis of sphingolipids, qualitative and quantitative TLC analysis of radiolabeled sphingolipids is also proposed. The post-translat ional effect on the Izhs produced upon addition of inhibitors like myriocin and fumonisin B1 can also be explored. To do this Western blot analysis is an appropriate technique to be used.

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116 Table 5-1. Strains and genotyes in Chapter 5 Strain Mutation Source/Derivation Genotype BY4742 Wild type EUROSCARF MAT ; his3; leu2; ura3; lys2 ypc1ydc1 ypc1ydc1 Howard Reizman MAT a ; ypc1:: LEU2 ; ydc1:: TRP1; ade2; his3;leu2; trp1; ura3; can1-100c W303-1A Wild type Howard Reizman MAT a; ade2; his3;leu2; trp1 ura3; can1-100c

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117 HO H2N OH OH Phytosphingosine C18-PHS O HO H2N 3-Keto-dihydrosphingosine (KDS) HO H2N OH Dihydrosphingosine C18-DHS HO H2N OH OH Phytosphingosine C20-PHS OH OH OHNH O OH OH C26-Phytoceramide (C26-PHC) OH OH NH O OH OH C26-Dihydroceramide (C26-DHC) OH OH OH NH O C2-Phytoceramide (C2-PHC) OH OH NHOC26-Dihydroceramide (C26-DHC) NH2Sterylamine (sphingoid base-like compound) Figure 5-1. Chemical structures of the sphingoid bases, ceramides, and sterylamine a structural sphingoid base homolog.

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118 OH OH (OH) NH O OH OH (Phyto)ceramide OH OH (OH) NH2(Phyto)sphingosine O OH OH Fatty acid O-+Ceramide synthases Lag1p, Lac1p, Lip1p, Ydc1p, Ypc1p Alkaline ceramidases Ydc1p, Ypc1p Izh1-4p ? Figure 5-2. Synthesis and hydrolysis of the yeas t ceramides. The alkaline ceramidases Ydc1p and Ypc1p catalyze the hydrolysis of th e yeast ceramides (dihydroceramide and phytoceramide, respectively) to produce the sphingoid base and a fatty acid. On the other hand, the ceramide synthases Lag1p, Lac1p, Lip1p, Ydc1p and Ypc1p catalyze the reverse alkaline ceramidase reaction. Ydc1p and Ypc1p are implicated in both reactions. The Izh1-4 proteins are pr oposed to be alkaline ceramidases.

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119 SOOHO NH2OH+O HO H2N HO H2N OH HO H2N OH OH HO HN OH HO HN OH23OHO23OMyriocin Serine palmitoyltransferase LCB1, LCB2, TSC3 Palmitoyl-CoA Serine 3-Keto-dihydrosphingosine TSC10 Dihydrosphingosine Phytosphingosine FB1Alkaline ceramidase YDC1 Alkaline ceramidase YPC1 Ceramide synthase Dihydroceramide LIP1, YDC1 FB1Ceramide synthase LAG1, LAC1, LIP1 YPC1 Phytoceramide LAG1, LAC1,CoAOH NH2COOH OH OH CH3OR OR CH3OH OHOH NH3+R = COCH2CH(COOH)CH2COOHOMyriocin Fumonisin B1 (FB1)A BSUR2 Figure 5-3. Overview of de novo biosynthetic pathway in yeast. In scheme A, straight arrows represent the stages of de novo sphingolipid biosynthesis with the respective genes enzymes and their inhibitors; represents the blockage of sphingolipid biosynthesis produced by myriocin and fumonisin B1 (FB1). B shows the chemical structures of the mycotoxins, myriocin and and fumonisin B1 (FB1).

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120 pmol lipid / noml phosphate 0 5 10 15 20 25 Vector YPC1 IZH2 IZH3 C18-PHS C18-DHS Figure 5-4. Overexpression of IZH2 and IZH3 produces an increase in the basal levels of the sphingoid bases C18-PHS and C18-DHS. The empty vector pRS316GAL1 (Vector), or the vector containing either of the YPC1, IZH2, or IZH3 genes (e.g. pRS316GAL1-YPC1, pRS316GAL1-IZH2 and pRS316GAL1-IZH 3 ) were transformed with the the BY4742 wild type strain. The figure shows the basal levels of C18-PHS and C18-DHS. Data represent the mean sta ndard deviation of three independent experiments. PHS, phytosphingosin e; DHS, dihydrosphingosine; C18represents the number of carbon atoms of the sphingoid base backbone.

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121 Figure 5-5. Overexpression of YPC1 and IZHs induces the increase in the levels of C18-PHS, C20-PHS, and C18-DHS. Total lipids were extract ed from the double mutant strain ypc1ydc1containing the empty vector pRS316GAL1 (Vector), or expressing the YPC1 and IZH1-4 (pRS316GAL1-YPC1 and pRS316GAL1-IZH1-4 respectively). A, B, and C show the basal levels of sphingoi d bases. D, E, and F show the levels of sphingoid bases after spiking with 278 nM C2-PHC.

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122 N-C12:0-NBD-phytoceramide HO HN (CH3)7NH N O N OH OH NO2O HO HN (CH3)7NH N O N OH NO2O N-C12:0-NBD-dihydroceramide HOOC (CH3)7NH N O N NO2O N-C12:0-NBD-dodecanoic acid Figure 5-6. The fluorescent ceramide substrat es and the fatty acid pr oduct used during the in vitro ceramidase assays. The scheme shows th e substrates for alkaline ceramidases N-C12:0-NBD-phytoceramide and N-C12:0-NBD-dehydroceramide, and N-C12:0NBD-dodecanoic acid, the ceramidase reaction product.

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123 Figure 5-7. TLC analysis of in vitro ceramidase activity at different pHs. In panels A and B, the empty vector (pRS316GAL1 ) or the vector expressing Ypc1p and Izh1-4p, were assayed for ceramidase activity toward the fluorescent substrates C12-NBD-PHC (left) and C12-NBD-DHC (right), at pH 8.6. Panels C and D, the empty vector and Ypc1p, Izh2p, and Izh3p were tested for ceramidase activity at pH 9.4 and 7.0, respectively. All panels show the TLC analysis the alka line ceramidase reaction. In the figure, C12NBD-FA represents the fluorescent dod ecanoic acid, PHC is phytoceramide and DHC is dihydroceramide.

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124 Figure 5-8. TLC-autoradiograph of radiolabeled lipids shows increased levels of PHS and DHS when IZHs and YPC1 are overexpressed Cells were labeled with [3H]serine and their total lipids were extracted as described under materials and methods. Radiolabeled lipids were resolved by TLC. Most of the sphingolipids were identified according to authentic standards like PHS, physto sphingoisine; PHC, phytocceramide; DHS, dihydrosphingosine; DHC, dihydroceramide; PE, phosphatidylethanolamine; and IPC, inositol phosphoceramide.

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125 C 18 -PHS 0 20 40 60 80 100 120 Vector YPC1 IZH2 IZH3 pmol lipid / nmol phosphate A 0 20 40 60 80 100 120 Vector YPC1 IZH2 IZH3 pmol lipid / nmol phosphate C 20 -PHSB 0 2 4 6 8 Vector YPC1 IZH2 IZH3 pmol lipid / nmol phosphateC 18 -DHS CFB 1 + FB 1 FB 1 FB 1 + FB 1 + FB 1 Figure 5-9. Effect of fumonisin B1 on the production of sphingoid bases. The figure shows the HPLC analysis of fluorescent sphingoid ba ses. Data are the mean standard deviation of two experiments. FB1, fumonisin B1; PHS, phystosphingosine, DHS, dihydrosphingosine; C18or C20-, are number of carbon atoms of each sphingolipid backbone.

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126 Figure 5-10. Effect of myri ocin on the growth and the s phingolipid biosynthesis of IZH2 and IZH3. A shows the phenotypic response of vector control, IZHs and YPC1 in presence of two different concentrations of my riocin. B shows the quantitation of C18-PHS in cells gown in +/myriocin by HPLC. Th e results shows a significant decrease on levels of C18-PHS is observed for IZH2 and IZH3 upon addition of 1.0 M myriocin.

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127 Figure 5-11. Overexpression of IZH2 and IZH3 produces an increase in the levels of C26-PHC and C26-DHC. The figure shows mass spectrometric analysis of the sphingolipids; C26-PHC; and C26-DHC; C26, a fatty acid with 26 carbon atoms. Data represent the mean standard deviation of two independent experiments.

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128 CHAPTER 6 CELLULAR LOCALIZATION OF THE Izh2 AND Izh3 P ROTEINS BY MEMBRANE FRACTIONATION Introduction The IZH genes encode p roteins with seven transmembrane domains and little amino acid conservation in the loop regions on the extracel lular face of the membranes where external ligands might make contact. In previous chapte rs, we demonstrated that certain nutritional conditions can affect the expressi on of these proteins, as well as their transcriptional response. Despite the progress reached in this regard, ma ny gaps concerning the function(s) of the Izhp remain to be uncovered. Perhaps one of the most important aspects that needs to be expl ored in order to gain more insights regarding the role(s) exer ted by proteins is to determine their cellular lo calization. With this in mind, our aim was to investigate the loca lization of Izh2p and Izh3p. As we have reported before, the expression of these two proteins is regulated by certain fatty acids (Chapter 3). In addition to this, when IZH2 and IZH3 are overexpressed, the sphingol ipid content is altered (Chapter 5). The fact that sphingolipids are f ound in plasma membranes led us to hypothesize that the IZH2 and IZH3 products are in plasma membrane possibly associated with lipid microdomains termed lipid rafts. Lipid rafts are formed by the lateral associa tion of sphingolipids and sterols (e.g. ergosterol in yeast, and cholesterol in mamma ls) (Dickson and Lester, 2002; Bagnat et al., 2000; GmezMountn et al., 2004). In addition to lipids, rafts also contain proteins (Grossmann et al., 2006) (Figure 6-1). Because of their co mposition, rafts were originally implicated in maintaining the optimal fluidity and integrity of plasma membranes (Grossmann et al., 2007). Nevertheless, recent studies have indicated that lipid rafts are also implicated in intracell ular protein trafficking and signaling processes (D upre and Haguenauer-Tsapis, 2003). Other studies have also revealed

