Post-translational Modification and the 20S Proteasome System of the Haloarchaeon Haloferax volcanii

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Post-translational Modification and the 20S Proteasome System of the Haloarchaeon Haloferax volcanii
Humbard, Matthew
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[Gainesville, Fla.]
University of Florida
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Doctorate ( Ph.D.)
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University of Florida
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Microbiology and Cell Science
Committee Chair:
Maupin, Julie A.
Committee Members:
Preston, James F.
De Crecy-Lagard, Valerie
Shanmugam, Keelnatham T.
Denslow, Nancy D.
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Subjects / Keywords:
Acetylation ( jstor )
Amino acids ( jstor )
Archaea ( jstor )
Enzymes ( jstor )
Haloferax volcanii ( jstor )
Ions ( jstor )
Mass spectroscopy ( jstor )
Phosphorylation ( jstor )
Proteins ( jstor )
Yeasts ( jstor )
Microbiology and Cell Science -- Dissertations, Academic -- UF
acetylation, acetyltransferase, activating, archaea, atpase, dimensional, electrophoresis, haloarchaea, haloferax, knock, mass, methylation, modification, ms, nucleotidase, out, pan, phosphorylation, posttranslational, protease, proteasome, proteome, samp, spectrometry, two, ubiquitin, volcanii
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Microbiology and Cell Science thesis, Ph.D.


While much of the study of proteasome systems in all organisms, eukaryotes and prokaryotes, focuses on substrate recognition, ubiquitin-like modifiers, or protein targeting, the 20S core particle and regulatory ATPases are subject to a wide variety of modifications that include, but are not limited to, phosphorylation and acetylation. Despite the large number of modifications known, the roles these modifications play within various proteasomes remain unclear, as they remain relatively unstudied. This study focused on identifying and studying specifically the sites of modification at the residue level, including mapping and altering these sites in order to better understand how proteasomes themselves are assembled and controlled. Through the use of proteomic approaches, we have identified post-translational modification on all three subunits of 20S core particles in Haloferax volcanii as well as on one of the proteasome activating nucleotidases, PanA. In addition to mapping phosphorylation sites to the alpha1, alpha2, beta subunits of the 20S core particle, and PanA, both alpha-type subunits are also N-terminally acetylated. A novel post-translational modification, methylesterification, was also identified on aspartic and glutamic acid residues of the alpha1 protein. This is the first report of methylation of a proteasomal subunit. Phenotypic characterization of variant proteins unable to undergo N-terminal acetylation revealed that the N-terminus of alpha1 plays a role in the stability of the protein, and that this can alter the hypo-osmotic response, growth rate and yield. Further investigation into N-terminal acetylation in Hfx. volcanii revealed this is a generalized pathway and nearly 30 % of soluble proteins are modified in this manner. Additionally, variant alpha1 proteins that were unable to undergo phosphorylation severely inhibited growth of Hfx. volcanii cells and altered their pigmentation. A candidate serine/threonine kinase from the atypical Rio-kinase family, was shown to catalyze autophosphorylation in vitro and phosphorylate recombinantly purified alpha1 proteins in vitro. Altogether, this study demonstrates that the proteins that make up the proteasomal system in Hfx. volcanii are heavily modified post-translationally, there is a generalized protein N-terminal acetylation pathway at work in the haloarchaeon, and a Rio type-I kinase autophosphorylates as well as phosphorylates alpha1 in vitro. ( en )
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Thesis (Ph.D.)--University of Florida, 2009.
Adviser: Maupin, Julie A.
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by Matthew Humbard.

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Copyright Humbard, Matthew. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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2 2009 Matthew A. Humbard