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129 the specific lipid raft associati on of GPI-anchored proteins (Moffett et al., 2000) as well as transmembrane proteins whose transmembrane doma ins are thought to have certain affinity for rafts (Simons et al., 1997). One interesting characteristic that has allowed the isolation and analysis of lipid rafts is their low density and insolubility in the mild nonionic detergent 1% Triton X100, at 4 oC (Pike, 2003; Grossmann et al., 2006) Therefore, we took advantage of these properties to isolate lipid rafts from the rest of the yeast membranes. To our knowledge, there is only one report published by Narasimhan et al., 2005, suggesting the presence of Izh2p in plasma membrane s. In this chapter, we have broadened the scope of that finding by localizing Izh2p and th e paralogous Izh3p in plasma membrane and within lipid rafts. In fact, we present evidence that suggests Izh2p and Izh 3p are co-localized in plasma membrane and lipid rafts. In addition to this, our results suggest that a fraction of these two proteins can eventually be dissociated from lipid rafts. In this scenario, Umebayashi et al., 2003 have proposed that lipid rafts serve as sorting platforms for proteins. Although still preliminary, the data presented herein, constitute an important framework that strongly contributes to elucidate the functio n(s) of the Izhs. Our findings certainly illuminate our original premise, implica ting Izh2p and Izh3p in different lipid biosynthetic pathways. Materials and Methods Plasmids and Yeast transformations For this study, the wild type strain B Y4742 (mating type obtained from EUROSCARF), (http://web.uni-frankfurt.de /fb15/mikro/euroscarf/) was used. Plas mids used in this study were generated by Dr. Thomas Lyons as described in Chapter 2. For the experiments described in this chapter, we used plasmids where the IZH2 and IZH3 genes had their own promoters. The

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130 plasmids pIZH23xHA and p IZH33xHA were introduced into the wild type strain BY4742 as described in Chapter 2. Growth Conditions Cells containing the p IZH2 -3xHA a nd p IZH3 -3xHA plasmids were grown in identical fashion as described in Chapter 2. Cells were grown to logarithmic phase (OD600 of 0.8) followed by harvesting at 3,000 rpm, for 5 min at 4oC. Pellets were washed twice with cold sterile nano pure water. Resultant pellets were then used to isolate Izh2-3xHA and Izh3-3xHA proteins. Isolation Purification and Characteri z ation of Yeast Plasma Membranes Unless otherwise indicated, the entir e procedure was carried out at 4oC. Reagents used were of high quality and obtained from Fisher or Sigma, Protease inhibitors and glass beads were obtained from Sigma. The TED buffer (10 mM Tris adjusted to pH 7.5 with HCl, 0.2 mM EDTA, pH 7.5, and 2 mM dithioth reitol, DTT) is present in all the sucrose and glycerol solutions. Pellets were re-suspe nded manually with a plastic stir ring rod. Plasma membrane was isolated using a combined method of differentia l and density gradient centrifugations according to Serrano, 1988, and with some minor modifications as follows. Portions of 25 g of cells (fresh wet weight) were diluted with nano pure water to 80 mL. Aliquots of 20 mL of the diluted cells were transferred to 50 mL sterile polypropylene centrifuge tubes, (Fishe r), and suspended with lysis buffer, (0.5 M Tris, pH 8.5; 6 mM EDTA pH 8.0; 0.6 mM phenylme thyl-sulfonyl fluoride (PMSF), 2 g/mL pepstatin A), and 15 mL of glass beads-acid washed, (Sigma). Cells were lysed by vortexing 10 times, one minute each time and with repe ated incubations on ice for 4 min between each vortexing. Lysates were transf erred to a thick wall 25 X 89 mm polycarbonate tube (from Beckman coulter), and centrifuged at 1,000 rpm, 4oC during 10 min in a Sorvall SS34 rotor to remove unbroken cells and debris. An aliquot of the low speed supernatant (lysate

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131 fraction or Lys) was saved for fu rther SDS-PAGE and we stern blot analysis, and the rest of the supernatant was further centrifuge d for 20 min at 20,000 rpm in a Sorvall SS34 rotor. An aliquot of this supernatant (Cytosolic fraction or Cy t) was saved for SDS-PA GE and Western blot analysis. The 20,000 rpm pellet, whic h is enriched in plasma membranes, was re-suspended with 7.5 mL of 20% glycerol, and pr otease inhibitors. Suspensions were homogenized by hand, applying 10 strokes in a 10 mL Potter-E lvehjem homogenizer. Ultra clearTM ultracentrifuge tubes (25 X 89 mm) from Beckman were used to prepar e a discontinuous gradient made of 8 mL 53% (w/w) sucrose and 16 mL 43% (w/w) sucrose. The homogenates were then applied to the gradient and centrifugated for 6 h at 25,000 rpm in a Beckman SW 28 rotor. Plasma membranes were recovered at the 43/53 inte rface. The band was collected with a Pasteur pipette, diluted with 4 volumes of water, and pelleted by centrif ugation at 35,000 rpm for 20 min, using a Beckman 70.1 Ti rotor. Protein concentra tion of all the protein fractions was determined by the Markwell assay (Marwell et al., 1981), and using lyophilized bovine seru m albumin (Bio Rad) as standard. Analysis of each protein fraction (100 g of protein) was performed by SDS-PAGE and western blot analysis. To do this, a 10% SDS-PAGE gel was run overnight at 45 volts, at room temperature, and then transferred to a nitro cellulose membrane (Bio Rad) by applying 200 mA for 2 h, and at 4oC. The Izh2and Izh3-3xHA-tagged pr oteins were analyzed incubating the membranes during 1 overnight, at 4oC with a 1/1000 and 1/500 (v/v) dilution, respectively of the primary antibody rabbit polyclonal anti-HA, ( HA-probe (Y-11)sc-805, from Santa Cruz), followed by incubation during 1 h, at room temper ature with the secondary antibody goat anti rabbit-Horse Radish Peroxidase-conjugate, (HRP -) (Santa Cruz) at a dilution of 1/10000, v/v. Likewise, the mouse monoclonal [40B7] to th e plasma membrane marker Pma1p (Abcam), (1/8000, v/v dilution), was used as primar y antibody. Pma1p is a proton-pumping H+-ATPase

PAGE 132

132 that is an abundant and very l ong lived protein of th e yeast plasma membrane. Pmap associates with lipid rafts (detergent-resi stant membrane domains) (Gaigg et al., 2006). Immunoreactive proteins were visualized in an X-ray film (Pierce) and by using th e pico super signal ECL detection kit (Pierce). Isolation of Lipid Rafts from Ye ast Plasma Membrane: Procedure 1 Once prepared, the Izh2p and Izh3p plasm a membra ne fractions were used for the isolation of lipid rafts according to Kumar et al., 2004 as follows. Purified yeast plasma membranes (1 mg) were extracted in MBS buffer (25 mM MES and 150 mM NaCl, pH 6.5) containing 0.2% TX100 obtained from Fisher, and supplemented with protease inhibito r mix (Sigma). The samples were mixed end-over-end in a rotator for 20 min at 4oC, and then homogenized by hand, with 10 strokes of a Dounce homogenizer. The homogenizer was rinsed with 500 L of MBS0.2% Triton and then combined with the 1 mL extract. These extracts were mixed with 1.5 mL of 80% sucrose (w/v) in MBS. Homogenates (now in 40% sucrose) were placed at the bottom of 14 X 89 mm ultra clearTM ultracentrifuge tubes (B eckman Coulter), and overlaid with 6 mL of 30% sucrose and 3 mL of 5% sucr ose (in MBS). After centrifugati on at 240,000 X g (37,400 rpm) in a Beckman SW41 rotor for 18 h, 1.0 mL fractions were collected by upward displacement using 60% (w/v) sucrose solution (prepared in the MBS buffer) as the displacement fluid at 4oC. Protease inhibitor mix (Sigma) was added to each fraction. Protein was precipitated by adding 500 l of 30% icecold trichloroacetic aci d (TCA), followed by incubation at 4oC for 30 min. The protein precipitate was collected at 13,200 rpm at 4oC, for 10 min, after which it was washed two times with 500 L of ice-cold acetone. The precipitate was then dissolved in 120 L 1X SDS-loading buffer, and the protein (100 g) was resolved on a 10% SDS-PAGE gel under reducing conditions. Gels were th en transferred onto nitrocellulose membranes (Bio Rad) for

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133 protein analysis. After transfer, nitrocellulose membranes were stained with a solution of 10% (v/v) amido black to visualize protein bands. The amido black solution was prepared mixing 1 volume of amido black stock, [(0.1% naptol blue black, 50% methanol, 10% glacial acetic acid, and 40% nano pure water), and 9 volumes of ami do black destain, (50% methanol, 10% glacial acetic acid, and 40% ultra pure water)]. Western blot analysis was performed in identic al fashion as describe d in former section. A rabbit polyclonal IgG antibody against the HA-tag (Santa Cruz) was used at a dilution of 1/1000 (v/v) for Izh2-3xHAp and at a dilution of 1/500 (v/v) for Izh3-3xHAp. Membranes were then incubated with the secondary antibody goat anti-ra bbit IgG HRP-conjugate (Santa Cruz) used at a dilution of 1/10000 (v/v). Pma1p, an associated yeas t lipid raft protein was used as a marker of the lipid raft fraction (Bagnat et al., 2000). This protein was identified with a 1/8000 (v/v) dilution of a mouse polyclonal IgG anti Pma1p (primary antibody) obtained from Abcam, and followed by incubation with 1/10000 (v/v) dilu tion of goat anti mouse IgG HRP-conjugate (secondary antibody) obtained from Bio Rad. Like wise, the vacuolar alkaline phosphatase (VALP), a marker for plasma membra ne proteins that are not associ ated with lipid rafts (Bagnat et al., 2000; Nothwehr et al., 1995) was identified with a 1/ 100 (v/v) dilutio n of a mouse monoclonal IgG anti-alkaline phosphatase (Molec ular Probes), followed by incubation with 1/10000 (v/v) dilution of goat anti mouse IgG HR P-conjugate (secondary antibody) obtained from Bio Rad. Immunoreactive proteins were visualized as described before. Isolation of Lipid Rafts from Total Membranes : Procedure 2 In this procedure, lipid rafts were isolated from total membranes according to Kbler et al., 1996 with some modifications as follows. Fresh pe llets (20 g of wet weight) were washed twice with cold sterile water, and then re-suspended in a total volum e of 100 mL of ice-cold lysis buffer (20 mM triethylethanolamine pH 7.2, 0.3 M sorbitol, 1 mM EDTA, 0.1 mM PMSF, 0.7

PAGE 134

134 mM pepstatin A, and 2 mL of protease inhibito r cocktail (Sigma). Por tions of 20 mL of cell suspensions were mixed with 15 mL of acid wa shed glass beads (Sigma) and lysed 10 times by vigorous vortexing, 1 min each time and with rep eated incubations on ice for 2-3 min between each vortexing. Unbroken cells were removed by a 1,000 rpm spin for 10 min, and using a Sorvall SS34 rotor. The resultant supernatant (total cell lysate fraction) was centrifuged for 1 h to pellet cellular membranes and using a Beckma n SW28 rotor at 27,500 rpm. The supernatant (Cytosolic fraction or Cyt) was discarded, and the resultant pellet is referred as total membrane fraction (TM). Pellets corresponding to the total membrane fraction was suspended in 3 mL MBS buffer (25 mM Mes and 150 mM NaCl, pH 6.5), supplemen ted with protease inhibitor mixture from Sigma. Suspensions were homogenized by hand a pplying 10 strokes in a 10 mL Potter-Elvehjem homogenizer. 1.0 mL of the homogenate was tran sferred to a 1.5 mL eppe ndorf tube and treated with cold 1% TX100, followed by mixing end-over-end in a rotator for 30 min at 4oC. The extracts were brought to 1.5 mL by adding 500 L of a solution of cold MBS-1% Triton. These extracts were then mixed with 1.5 mL of 80% sucrose (w/v) in MBS. Homogenates (now in 40% sucrose) were pl aced at the bottom of 14 X 89 mm ultra clearTM ultracentrifuge tubes (Beckman Coulter); and overlaid with 6 mL of 30% sucrose and 3 mL of 5% sucrose (in MBS, pH 6.5). After centrifugation in a Beckman SW41 rotor at 240,000 X g (37,400 rpm) at 4oC for 18 h, 12 fractions of 1.0 mL each one were collected by upward displacement using 60% (w/v) sucrose solution (p repared in the MBS buffer, pH 6.5) as the displacement fluid. Protease inhi bitor mixture (1/1000, v/v dilution) from Sigma, was added to each fraction.

PAGE 135

135 Proteins were precipitated by adding 500 L of 30% icecold tric hloroacetic acid (TCA), followed by incubation at 4oC for 30 min. Precipitates were collected at 13,200 rpm, 4oC for 10 min. Precipitates were thor oughly washed twice with 500 L of ice-cold acetone. Resultant protein pellets were suspended in 120 L of loading buffer prepared by mixing 1 part of sample dilution buffer (2X SDB) consisting of SDS, D TT, bromophenol blue, glycerol, and Tris-base, pH 6.8 from Fluka) with 1 pa rt of 0.1% SDS and 1% of -mercaptoethanol ( ME). After determination of total membrane protein concen trations by using the Markwell assay, 1 mg of each protein was suspended in 2X SDB and 2% ME (1:1, v/v). Proteins were then analyzed by 10% SDS-PAGE and Western blotti ng in identical fashion as described before (procedure 1). Results Izh2p and Izh3p Were Found Enriched in Plasma Membrane After synthesis occurs in th e endoplasm ic reticulum, some sphingolipids are trafficked to the Golgi apparatus becoming structurally more co mplex, whereas others di rectly can be sorted to the plasma membrane to serve as structur al and signaling molecule s. Because Izh2p and Izh3p are predicted to be involved in the sphingolipid metabolic pathwa y, our initial rationale was that these two proteins could be embedded in the plasma membrane, modulating role(s) in the sphingolipid pathway. So far, however, there is ju st one report suggesting that Izh2p is a plasma membrane protein (Narasimhan et al., 2005), and no precedent exists regarding the localization of Izh3p. Therefore, our aim was to investigate if Izh2p and Izh3p are in fact plasma membrane proteins. To do this, a standard procedure developed by Serrano, 1988 to isolate and purify plasma membrane from yeast cells was followe d. This method, which is based on differential centrifugation of discontinuous sucrose gradients allowed us to detected Izh2p and Izh3p tagged with the HA-epiptope in plasma membranes. Isolation and purification of plasma membrane was

PAGE 136

136 carried out by conducting two independent experi ments. Yeast cells were first lysed, and an aliquot of the resultant lysis fraction (Lys in Figures 6-2 and 6-3) was used for SDS-PAGE and Western blot analysis. The rest of this fraction was centrifuged at 20,000 rpm to generate a pellet enriched in plasma membranes, and a supernatant composed mainly of soluble proteins (Cyt in Figures 6-2 and 6-3). The 20,000 rpm pellet was ho mogenized and then applied to a sucrose gradient and centrifuged at 25,000 rpm, which allowed the isolation of the pure plasma membranes at the interface of th e 43% and 53% sucrose gradient (PM in Figures 6-2 and 6-3). A layer at the top of the 43% sucr ose gradient was also isolated and designated as the microsomal fraction (M, in Figure 6-3) because it contains proteins from different organelles like mitochondria, ER, Golgi, vacuole, etc (Serrano 1988). The purity of the resulting plasma membrane isolate was tested by Western blotting, and using the yeast proton-ATPase protein, Pma1p as a marker. Pma1p is an appropriate marker, not only because it is an essential protein in the in plasma membrane where comprises > 25% of the total protein at steady state (Lee et al., 2002), but also due to the comercial availability of antibodies against it. On the other hand, the presence of th e HA-tagged Izh2p and Izh3p was tested by using an antibody against the HA epitope tag. Our results suggest that both proteins Izh2p and Izh3p are in plasma membrane. Howeve r, Izh3p showed a particular pattern of migration with bands at higher mo lecular weight than that one predicted by its molecular weight (at 63.0 KDa), (e.g. a band close to 97.4 KDa and a nother one close to 66.2 KDa ) (Figure 6-2 B, and F). We could not determine the identity of these bands. However, we speculate that are the results of a post-tranlational modification for the Izh3p, like glycosilation. Finally, a band observed just below 63.0 KDa (Figure 6-2 F) might correspond to degradat ed protein.Thus, more

PAGE 137

137 experiments need to be conducted to elucidate th e identity of those un-ex pected bands, and to improve the results presented in this study. Izh2p and Izh3p Are Associated with Lipid Rafts To address the possibility that Izh2p and Izh3p are localized in lipid rafts, we first adapted a well established experim ental procedure used to isolate and characterize lipid rafts from mammalian cells (Kumar et al., 2004). In our case, we first isolated and purified plasma membrane fractions as described previously in materials and methods, followed by treatment with cold 1% TX100, and detergent-lysate homogenizing. Homogenized samples were then adjusted to 40% sucrose and load ed to the bottom of a step-den sity gradient (40%, 30%, 5% sucrose) followed by ultracentrifugation. A successf ul isolation of lipid rafts produces a low density layer floating at the top of the step-sucrose gradient. A lthough we could not detect such a layer, 12 fractions (1 mL each one) were collect ed and analyzed by SDS-PAGE and Western blot (Figure 6-3). Usually, fractions 1-5 correspond to lipid rafts if any, and fractions 6-12 contains membranes proteins that have been dissociat ed from membranes by treatment with cold TX100 (Bagnat et al., 2000; Kumar et al., 2004). The results obtained suggest, at first glance, that Izh2p (Figur e 6-3 A, top panel) and Izh3p (Figure 6-3 A Bottom panel) are fully soluble under the conditions used in this procedure (Figure 6-3, fractions 10 and 11 of the sucrose gradie nt), and that although localized in plasma membrane, these proteins are not present in lipid rafts at all. We confir med that fractions 10-11 were indeed soluble due to the identification of V-ALP in such fractions (Figure 6-3 B, bottom panel). At this point is pertin ent to remember that V-ALP (a vacuolar membrane protein) was used as a marker for membrane proteins that are soluble upon treatm ent with TX100 (Bagnat et al., 2000; Nothwehr et al., 1995). An interesting aspect that is necessary to mention is the absence of Pma1p from lipid rafts and its detec tion in the soluble fract ion (Figure 6-3 A, top

PAGE 138

138 panel). Besides being a plasma membrane protei n, Pma1p has also been co-localized in lipid rafts. Pma1p has been found in association with lipid rafts (Bagnat et al., 2000; Lauwers et al., 2006). Therefore, our results suggest a possible disruption of lipid rafts under the conditions used. In fact, it has been reported that the extent of physical manipulations of detergent lysates, in some cases, disrupt lipid rafts a nd thus thir components (Pike, 2003). In an effort to solve experimental proble ms associated with the procedure 1, a second procedure, based on the isolation of lipid raft s from total membranes, was conducted. To do so, yeast cells were lysed and then centrifuge d at 100,000 X g (25,700 rpm) to obtain the total membranes. These membranes were homogeni zed by hand as describe d in materials and methods, followed by incubation in cold 1% TX 100 for 30 min and further ultracentrifugation at 240,000 X g (37,400 rpm). A thick layer floating at the top of the sucrose-step gradient was observed. Twelve fractions of 1 mL each one we re collected as described under materials and methods and analyzed by SDS-PA GE and Western blot. This purif ication scheme allowed us to identify Izh2p and Izh3p in lipid rafts by their distribution along the density gradient (Figure 6-4, panels A and B, respectively). The successful isol ation of lipid rafts was tested by the presence of Pma1p in fraction 4 of the gradient (Figur e 6-4 C, top panel). Interestingly, Izh2p, Izh3p, and the marker Pma1p were also observed in the soluble fractions of the gr adient suggesting that these proteins although associate d, are not permanent residents of lipid rafts. However, more experiments need to be perfomed to prove th is hypothesis. Finally, as expected the V-ALP protein was localized in the soluble fractions of the sucrose gradient (Figure 6-4 C, bottom panel). Discussion The aim of this study was to investigate the co-localization of Izh2p and Izh3p in plasma membranes and lipid rafts. By using differe ntial centrifuga tion of discontinuous sucrose