3 To my wife for her love, understanding, and patience


4 ACKNOWLEDGMENTS I would like to acknowledge and thank Dr. Julie MaupinFurlow for the opportunity to pursue this project in her lab. Without the independence she granted me to pursue side projects and satisfy my intellectual curiosity I would have never made it. Her constant guidance a nd support was the one thing I knew I could count on throughout my graduate education. I would also like to thank the members of my committee Drs. Valerie d Crecy Lagard, K. T. Shanmugam, James Preston, and Nancy Denslow for their advice attention, and t ime. I would also like to thank Dr. Rasche for her helpful input on my project and insights into archaeal physiology. I would like to thank my coworkers in Dr. MaupinFurlows lab especially Drs. Zhou, Reuter, and Kirkland. Dr. Zhou was instrumental in the creation of acetyl tranfer ase knockout strains and the proteasomal knockout strains, Dr. Reuter assisted me with protein chromatography and activity assays, and Dr. Kirkland helped me with proteomics and held insightful discussions concerning my project and related side projects. I would also like to thank some undergraduate researchers, Chris Tzikas, Kristin Toscano, Steve Garret, Jessica Coleman, Dave Krause, Jonathan Pritz and Cortlin Phillips. Chris and Kristin cloned and expressed different membrane bound proteases in Haloferax volcanii Steve Garret and Jessica Coleman II construct that was instrumental to the development of this work. I am especially indebted to Drs. Stan Steven s Sixue Chen, and Alfred Chung as well as Scott McClung from the ICBR protein chemistry core for their support with my project. Dr. Stevens experience and knowledge of mass spectrometry protocols and data interpretation were integral in the genesis of my graduate project. Dr. Alfred Chung synthesized artificial peptides for confirmation of phosphopeptides and AQUA studies. Scott McClung trained me to use the LCQ Deca for personal projects and taught me how to set up and use the specific equipment, one of the most valuable parts of my graduate education. I also want to thank Dr. Jennifer Busby at


5 the Scripps Research Institute in Jupiter, Florida for allowing me to come and visit her laboratory to learn about mass spectrometry and run samples on her equipment. This was a valuable trip and set the tone for my graduate project. I would also like to thank Dr. Sa vita Shanker at the ICBR DNA sequencing core for her quality work sequencing DNA of constructs and clones. My graduate career would have never been possible without the advice of Dr. Rajeev Misra at Arizona State University who gave me my first scientific job and convinced me not to drop out of college. I can not express how much that has changed the arc of my life and how that dec ision has led me to this point. Finally, I need to thank my family for all their support. I would like to thank my grandfather D r. Ralph Kelting who exposed me to biology and the university environment at a young age. I have aspired to emulate him in as many ways as possible. I want to thank my wife for her support and patience. I would not have completed this task without her love and understanding.


6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGU RES .......................................................................................................................11 LIST OF ABBREVIATIONS ........................................................................................................13 ABSTRACT ...................................................................................................................................17 CHAPTER 1 LITERATURE REVIEW .......................................................................................................19 Introduction .............................................................................................................................19 Proteasome Structure and Function ........................................................................................19 20S Core Particle Structure and Subunit Arrangement ...................................................20 Assembly of 20S Core Particle ................................................................................22 subunit Processing ................................................................................................26 subunits .........................................................................29 Proteasome Activation ............................................................................................................31 19S Regulatory P article ...................................................................................................31 11S Regulator / PA28 ......................................................................................................32 PA200 (Blm10) ...............................................................................................................34 Proteasome A ctivating Nucleotidases (PAN) .................................................................35 Targeting of Proteins for Degradation ....................................................................................36 SsrA tagging ....................................................................................................................37 N end Rule .......................................................................................................................38 Ubiquitin Conjugation System and Polyubiquitin Chains ...............................................40 Pupylation ........................................................................................................................42 Co and Post translational Modification .................................................................................43 N terminal Processing and Maturation ............................................................................44 Deformylation of methionine ...................................................................................44 Methionine removal .................................................................................................45 N terminal acetylation ..............................................................................................46 Acylamino acid hydrolysis .......................................................................................49 Phosphorylation ...............................................................................................................50 Glycosylation ...................................................................................................................53 O linked glycosylation .............................................................................................53 N linked glycosylation .............................................................................................54 Other Modifications .........................................................................................................55 Methylation ..............................................................................................................55 Acylation ..................................................................................................................57 Lipidation .................................................................................................................57