PAGE 139

139 gradients, we were able to isolate and partially purify plasma membrane fractions. In addition to this, we isolated lipid rafts from total membrane s by flotation and after treatment with 1% Triton X100 at 4oC. Although Izh2p and Izh3p were localized in plas ma membrane, both proteins were also colocalized in other fractions. For instance, the first experiment shown in Figure 6-2, panels A, B, and C, clearly shows that Izh2p is mainly enriched in plasma membrane. However, Izh3p was compartmentalized in the microsomal and plasma membrane fractions. In addition to this, other bands, at higher molecular wei ghts than the pr edicted one (at 63.0 KDa), were also observed. One possible explanation for this migration pa ttern is that Izh3p could be undergoing posttranslational modifications, whic h yield a protein with a high er molecular weight. Future experiments are required in or der to test this hypothesis. A second experiment was performed to confirm the results obtained in the first one. This experiment showed that Izh2p was also co-localiz ed in the microsomal fraction (Figure 6-2 D). Izh3p showed more than one band, confirming th e results obtained in the first experiment (Figure 6-2 F). The result obtained for Izh2p, sugge sts an incomplete pur ification of plasma membranes. This result was confirmed with the co-localization of Pma1p in plasma membrane and microsomal fraction (Figure 62 E). This is attributed to c ontamination generated during the isolation of plasma membrane from the sucrose gradient. Although two procedures were followed to isolate lipid rafts, only one of them was effective. In a first approach, plasma membrane s were isolated and purified as describedc under materials and methods. After that, we tried to isolate lipid rafts, from purified plasma membranes, by treatment with cold TX100, followed by homogenizing. This procedure failed to

PAGE 140

140 yield lipid rafts (Figure 6-3). We attributed this to possible disruption of lipid rafts during the treatment of the samples with dete rgent and followed by homogenizing. In a second approach, lipid rafts were su ccessfully isolated from total membranes by flotation. This procedure allowe d us to identify Izh2p and Izh 3p in the detergent resistant fractions (Figure 6-4), suggesting th at these proteins are associated with lipid rafts. Interetingly, we also found that Izh2p, Izh3p, and the Pma1p (use d as positive control) are fractionated with detergent sensitive membranes (Figure 6-4, fractions 8-11). One possible reason that would support our results is an inherent sensitivity of the tested proteins to the TX100 extraction. In fact, Umebayaski, 2003 has reported that certain lipid raft proteins are resi stant to solubilization by detergents like CHAPS, but are highly sens itive to the treatment with TX100. Regarding Pma1p, Bagnat et al., 2000 have argued the possibility that th e lack of association of this protein to lipid rafts arises from the pelleting step, just before the floatation in 60% sucrose. When total membranes are pelleted from the cleared lysate, th e pellet likely contains the remainder of cell walls, debris, and cytoskeletal el ements that result in the trapping of material, that otherwise would have a lower density. Like wise, studies performed by Bagnat et al., 2001; Lawrens and Andr, 2006 have indicated that, upon treatmen t with TX100, Pma1p can dissociate from lipid rafts and missort to the vac uole for further degradation. Overall, the results obtained are consiste nt with the hypothesis that Izh2p and Izh3p proteins are colocalized with sterols and sphingolipids. The f act that the ove rexpression of IZH2 and IZH3 induces an increase in the levels of sphingoid bases (Chapter 5), and the localization of these two IZH gene products in plasma membrane and lip id rafts constitute additional and robust evidence indicating a potential role of the Izhp family in sphingolipid metabolism.

PAGE 141

141 The results presented in this ch apter are very valuable since thery have started to shed light into the cellular localization of two members of the Izh family. In addition to this, we demonstrate that the metodologies used to isolat e plasma membrane and lipid rafts are powerful approaches to study membrane proteins.

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142 Figure 6-1. Lipid composition of th e yeast lipid rafts. In the figure sterols and sphingolipds associate to form detergent-re sistant microdomains or lipid rafts. Lipids rafts are in plasma membrane where membrane proteins are also localized. In the figure black circles (A, B, C, and D) ar e highly conserved motifs of a membrane protein with seven tansmembrane domains.

PAGE 143

143 Figure 6-2. Localization of Izh2p and Izh3p in plasma membrane. Two independent experiments are shown in this figure. The fi rst one is represented by panels A, B, and C. A shows that Izh2p is mainly localized in plasma membrane. B shows that Izh3p, although localized in plasma membrane, it is also co-localized in the cytosolic fraction (Cyt). C shows that the plasma membrane marker Pma1p is localized in lysate (Lys) and plasma membrane (PM) fr actions, but not in the soluble cytosolic fraction (Cyt). In a second experiment, D shows that Izh2p is co-localized in the microsomal and the plasma membrane fractions. E confirms that Pma1p is mainly localized in plasma membrane. F, Izh3p is co -localized in all the isolated fractions. Figure shows Western blots. HA-tagged Izh2p and Izh3p were recognized with antiHA antibody. Pma1p was recognized with an anti-Pma1p antibody.

PAGE 144

144 Figure 6-3. Izh2p and Izh3p are disso ciated from lipid rafts prepared from plasma membranes. Panel A-top shows that although Izh2p is pr esent in plasma membrane, it was fully solubilized under treatment with cold TX100. Likewise Izh3p was primarily detected in the soluble fractions of the sucrose gr adient panel A-bottom. Panel B-top shows that the plasma membrane marker Pma1p although enriched in this fraction, it also undergoes dissociation of lipid rafts under our experimental conditions. V-ALP (V acuolar Al kaline P hosphatase), a soluble protein under treatment with cold TX100, was recovered from fractions 10 and 11 (panel B-bottom). To generate the panels shown in the figure, 12 fractions were collected from the top of the step sucrose gradient. Proteins were TC A-precipitated and analyzed by Western blot, using with anti-HA, anti-Pma1p, and anti-V-ALP antibodies. PM stands for plasma membrane.

PAGE 145

145 Figure 6-4. Localization of Izh2p a nd Izh3p in lipid rafts prepared from total membranes. Panel A from top to the bottom, shows that Iz h2p and Izh3p are distributed between lipid raft and soluble fractions indi cating that these proteins are associated to lipid rafts but are not permanent residents of these memb rane microdomains. In a similar way, Pma1p was barely localized in lipid raft, as well as in soluble fractions (panel B-top), indicating that this pr oteins is also undergoing dissocia tion from lipid rafts. On the other hand, V-ALP was strictly recovered in the lowermost fractions, corresponding to soluble proteins (panel B-bottom). In the figure, proteins shown in each Western blot were recognized using specific antibodies against each of the proteins analyzed. In the figure TM, stands for total membrane.

PAGE 146

146 APPENDIX A YEAST GROWTH MEDIA AND PROCEDURE Yeast Peptone Dextrose (1X-YPD) 5 g/L yeast extra ct [Fisher] 10 20 g/L peptone [Fisher] 2%, w/v alpha (+) glucose (99% anhydrous) [Across Organics] Nano pure water to 1 L Before being used, the solution was sterilized Synthetic Medium Supplemented with Dextrose (SD) 1.7 g/L YNB without amino acids a nd ammonium sulfate [Fisher] 2%, w/v alpha (+) glucose (99% anhydrous) [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] Nano pure water to 1 L Before being used, the solution was sterilized Synthetic Medium Supplemented with Galactose (SGal) 1.7 g/L YNB without amino acids a nd ammonium sulfate [Fisher] 2%, w/v D (+) galactose [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] Sterile nano pure water Chelexed Synthetic Medium Supplemented with Dextrose (CSD) 5.1 g/L YNB without divalent cations, ami no acids, ammonium sulfate, and phosphates [Qbiogene] 2%, w/v alpha (+) glucose (99% anhydrous) [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] Sterile water to 850 mL The mixture was stirred for 1 overnight at 4oC with 25 g Chelex-100 ion exchange resin [Sigma] The resin was removed and then the pH was adjusted to 4.0 with HCl

PAGE 147

147 The mixture was supplemented with 10 mL 100g/L potassium phosphate monobasic (KH2PO4), 24 L 100 mM manganese sulfate (MnSO4), 10 L 4 g/L copper sulfate (CuSO4), 1 mL 100 g/L calcium chloride (CaCl2), and 1 mL 500 g/L magnesium sulfate (MgSO4). Either of the following solutions was added to the indicated final concentration only if the medium did not have rest riction of each metal: 1.2 M iron chloride (FeCl3) final concentration 2.2 M zinc chloride (ZnCl2) final concentration Sterile nano pure water was added to complete 1 L For either zinc or iron deficiency ZnCl2 or FeCl3 was not added back to CSD. For a mdium replete of zinc or iron 10 M of either ZnCl2 or FeCl3 was adde d back to the growth medium. The solution was then filter-sterilized into pol ycarbonate flasks. All plas tic used for CSD media preparation and cell culturing was washed with Acationox detergent (Baxte r Scientific Products, McGaw Park, IL) before being used. Chelexed Synthetic Medium Supplemented with Galactose (CSGal) 5.1 g/L YNB without divalent cations, ami no acids, ammonium sulfate, and phosphates [Qbiogene] 2%, w/v D (+) galactose [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] Sterile water to 850 mL The mixture was stirred for 1 overnight at 4oC with 25 g Chelex-100 ion exchange resin [Sigma] The resin was removed and then the pH was adjusted to 4.0 with HCl The mixture was supplemented with 10 mL 100g/L potassium phosphate monobasic (KH2PO4),

PAGE 148

148 24 L 100 mM manganese sulfate (MnSO4), 10 L 4 g/L copper sulfate (CuSO4), 1 mL 100 g/L calcium chloride (CaCl2), 1 mL 500 g/L magnesium sulfate (MgSO4). Either of the following solutions was added to the indicated final concentration, only if the medium did not have restriction of each metal: 1.2 M iron chloride (FeCl3) final concentration 2.2 M zinc chloride (ZnCl2) final concentration Sterile nano pure water was added to complete 1 L. Either, zinc or iron was added back to CSGal to a final concentration of 50 nM (deficiency), and 10 M (repletion). The solution was then filter-sterilized into polycarbonate flasks. All plastic used for CSD media prepara tion and cell culturing was washed with Acationox detergent (Baxter Scientific Products, McGaw Park, IL) before use. Low Iron Medium Supplemented with Dextrose (LIMD) 1.7 g/L of YNB without amino acids and ammonium sulfate [Fisher] 2%, w/v alpha (+) glucose (99% anhydrous) [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] 20 mL 1.0 M sodium citrate, pH 4.2 (sodi um citrate was obtained from Fisher] 1 mM, (final concentration) of EDTA, pH 8.0 [EDTA was obtained from Sigma] MnCl2 was added back to LIM to a final concentration of 20 M ZnSO4 was added to a final concentration of 0.8 g/mL, (5 M) Iron deficiency, or iron repletion were generated by adding either, 1 M or 1 mM FeCl3, respectively. Sterile nano pure water was added to 1 L The solution was then filter ster ilized into polycarbonate flasks.

PAGE 149

149 Low Iron Medium Supplemented with Galactose (LIMGal) 1.7 g/L of YNB without amino acids and ammonium sulfate [Fisher] 2%, w/v D (+) galactose [Across Organics] 5 g/L ammonium sulfate [Fisher] 0.01%, w/v appropriate ami no acids [Sigma-Aldrich] 20 mL 1.0 M sodium citrate, pH 4.2 (sodi um citrate was obtained from Fisher] 1 mM (final concentratio n) of EDTA, pH 8.0 [EDTA was obtained from Sigma] MnCl2 was added back to LIM to a final concentration of 20 M ZnSO4 was added to a final concentration of 0.8 g/mL, (5 M) Iron deficiency, or iron repletion were generated by adding either, 1 M or 1 mM FeCl3, respectively. Sterile nano pure water was added to 1 L The solution was then filter ster ilized into polycarbonate flasks.

PAGE 150

150 150 APPENDIX B YEAST TRANSFORMATION Protocol Single co lonies of a S. cerevisiae yeast strain (e.g. the BY4742 wild type or the double mutant ypc1ydc1 strain) were grown until stationary phase (OD600 of 3) during one overnight (betweeen12-18 h) in 1X-Yeast Peptone Dextrose medium (1X-YPD) at 30oC and with contant agitation at 250 rpm. Fresh YPD (5 mL) was inoculat ed with the overnight culture and cells were then grown to logarithmic phase (OD600 of 1.0). Cells were harveste d at 3,500 rpm for 5 min and resultant pellets were washed with 5 mL of LiTE solution. The suspension was spun down and most of the supernatant was discarded (50 L of cell suspension in LiTE solution is enough for each transformation). Yeast transformation was performed as follows. Cell suspension (50 L) was aliquoted in a 1.5 mL eppendorf tube and mixed with 400 g of appropriate plasmid DNA (approximately 2 L), and 10 L of carrier DNA (10 mg/ml salmon testes DNA stock, which must be boiled for 5 min before each use and flash cooled on ice before addition to the cells). 500 L of PEG-LiTE solution was added to the mixtures and suspe nded by vortexing. The suspension was incubated at 30oC for 1 h. This was followed by heat-shock at 42oC for 12 min. Cells were then spun down at 4,000 rpm in a microcentrifuge and the supernatan t wasaspirated. The pellet was suspended in 200 L of LiTE solution. Aliquots of 50 L of this suspension were plated onto solid agar synthetic media plates supplemented with the ap propriate carbon source and amino acids. Plates were incubated at 30oC until colonies were visible (usually 3 to 4 days after transformation).

PAGE 151

151 151 Solutions Litium Acetate Tris Base Ethylenedia mmine tetraacetic Acid (LITE) Solution 5 mL, sterile 10X-TE stock (100 mM Tris pH 7.5, 10 mM EDTA, pH 7.5); 5 mL, sterile 10X-litium acetate stock solution (sterile 1.0 M lithium acetate); 40 mL sterile ultrapure water. Store this solution at room temperature. Polyethylenglycol Litium Acetate Tris Base Ethylenediamminetetr a acetic Acid (PEGLiTE) Solution 5 mL, sterile 10X-TE stock; 5 mL, sterile 10X-litium acetate stock; 40 mL, sterile 44%, w/v PEG-3350 stock. Store this so lution at room temperature. Tris Base Ethylenediamminetetraa cetic Acid (10X-TE) Solution 25 m L of 1 M tris stock, pH 7.5; 5 mL 500 mM EDTA st ock, pH 7.5; 250 mL ultrapure water. Sterilize the solution and store it at room temperature. Carrier DNA 10 m g salmon testes DNA and 10 mL ultrapure water. Shear by drawing up into a 10 mL syringe with 18 g during 15 times. Boild, a nd restore volume to 10 mL. Make 10x-(1mL) aliquots and store them at -20oC.

PAGE 152

152 APPENDIX C GAS-CHROMATOGRAMS AND MASS SPECTRA OF TOTAL STEROLS Figure C-1. Gas chromatography-el ectron ionization in tandem with mass spectrometric analysis (GC-EI-MS). TIC of internal standards. Panel A, GC-chromatograms, show that the TMS derivative of cholestero l (I) eluted first, followed by those for ergosterol (II), dihydrolanosterol (III) and la nosterol (IV). B, mass spectra of I, II and IV were similar to those found in the NIST EI spectral library.

PAGE 153

153 (mainlib) Cholesterol trimethylsilyl ethe r 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 0 50 100 43 57 61 69 73 81 95 107 121 129 145 159 173 189 213 233 247 275 283 301 313 329 340 353 368 443 458 O Si (mainlib) Silane, (ergosta-5,7,22-trien-3-yloxy)trimethyl 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 0 50 100 61 69 81 91 105 119 131 143 157 169 185 199 211 226 253 267 279293 337 363 378 453 468 O Si (mainlib) Silane, [[(3)-lanosta-8,24-dien-3-yl]oxy]trimethyl 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370 390 410 430 450 470 490 510 0 50 100 55 69 73 81 95 109 119 135 145 161 173 187 201 215 227241 255 269283 297 311 393 483 498 O Si Figure C-2. The NIST EI mass spectra for the TM S derivatives of choles terol (top), ergosterol (middle) and lanosterol (bottom).

PAGE 154

154 Figure C-3. Total ion chromatogr ams (TICs) and electron ionizat ion chromatograms (EICs) of sylilated sterols. A represen ts wild type and B, is izh3.

PAGE 155

155 APPENDIX D CHROMATOGRAMS AND MASS SPECTRA OF ERGOSTEROL ANALYZED BY HPLCAPCI-MS Figure D-1. High perfomace liquid chromatography-atmospheric pressure chemical ionization in tandem, with mass spectrometric analysis (HPLC-APCI-MS) of standards. The HPLC-APCI-MS BPC (topmost) shows that er gosterol (II) eluted first, followed by lanosterol (IV) and dihydrolanosterol (III).

PAGE 156

156 Figure D-2. Atmospheric pressure chemical i onization chromatogram (APCI)-mass spectra for the sterol standards. The figure shows the [(M+H)-H2O]+. ion as the base peak for ergosterol (II), lanosterol (IV), dihydrolanosterol ( III) and cholesterol (I).

PAGE 157

157 Figure D-3. Base peak chromatogram (BPC) an d electron ionization chromatograms (EICs) for wild type. The chromatograms indicate th at ergosterol (II) is present in higher concentration than lanosterol (IV).

PAGE 158

158 Figure D-4. Base peak chromatogram (BPC) an d electron ionization chromatograms (EICs) for izh3Chormatograms show that ergostero l (II) was also present in higher concentration than lanosterol (IV).