7 Post translational Modification of Proteasomal Proteins ................................................58 Mass Spectrometry .................................................................................................................60 Phosphorylation Site Identification by Ta ndem Mass Spectrometry ..............................62 Quantitative Mass Spectrometry .....................................................................................63 Label free methodology ...........................................................................................63 Label based methodology ........................................................................................66 Project Rational and Design ...................................................................................................69 2 MATERIALS AND METHODS ...........................................................................................75 Chemicals, Strains and Media ................................................................................................75 Materials ..........................................................................................................................75 Strains, Media, and Plasmids ...........................................................................................75 DNA Techniques ....................................................................................................................76 Cloning ............................................................................................................................76 DNA Electrophoresis .......................................................................................................77 Plasmid Isolation and Transformation .............................................................................77 Site directed Mutagenesis ...............................................................................................78 Generation of N terminal Acetyltransferase Knockouts .................................................78 Genome Analysis .............................................................................................................79 RNA Techniques ....................................................................................................................79 RNA Isolation and RT PCR ............................................................................................79 qRT PRC .........................................................................................................................80 Protein Techniques .................................................................................................................80 Protein Expression in Escherichia coli ............................................................................80 Protein Expression in Haloferax volcanii ........................................................................80 Protein Quantification .....................................................................................................81 Protein Separation and Chromatography .........................................................................81 Nickel affinity chromatography ...............................................................................81 Strep tactin chromatography ....................................................................................82 Gel filtration chromatography ..................................................................................82 Peptide Synthesis .............................................................................................................82 Two Dimensional Gel Electrophoresis ...........................................................................83 Immunoblotting ...............................................................................................................83 Labeling Hfx. volcanii Cells with Orthophosphate in Minimal Media ...........................84 Pulse Chase .....................................................................................................................84 Immunoprecipitation (IP) ................................................................................................85 Molecular Modeling ........................................................................................................86 Enzyme Assays .......................................................................................................................86 Peptide Hydrolyzing Activity ..........................................................................................86 Kinase Assays ..................................................................................................................87 Mass Spectrometry .................................................................................................................87 Sample Preparation ..........................................................................................................87 Three Dimensional Ion Trap (LCQ Deca) .......................................................................88 Hybrid ESI Q ToF (ABI QSTAR) ..................................................................................88 Triple Quadrupole (ABI QTRAP) ...................................................................................88 Database Searching and Software ...................................................................................89


8 3 N TERMINAL ACETYLATION OF ALPHA TYPE SUBUNITS OF 20S PROTEASOMES IN Haloferax volcanii ...............................................................................90 Introduction .............................................................................................................................90 Results .....................................................................................................................................93 Isoforms of 20S Proteasome Subunits .............................................................................93 Analysis of Proteasome Isoforms by Mass Spectrometry ...............................................95 terminally Acetylated .........................................................................................96 ...........................................................................97 ..................................................................97 Amino Acid Substitutions in th Cleavage of the Initiator Methionine Residue .............................................................98 Co purifying Nonproteasomal Proteins .........................................................................99 20S Proteasomes Rapidly Isolated by Twostep Affinity Purification from Hfx. volcanii .......................................................................................................................100 cell Lysates .......................................................................................................................101 212 Modifications ..........101 americ Rings in Haloferax volcanii .......................................................................................................................102 Complementation of the Hypoosmotic Stress Phenotype of psmA Mutant ..................103 1 Q2T Display a More Thermotolerant Phenotype .........................104 terminally Acetylated .......................................................................................106 Discussion and Conclusions .................................................................................................107 4 N TERMINAL PROTEOME AND ACETYLTRANSFERASES IN Haloferax volcanii ..131 Introduction ...........................................................................................................................131 Results and Discussion .........................................................................................................133 Phylogenetic Distribution of GNAT Family in Haloferax volcanii ..............................133 Other Nacetylated Proteins in Haloferax volcanii ......................................................136 Occurrence of Different Amino Acids in the Penultimate and Antepenultimate Positions .....................................................................................................................139 Conclusions ...........................................................................................................................140 5 PHOSPHORYLATION AND METHYLATION OF SUBUNITS FROM 20S PROTEASOMES AND PROTEASOME ACTIVATING NUCLEOTIDASE A IN Haloferax volcanii ................................................................................................................160 Introduction ...........................................................................................................................160 Results and Discussion .........................................................................................................161 phase Transitions ........................................161 ..........................................................................................163 PanA is Phosphorylated .................................................................................................165 ........................................................................................166 ........................................................................................166 Variant ......................167