PAGE 159

159 LIST OF REFERENCES Arthinggton-Skaggs BA, Crowell DN, Yang H, Sturley SL, and Bard M (`1996) Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast ergoste rol pathway. FEBS Lett 392: 161-165 Arthington-Skaggs BA, Jradi H, Desai T, Mo rrison, CJ (1999) Quantitation of ergosterol content: Novel method for determination of fluconazole susceptibility of Candida albicans J Clin Microbiol 37: 3332-3337 Bagnat M, Kernen S, Shevchenko A, Shevchenko A, and Simons K (2000) Lipid rafts function in biosynthetic delivery of protei ns to the cell surface in yeast. Proc Natl Acad Sci USA 97: 3254-3259 Bagnat M, Chang A, and Simons K (2001) Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast. Mol Biol Cell 12 : 4129-4138 Baida GE, Kuzmin NP (1995) Cloning and primar y structure of a new hemolysin gene from Bacillus cereus Biochim Biophys Acta 1264: 151-154 Baida GE, KUzmin NP (1996) Mechanism of action of hemolysin lll from Bacillus cereus. Biochim Biophys Acta 1284: 122-124 Bailey RB, and Parks LW (1975) Yeast sterol esters and their relationship to the growth of yeast. J Bacteriol 124: 606-612 Baker RT, and Board PG (1989) Unequal crossove r generated variation in ubiquitin coding unit number at the hyman Ubc polyubiquitin. Am J Hum Genet 44: 534-542 Baudry K, Swain E, Rahier A, Germann M, Batta A, Ronde S, Mandal S, Henry K, Tint GS, Edlind T, Kurtz M, and Nickels Jr JT (2001) The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cersevisiae. J Biol Chem 276: 12702-12711 Baumgartner U, Hamilton B Piskacek M, Ruis H, Rottensteiner, H. (1999) Functional analysis of the Zn2Cys6 transcription factors Oaf1p and Pip2p. J Biol Chem 274: 22208-22216 Bhuiyan MSA, Ito Y, Nakamura A, Tanaka N, Fujita K, Fukui H, and Takegawa K (1999) Nystatin effects on vacuolar function in Saccharomyces cerevisiae. Biosc Biotechnol Biochem 63: 1075-1082 Bielawski J, Szulc ZM, Hannun YA, Bielawska A (2006) Simultaneous quantitative analysis of bioactive sphingolipids by high-pe rformance liquid chromatography-tandem mass spectrometry. Methods 39 : 82-91 Black PN, Frgeman NJ, and DiRusso CC (2000) Long-chain acyl-CoA-dependent regulation of gene expression in bacter ia, yeast and mammals. J Nutr 130: 305S-309S

PAGE 160

160 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37 : 911-917 Bolard J (1986) How do the polyene macrolide antibiotics affect the cellular membrane properties?. Biochim Biophys Acta 864: 257-304 Breivik ON, Owades JL (1957) Spectrophotometric semi-microdetermination of ergosterol in yeast. Agric Food Chem 5: 360-363 Carrillo-Muoz AJ, Giusiano G, Ezkurra PA, Quinds G (2006) Antifunga l agents: mode of action in yeast cells. Rev Esp Quimioter 19: 130139 Cherry JM, Alder C, Ball C, Chervitz SA, Dwight, SS, Hester, ET, Jia Y, Juvik G, Roe T, Schroeder M, Weng SA, Botstein D (1998) Sacchraromyces genome database. Nucleic Acids Res 26: 73-79 Choi JY, Stukey J, Hwang SY, and Martin CE (1996) Regulatory elements that control transcription activation a nd unsaturated fatty acid-m ediated repression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 27: 3581-3589 Chung CY, and Obeid LA, (1999) Use of yeast as a model system for studies of sphingolipid metabolism and signaling. Methods Enzymol 311: 319-331 Cowart LA, Obeid L (2007) Yeast sphingolipid s, recent developments in understanding biosynthesis, regulation, and function. Biochim Biophys Acta 1771: 421-431 Daum G, Tuller G, Nemec T, Hrastnik C, Ballia nao G, Cattel L, Milla P, Rocco F, Conzelmann A, Vionnet C, Kelly DE, Kelly S, Schweizer E, Schuller HJ, Hojad U, Greiner E, Finger K (1999) Systematic analysis of yeast strains with possible defects in lipid metabolism. Yeast 15: 601-614 DeRusso CC, Black PN, and Weimar JD (1999) Molecular inroads into the regulation and metabolism of fatty acids lessons from bacteria. Prog Lipid Res 38: 129-197 Dickson R (1998) Sphingolipid functions in Saccharomyces cerevisiae : Comparison to mammals. Annu Rev Biochem 67: 27-48 Dickson RC, Lester RL, (2002) Sphingolipid functions in Saccharomyces cerevisiae. Biochim Biophys Acta 1583: 13-25 Dupont D ( 2006) Use of topical antifungal agents. Therapie 61 : 251-254 Dupre S, Haguenauer-Tsapis R (2003) Raft partitioning of the yeast uracil perm ease during trafficking alonf the endocytic pathway. Traffic 4: 83-96 Edsall LC, Pirinanow GG, Spiguel S (1997) Invo lvement of Sphingosine 1-phosphate in nerve growth factor-mediated neurona l survival and differentiation. J Neurosci 17 : 6952-6960

PAGE 161

161 Eide D, Broderious M, Fett J, Guerinot ML (199 6) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 93: 5624-5628 Eide D, Zhao H, Butler E, and Rodgers J (1999) Regulation of zinc homeostasis in yeast by the ZAP1 transcriptional activator. Metals and Genetics 24: 325-338 Einerhand AWC, Kos WT, Distel B and Tabl HF (1993) Character ization of a tr anscriptional control element involved in pr oliferation of peroxisomes in yeast in response to oleate. Eur J Biochem 314: 323-331 Eisenkolb M, Zenzmaier C, Leitner E, and Schneite r R (2002) A specific structural requirement for ergosterol in long-chain fatty acid synthesi s mutants important for maintaining raft domains in yeast. Mol Biol Cell 13 : 4414-4428 Fujita T, Inoue K, Yamamoto S, Ikumoto T, Sasaki S, Toyama R, Chiba K, Hoshino Y, Okumoto T (1994) Fungal metabolites. Part 11. A potent im munosuppressive activity found in Isaria sinclairii metabolite. J Antibiot 47: 208-215 Gable K, Slife H, Bacikova D, Monaghan E, and Dunn TM (2000) Tsc3p is a 80-amino acid protein associated with serine palmitolyltransfe rase and required for optimal enzyme activity. J Biol Chem 276: 7597-7603 Gachotte D, Pierson CA, Lees ND, Barbuch R, Koegel C, and Bard M (1997) A yeast sterol auxotroph ( erg25) is rescued by addition of azole antif ungals and reduced levels of heme. Proc Natl Acad Sci USA 94: 11173-11178 Gaigg B, Toulmary A, and Schneiter R (2006) Very long-chain fatty acid-containing lipids rather than sphingolipids per se are required for raft as sociation and stable surface transport of newly synthesized plasma membrane ATPase in yeast. J Biol Chem 281: 34135-34145 Gaither LA, and Eide D (2001) Eukaryotic zinc transporters and their regulation. Biometals 14 : 251-270 Gietz RD, and Woods RA (1994) High efficiency transformation with lithium acetate. In JR Johnson (ed), Molecular Genetics of Yeast: A Practical Approach Oxford University Press, NY, 121-134 Gerst N, Ruan B, Pang J, Wilson WK, and Schr oepfer GJ Jr (1997) An updated look at the analysis of unsaturated C27 sterols by gas chromatogr aphy and mass spectrometry. J Lipid Res 38:1685-1701 Ghannowm MA, and Rice LB (1999) Antifungal ag ents: Mode of acti on, mechanisms of resistence, and correlation of these m echanisms with bacterial resistence. Clin Microbiol Rev 12 : 501-517 Gitan RS, and Eide D (2000) Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter. Biochem J 346: 329-336

PAGE 162

162 Goldstein AL, Pan X, & McCusker JH (1999) Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. Yeast 15: 507-511 Goldstein AL, and McCusker JH (1999) Three ne w dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae Yeast 15 : 1541-1553 Gmez-Mountn C, Lacalle RA, Mira E, Jimnez-Barada S, Barber DF, Carrera AC, Martnez-A C, and Maes S (2004) Dynamic re distribution of raft domains as an organizing platform for signaling during cell chemotaxis. J Chem Biol 164 : 759-768 Gong P, Hu B, Stewart D, Ellerbe M, Figuero a YG, Blank V, Beckman BS, and Alam J (2001) Cobalt induces heme oxygenase-1 expression by a hypoxia-inducible factor-independent mechanism in chinese hamster ovary cells. J Biol Chem 276: 27018-27025 Grossmann G, Opekarova M, Novakoca L, Stol z J, and Tanner W (2006) Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae Erukatyot Cell 5 : 945-953 Grossmann G, Opekarov M, Malinsky J, Weig-Meckl I, and Tanner W (2007) Membrane potential governs lateral segreg ation of plasma membrane pr oteins and lipids in yeast. EMBO J 26: 1-8 Guarente L (1983) Yeast promoters and lacZ fusion designed to study expression of cloned genes in yeast. Methods Enzymol 101: 181-191 Han, G, Gable K, Kohlwein SD, Beaudoin F, Napier JA, and Dunn TM (2002) The Saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase. J Biol Chem 277: 35440-35449 Hannun YA, and Bell RM (1989) Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science 243: 500-507 Hannun YA, and Obeid L (2002) The ceramide ce ntric universe of lipid-mediated cell regulation: stress encoun ters of the lipid kind. J Biol Chem 277: 25847-25850 Hapala I, Klobu nikov V, Maz ov K, and Koht P (2005) Two mutants selectively resistant to polyenes revceal distinct mechanisms of an tifungal activity by nystatin and amphotericin B. Transations 33: 1206-1209 Hauwaerts D, Alexandre G, Da s SK, Vanderleyden J, Zhulin IB (2002) A major chemotaxis gene cluster in Azospirillum brasilense and relationships between ch emotaxis operon in alphaproteobacteria. FEMS Microbiol Lett 208: 61-67 Hertz GZ, and GD Stormo (1999) Identifying DNA and protein patterns with statistically significant alignments of multiple sequences. Bioinformatics 15: 563-577

PAGE 163

163 Hiltunen JK, Mursula AM, Rottensteiner H, Wier enga PK, Kastaniotis AJ, Gurvitz A (2003) The biochemistry of peroxisomal -oxidation in the yeast Saccharomyces cerevisiae. FEMS Miocrobiol Rev 27: 35-64 Ho E (2004) Zinc deficienc y, DNA damage and cancer risk. J Nutr Biochem 15: 572-578 Hsieh M-H, and Goodman HM ( 2005) A novel gene family in encoding putative heptahelical transmembrane proteins homologous to human adiponectin receptors and progestin receptors. J Exp Bot 56: 3137-3147 Huang LE, Arany Z, Livingston DM, and B unn F (1996) Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive st abilization of its subunit. J Biol Chem 271: 32253-32259 Jiang Y, Vasconcelles MJ, Wretzel S, Light A, Gilooly L, McDaid K, Oh C, Martin CE, and Goldberg, MA (2002) Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: Impact on LORE-dependent gene expression. Eukaryot Cell 1 : 481490 Jiang Y, Vasconvelles MJ, Wretzel S Light A, Martin CE, and Goldberg MA (2001) MGA2 is involved in the low-oxygen response elemen t-dependent hypoxic induction of genes in Saccharomyces cerevisiae. Mol Cell Biol 12: 6161-6179 Kandasamy P, Vemula M, Oh C, CHellappa R, and Martin CE (2004) Re gulation of unsaturated fatty acid biosynthesis in Saccharomyces J Biol Chem 279: 36586-36592 Karpichev IV, Luo Y, Marians RC, and Sm all GM (1997) A comp lex containing two transcription factors regulate s peroxisome proliferation a nd the coordinate induction of oxidation enzymes in Saccharomyces cerevisiae Mol Cell Biol 17: 69-80 Karpichev IV, and Small GM (1998) Global regu latory functions of Oaf12p and Pip2p (Oaf2p) transcription factors that regulate ge nes encoding peroxisomal proteins in Saccharomyces cerevisiae. Mol Cell Biol 18: 6560-6570 Karpichev IV, Cornivelli L, and Small GM (2002) Multiple regulatory roles of a novel Saccharomyces cerevisiae protein, encoded by YOL002c in lipid and phosphate m etabolism. J Biol Chem 277: 19609-1961 Kastaniotis AJ, and Zitomer RS (2000) Rox1 mediated repression: Oxygen dependent repression in yeast. Adv Exp Med Biol 475: 185-195 Keen CL, Peters JM, Hurley LS (19 89) The effect of valproic acid on 65Zn distribution in the pregnant rat. J Nutr 119: 607-611 Kerridge D (1986) Mode of action of c linically important antifungal drugs. Adv Microb Physiol 27: 1-72

PAGE 164

164 Kerridge D, Koh TY, Marriott MS and Gale EF (1976) Microbiology and plant protoplasts. In J. F. Peberdy, A. H. Rose, H. J. Rodger, and E. C. Cocking (ed.), Microbiology and plant protoplasts Churchill Livingst on, London, England 23-38 Kim, H, Melen, K. & von Heijne G (2003) Topology models for 37 Saccharomyces cerevisiae membrane proteins based on C-terminal reporter fusions and predictions. J Biol Chem 278: 10208-10213 Koh JY, Suh SW, Gwang BJ, He YY, Hsu CY, Choi DW (1996) The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272: 1013-1016 Kontoyiannis DP, Lewis RE (2002) Antifungal drug resistance of pathogenic fungi. Lancet 359: 1135-1144 Kos W, Kal, AJ, van Wilpe S, and Tabak HF (1995) Expression of genes encoding peroxisomal proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control. Biochim Biophys Acta 1264: 79-86 Kbler E, Dohlman HG, and Lisanti, MP ( 1996) Identifiaction of triton X-100 insoluble membrane domains in the yeast Saccharomyces cerevisiae J Biol Chem 271: 332975-32980 Kumar A, Xia YP, Laipis PJ, Fletcher BS, and Frost SC (2004) Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adypocytes. Am J Physiol Endocrinol Metab 286: 568-576 Kupchak BR, Garitaonandia I, Villa NY, Mulle n MB, Weaver MG, Regalla LM, Kendall EA, Lyons TJ (2007) Probing the mechanism of FET3 repression by Izh2p overexpression. Biochim Biophys Acta 1773: 1124-1132 Kwast KE, Burke PV, Staahl BT, and Poyton RO (1999) Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcripti on of a subset of hypoxic genes. Proc Natl Acad Sci USA 96: 5446-5451 Lampen JO, Arnow PM, and Safferman RS (1960) Mechanism of protection by sterol against polyene antibiotics. J Bacteriol 80: 200-206 Lauwers E, and Andr B (2006) Association of yeast transporters with detergent-resistant membranes correlates with their cell-surface. Traffic 7: 1045-1059 Leber A, Fischer P, Schneiter R, K ohlwein SD, Daum G (1997) The yeast mic2 mutant is defective in the form ation of mannosyl-diinositolphosphorylceramide FEBS Lett 411: 211-214 Lederberg J (1950) The -galactosidase of Escherichia coli strain K-12. J Bacteriol 60 : 381-392 Lee MCS, Hamamoto S, and Schekman R (2002) Ceramide biosynthesis is required for the formation of the oligomeric H+-ATPase Pma1p in the yeast endoplasmic reticulum. J Bol Chem 277: 22395-22401

PAGE 165

165 Lester RL, and Dickson RC (1993) Sphingolip ids and inositol c ontaining headgroups. Ad Lipid Res 26: 253-274. Luo Y, Karpichev IV, Kohanski RA, and Sma ll GM (1996) Purificati on, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involve d in the activation of POX1 J Biol Chem 271: 12068-12075 Lyons TJ, Gasch AP, Gaither LA, Botstein D, Brown PO, and Eide DJ (2000) Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast. Proc Natl Acad Sci USA 97: 7957-7962 Lyons TJ, Villa NY, Regalla LM, Kupcha k BR, Vagstad A, and Eide DJ (2004) Metalloregulation of yeast membrane steroid receptor homologs. Proc Nat Acad Sci USA 101: 5506-5511 MacDiarmid CW, Gaither LA, and Eide D (2000) Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae 19: 2845-2855 MacDiarmid CW, Milanick MA, and Eide DJ (20 02) Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J Biol Chem 277: 39187-39194 Mandala SM, Thornton RA, Frommer BR, Curotto JE, Rozdilsky W, Kurtz MB, Giacobbe R A, Billis GF, Cabello MA, Martin I, Pelaez, F, Harris, GH (1995) The discovery of australifungin, a novel inhibitor of sphinganine N-acyltransfer ase from Sporormiella australis. Producing organism, fermentation, isolat ion, and biological activity. J Antibiot 48: 349-356 Mao C, Xu R, Bielawska A, Szulc ZM, Obei d LM (2000a) Cloning and characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide. J Biol Chem 275: 31369-31378 Mao C, Xu R, Bielawska A, Obeid LM (2000b) Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA -independent) ceramide synthase activity. J Biol Chem 275: 6876-6884 Markwell MAK, Haas SM, Tolber t NE, and Bierber LL (1981) Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol 72: 296-303 Marini F, Arnow P, and Lampen JO (1960) The e ffect of monovalent cati ons on the inhibition of yeast metabolism by nystatin. J Gen Microbiol 24 : 51-62 McDounough VM, Stukey JE, and Martin CE (1992) Specificity of unsaturated fatty acidregulated expression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 267: 5931-5936 Megumi S, Shin-ichiro M, Mariko A, Tsutoma F, Kazuhiro I, and Haruyk i I (2005) Effects of culture conditions on Ergosterol biosynthesis by Saccharomyces cerevisiae Biosci Biotechnol Biochem 69: 2381-2388.

PAGE 166

166 Merrill AH Jr, Wang E, Mullins RE, Jamison WC, Nimkar S, and Liotta DC (1988) Quantitation of free sphingosine in liver by highperformance liquid chromatography Anal Biochem 171: 373-381 Merrill AH Jr, Caligan TB, Wang E, Pe ters K, and Ou J (2000) Analysis of sphingoid bases and sphingoid base-1-phosphates by high performance liquid chromatography. Methods Enzymol 312: 3-9 Merrill AH Jr, Schmelz EM, Dillehay DL, Spigel S, Shayman JA, Schroeder JJ, Riley RT, Voss KA, and Wang E (1997) Sphingolipids-The Enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 142: 208-225 Merrill AH Jr, Van-Echten G, Wang E, and Sandhoff K (1993) Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingoilipid biosynthesis in cultured neurons in situ J Biol Chem 268: 27299-27306 Menaldino DS, Bushnev A, Sun A, Liotta DC, Symolon H, Desai K, Dillehay DL, Peng Q, Wang E, Allegood J, Trotman-Pruett S, Sullards MC, Merrill AH Jr (2003) Sphingoid bases and de novo ceramide synthesis: enzymes involved, pharmacology and mechanisms of action. Pharmacol Res 47: 373-381 Mo C, Valachovic M, Randall SK, Nickels JT, a nd Bard M (2002) Proteinprotein interactions among C-4 demethylation enzymes involve d in yeast sterol biosynthesis. Proc Natl Acad Sci USA 99: 9739-9744 Moffett S, Brown DA, Linder ME (2000) Lipid-depe ndent targeting of G pr oteins into rafts. J Biol Chem 275: 2191-2198 Mukhopadhyay K, Kohli A, and Prasad R (2002) Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46: 3695 Mllner H, Deutsch G, Leitner E, Ingolic E, and Daum G (2005) YEH2/YLR020c encodes a novel steryl ester hydro lase of the yeast Saccharomyces cerevisiae. J Biol Chem 280: 1332113328 Nakagawa Y, Sugioka S, Kaneko Y, and Harash ima S (2001) O2R, a novel regulatory element mediating Rox1p-independent O2 and unsaturated fatty acid repression of OLE1 in Saccharomyces cerevisiae J Bacteriol 183: 745-751 Narasimhan ML, Damsz B, Coca MA, Ibeas JI, Y un D-J, Pardo JM, Hasegawa PM, and Bressan RA (2001) A plant defense protei n induces microbial apoptosis. Mol Cell 8: 921-930 Narasimhan ML, Coca MA, Jin J, Yamauchi T, Ito Y, Kadowaski T, Kin KK, Pardo JM, Damsz B, Hasegawa PM, Yun DJ, and Bressan RA (2005) Osmotin is a homolog of mammalian adiponectin and controls apopt osis in yeast though a homol og of mammalian adiponectin receptor. Mol Cell 17 : 171-180