9 Haloferax volcanii Encodes a Number of Putative Protein Kinases .............................168 Rio type I Kinase from Hfx. volcanii Catalyzes Autophosphorylation in vitro ............169 Rio type in vitro ...............................................................170 ......................................................................................170 Conclusions ...........................................................................................................................171 6 SUMMARY AND CONCLUSIONS ...................................................................................198 Summary of Findings ...........................................................................................................198 Future Directions ..................................................................................................................201 APPENDIX A PRIMERS USED IN THIS STUDY ....................................................................................202 LIST OF REFERENCES .............................................................................................................206 BIOGRAPHICAL SKETCH .......................................................................................................239


10 LIST OF TABLES Table page 11 List of known post translational modifications of proteasome subunits. ..........................73 31 Strains and plasmids used in this study ............................................................................113 32 ............115 33 n and chymotryps in digest ..................................................................................................116 34 Summary of MS/MS spectral counting and integration with a calculated ratio of Ac1 ............................................................................................................117 35 N acetylation and MAP cleavage pattern in 20S proteasomes and whole cells and influence peptidase activity. ................................118 36 Three additional proteins identified by ESIQTOF MS scans of proteasomeenriched samples. ............................................................................................................................119 37 MS/MS analysis of 20S proteasomes purified by twostep affinity purification. ............120 38 MS/MS identification of the 30 kDa isoform chain predominant in Hfx. volcanii ..........................................................121 41 Strains and plasmids used in this study ............................................................................141 42 Methionine aminopeptidase and examples of acetyltransferase homologs proposed to play a role in N terminal cleavage and acetylation. .........................................................146 43 Summary of N terminal peptides and associated pro teins of Hfx. volcanii detected by MS/MS. ............................................................................................................................152 51 Strains and plasmids used in this study ............................................................................175 52 Site directed mutants and phenotypes. ............................................................................178 53 MS/MS results of Rio1p Strep fractions. .........................................................................179 A 1 Oligonucleotide primers used in t his study. .....................................................................202 A 2 Oligonucleotide primer pairs for site directed mutagenesis. ...........................................203 A 3 Oligonucleotide primer pairs for cloning of kinase genes from Hfx. volcanii ................204 A 4 Oligonucleotide primer pairs for N terminal acetyltransferase knockouts. .....................205


11 LIST OF FIGURES F igure page 11 Crystal structures of 20S proteasomes from Thermoplasma acidophilium ......................70 12 Ubiquitination and pupylat ion pathways. ..........................................................................71 13 Modified amino acid structures .........................................................................................72 31 I soforms of 20S proteasomal proteins. ............................................................................122 32 MS/MS Fragmentation of Acet MoxQGQAQQQAYDR, an N terminal fragment of ..................................................................................123 33 MS/MS Fragmentation of Acet MNRN(D)DKQAYDRGTSLFSPDGRIYQVE, an Nterminal fragment of the 2 protein of 20S proteasomes .................................................124 34 20S proteasome CPs are rapidly isolated by two step affinity purification from Hfx. volcanii .............................................................................................................................125 35 Quantitati terminal initiator methionine (Ac1) were dominant in wild type cells. ......................................126 36 in cells as heptameric rings. ........127 37 chromosomal psmA deletion or exacerbate it ..................................................................128 38 Hfx. volcanii phenotype .........................................................................................................................129 39 Two dimensional gel electrophoresis of cell lysate of Hfx. vo lcanii GZ130 ......................................130 41 GNAT protein phylogenetic tree. Haloferax volcanii proteins compared to bacterial and eukaryotic homologues .............................................................................................144 42 Frequency of amino acids in the penultimate position of the N termini of proteins from the deduced proteome of Hfx. volcanii ..................................................................150 43 MS/MS Fragmentation of Acet SSIELTSSQK, an Nterminal fragment of HVO_1577. ......................................................................................................................151 51 Phosphatase type 20S proteasomal proteins as a function of Hfx. volcanii growth .........................................................................................................181 52 MS/MS Fragmentation of RGEDMSMQALpSTL, an internal phosphopeptide of the it of 20S proteasomes ..........................................................................................182