PAGE 167

167 Nothwehr SF, Conibear E, and Stevens TH (1995) Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane. J Cell Biol 129: 35-46 Oteiza PI, Olin KL, Fraga CG, and Keen CL (1995) Zinc deficiency causes oxidative damage to proteins, lipids and DNA in rat testes. J Nutri 125: 823-829 Peng J, Schwartz D, Elias JE, Thoreen CC, Che ng D, Marsischky G, Roelofs J, Finley D, and Gygi S (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21: 921-926 Perry DK (2002) Serine palmitoyltr ansferase: role in apoptotic de novo ceramide synthesis and other stress responses. Biochim Biophys Acta 1585 : 145-152 Pike LJ (2003) Lipid rafts: bringing order to chaos. J lipid Res 44: 655-665 Pizzirusso M, and Chang A (2004) Ubiquitin-media ted targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast. Mol Biol Cell 15: 2401-2409 Rehli M, Krause SW, Schwarzfischer L, Kreutz M, Andreesen R (1995) Molecular cloning of a novel macrophage maturation-associated transcri pt encoding a protein with several potential transmembrane domains. Biochem Biophys Res Commun 217 : 661-667 Reiner S, Micolod D, and Schneider R (2005) Saccharomyces cerevisiae a model to study sterol uptake and transport in eukaryotes. Biochem Soc Trans 33: 1186-1188 Reiner S, Micolod D, Zellnig G, and Schneiter R (2006) A genomewide scr een reveals a role of mitochondria in anaerobic uptake of sterols in yeast Mol Biol Cell 17 : 90-103 Rodriguez RJ, Low C, Bottema CD, and Parks LW (1985) Multiple functions for sterols in Saccharomyces cerevisiae. Biochim Biophys Acta 837: 336-343 Rutherford JC, Jaron S, and Winge DR. (2003) Aft1 and Aft2 mediate iron-responsive gene expression in yeast through re lated promoter elements. J Biol Chem 278: 27636-27643 Rutherford JC, and Bird AJ (2004) Metal-responsive transcription factors that regulate iron, zinc and copper homeostasis in eukaryotic cells. Eukaryot Cell 3: 1-13 Sambrook J, and Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd. edition, Cold Spring Harbor Lab Pre ss, Cold Spring Harbor, NY Schnabl M, Daum G, Pichler H (2004) Multip le lipid transport pathways to the plasm a membrane in yeast. Biochim Biophys Acta 1687: 130-140 Serrano R (1988) H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: Purificatio n and reconstitution. Methods Enzymol 157: 533-544 Shapiro L, Scherer PE (1998) The crystal stru cture of a complement-1q family suggests an evolutionary link to tu mor necrosis factor. Curr Biol 8: 335-338

PAGE 168

168 Shimada K, Miyazaki T, Daida H (2004) Ad iponectin and atherosclerotic disease. Clin Chim Acta 344: 1-12 Simons K, and Ikonen E (1997) Func tional rafts in cell membranes. Nature 387: 569-572 Sims KJ Spassieva SD, Voit EO, and Obeid LM (2004) Yeast sphingolipid metabolism: clues and connection. Biochem Cell Biol 82 : 45-61 Smith SW, and Lester, RL (1974) Inositol phosp horylceramide, a novel substance and chief member of a major group of yeast sphingolipids containi ng a single inositol phosphate. J Biol Chem 249: 3395-3405 Streit F, Niedmann P-D, Shipkova M, ArmstrongVW and Oellerich M (2001) Rapid and Sensitive Liquid ChromatographyTandem Mass Spectrometry Method for Determination of Monoethylglycinexylidide. Clin Chem 47: 1853-1856 Swain E, Baundry K, Strukey J, McDonough V, Germ ann M, and Nickels JT Jr (2002) Steroldependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae J Biol Chem 277: 26177-26184 Taylor KM, Nicholson RI (2003) The LZT proteins, the LIV-1 subf amily of zinc transporters. Biochim Biophys Acta 78452: 1-15 Toulmay A, Schneiter R (2007) Lipid-depende nt surface transport of the proton pumping ATPase: A model to study plasma membrane biogenesis in yeast. Biochimie 80: 249-254 Umebayaski K (2003) The roles of ubiquitin a nd lipids in protein sorting along the endocytic pathway. Cell Struct Function 28 : 443-453 Umebayashi K, and Nakano A (2003) Ergosterol is required for targeti ng of tryptophan permease to the yeast plasma membrane. J Cell Biol 161: 1117-1130 Valachovi M, Hronsk L, Hapala I (2001) Anaerobios is induces complex changes in sterol esterification pattern in the yeast Saccharomyces cerevisiae. FEMS Microbiol Lett 197 : 41-45 Valle B, and Riezman H (2005) Lip1p: a nove l subunit of acyl-CoA ceramide synthase. EMBO J 24: 730-741 Vasconcelles MJ, Jiang Y, McDaid K, Gilooly L, Wretzel S, Porter DL, Martin CE, and Goldberg MA (2001) Identification and char acterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. J Biol Chem 276: 14374-14384 Veen M, Stahl U, Lang C (2003) Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Res 4: 87-95

PAGE 169

169 Veen M, and Lang C (2005) Interactions of the ergosterol biosynthetic pa thway with other lipid pathways. Biochem Soc Trans 33: 1178-1181 Veronese P, Ruiz M, Coca MA, Hernandez-Lop ez A, Lee H, Ibeas JI, Damsz B, Pardo JM, Hasegawa PM, Bressan RA (2003) In defense agai nst pathogens. Both plant sentinels and foot soldiers need to know the enemy. Plant Physiol 131: 1580-1590 Wach A, Brachat A, Pohlmann R, and Philli ppsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808 Watson PF, Rose ME, and Kelly SL (1998) Isolation and analysis of ke toconazole resistant mutants of Saccharomyces cerevisiae J Med Vet Mycol 3: 153-162 Woods RA (1971) Nystatin-resistant mutants of yeast: Alterations in sterol content. J Bacteriol 108: 69-73 Wu WI, McDonough VM, Nickels JT Jr, Jo J, Fischl AS, Vales TR, Merrill AH Jr, and Carman GM (1995) Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1. J Biol Chem 270: 13171-13178 Wu CY, Bird AJ, Winge DR, and Eide DJ (2006) Regulation of the yeast TSA1 peroxiredoxin by ZAP1 is an adaptive response to the oxidative stress of zinc deficiency. J Biol Chem 282: 2184-2195 Yamaguchi-Iwai Y, Stearman R, Dancis A, and Klausnerl RD (1996) Iron-regulated DNA binding by the AFT1 protein contro ls the iron regulaton in yeast. EMBO J 15: 3377-3384 Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yoko mizo T, Kita, S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami, K, Otheki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T (2003a) Cloning of adiponectin rece ptors that mediate antidiabetic metabolic effects. Nature 423: 762-769 Yamuchi T, Hara K, Kubota N, Terauchi Y, Tobe K, Froguel P, Nagai R, Kadowaki T (2003b) Dual role of adiponectin/Acrp30 in vivo as an anti-diabetic and anti-atherogenic adipokine. Curr Drug Targets Immune Endocr Metabol Disord 3 : 243-254 Yang HM, Bark DA, Bruner A, Gleeson RJ, Deckel baum G, Aljinovic TM, Pohl R, Rothstein R, and Sturley SL (1996) Ster ol sterification in yeas t: a two-gene process. Science 272 : 1353-1356 Yun D-J, Zhao Y, Pardo JM, Narasimhan ML, Damsz B, Lee H, Abad LR, Paino DUrzo M, Hasegawa PM, and Bressan RA (1997) Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proc Natl Acad Sci USA 94: 7082-7087 Zhang S, Burkett TJ, Yamashita I, and Garf inkel, DJ (1997) Gene tic redundancy between SPT23 and MGA2 : Regulators of Ty-induced mutati ons and Ty1 transcription in Saccharomyces cerevisiae. Mol Cell Biol 17: 4718-4729

PAGE 170

170 Zhang S, Skalsky Y, and Garfinkel DJ (1999) MGA2 or SPT23 is required for transcription of the 9 fatty acid desaturase gene, OLE1 and nuclear membrane integrity in Saccharomyces cerevisiae. Genetics 151 : 473-483 Zhao H, Eide D (1996a) The yeast ZRT1 gene encodes the zinc transporter protein of a highaffinity uptake system induced by zinc limitation. Proc Natl Acad Sci USA 93: 2454-2458 Zhao H, Eide D (1996b) The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae. J Biol Chem 271: 23203-23210 Zhao H, and Eide D (1997) Zap1p, a metalloregu latory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae. Mol Cell Biol 17: 5044-5052 Zhao H, Butler E, Rodgers J, Spizzo T, Duesterh oeft S (1998) Regulation of zinc homeostasis in yeast by binding of the ZAP1 tr anscriptional activator to zinc -responsive promoter elements. J Biol Chem 273: 28713-28720 Zhu Y, Bond J, Thomas P (2003a) Identification, classification, and partia l characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor Proc Natl Acad Sci USA 100: 2237-2242 Zhu Y, Rice CD, Pang Y, Pace M, Thomas P (2003b) Cloning, expression, and characterization of a membrane progestin recptor and evidence that it is an in termediary in meiotic maturation of fish oocytes. Proc Natl Acad Sci USA 100: 2231-2236 Zinser E, Paltauf F, and Dunn G (1993) Sterol composition of yeast organelle membranes and subcellular distribution of enzyme s involved in sterol metabolism. J Bacteriol 175 : 2853-285

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171 BIOGRAPHICAL SKETCH Nancy Y. Villa was born in La Union (Valle), a small town of Colombia. After receiving a B.S. in education of chemistry from the Universi dad Santiago de Cali, and a B.S. in chemistry from the Universidad del Valle, she joined the gr aduate program in department of chemistry of the University of Florida where she pursued a doctorate, working under th e supervision of Dr. Thomas Lyons. One of her major interests before starti ng her PhD was to investigate about the development and progression of cancer, at biochemical level. This interest was mainly motivated by familial reasons that put her close to the development and consequences of such a disease. After completing her PhD, she will join the rese arch group of Dr Grant McFadden, to work as a post doctoral fellow in the department of Molecu lar Genetics and Microbio logy in the school of medicine at the University of Florida. In this group, she will investigate biochemical aspects that determine the malignance of viru ses and their implications in the development of cancer.