12 53 MS/MS fragmentation of the de novo synthesized peptide RGEDMSMQALpSTL. Region corresponding to residues 119 ...........................183 54 A PanA specific phosphopeptide was reproducibly detected by ESI QTOF analysis of tryptic digestions of PanA purified from Hfx. volcanii DS70 cells .............................184 55 specific phosphopeptide detected in purified 20S proteasomes by MS/MS ........185 56 specific phosphopeptide was detected in purified 20S proteasomes by ESI Q ToF with precursor ion scanning .....................................................................................186 57 HypopanA cells expressing pJAM202c, panA panA t1018g (PanA S340A) and panB at different NaCl concentrations. .......................187 58 psmA type subuntis from Hfx. volcanii ....................................................................................................................188 59 Rio kinases in Hfx. volcanii .............................................................................................189 510 Strep_II tagged Rio1p purified from Hfx. volcanii His6 subunits purified from E. col i .........................................................................................................190 511 MS/MS spectra of a methylesteri residues 105 116, YGEPIGIEmethylTLTK .....................................................................191 512 residues 58 68, SPLMEmethylPTSVEK. .........................................................................192 513 residues 23 30, LYQVEmethylYAR. ...............................................................................193 514 residues 13 22, GITIFSPDmethylGR. ..............................................................................194 515 1 corresponding to residues 150 163, LYETDPSGTPYEmethylWK. ............................................................195 516 type 20S proteasome proteins .........................196 517 Multiple sequence alignment of the central region of select type 20S proteasome proteins .............................................................................................................................197


13 LIST OF ABBREVIATIONS 2DGE Two dimensional gel electrophoresis 5FOA 5Fluoroorotic acid AARE Acylamino ac id releasing enzymes AcP Acetyl phosphate ADP Adenosine 5 diphosphate AMC 7amido 4methyl coumarin AMP Adenosine 5'm onophosphate AP Alkaline phosphatase Apr Ampicillin resistance AQUA Absolute quantification ATCC American Type Culture Collection ATP Ade nosine 5 triphosphate BCIP 5bromo4chloro3 indolyl phosphate ptoluidine BLAST Basic Local Alignment Search Tool Degrees Celsius C Carboxyl CHAPS 3[(3 Cholamidopropyl)dimethylammonio] 1propanesulfonate CID Collision induced dissociation CP Core particle or 20S core particle Da Dalton (atomic mass unit) DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DTT Dithi othreitol DUB Deubiquitinating enzyme


14 Ec Escherichia coli EDTA Ethylenediaminetetraacetic acid emPAI Exponentially modified protein abundance indices ESI Electron spray ionization HABA hydroxyl azophenyl benzoic acid HPLC High pressure liquid chromatograph y HRP Horseradish peroxidase Hv Haloferax volcanii ICAT Isotope coded affinity tag IEF Isoelectric focusing IMAC Immobilized metalaffinity chromatography IPTG Is opropyl D thiogalactopyranoside iTRAQ Isobaric tag for relative and absolute quantification kDa Kilodalton Kmr Kanamycin resistance LB Luria Bertani broth LC Liquid chromatography Succ LLVY AMC N s uccinyl l eucine l eucine valine t yrosine 7amino 4methylc oumarin MALDI Matrix assisted laser desorption/ionization MAP Methionine aminopeptidase MIDAS MRM initiated detection and sequencing MRM Multiple reaction monitoring MS Mass spectrometry MS/MS (MS2) Tandem mass spectrometry N Amino


15 NBT Nitro blue tetrazolium NAT N terminal acetyltransferase NHS N hydroxysuccinimide Ni NTA Nickel nitrilotriacetic acid nm N anometer Nvr Novobiocin resistance O GlcNAc O linked N acetylglucosamine OD Optical density PAGE Polyacrylamide gel electrophoresis PAI Protein abundance indices PAN Proteasome activating nucleotidase PEP Phosphoenol pyruvate PCR Polymerase chain reaction PGPH P eptidylglutamyl peptide hydrolyzing Pup Proka ryotic ubiquitinlike protein PVDF Polyvinyldiflouride Q Quadrapole qRT PCR Quantitative real time polymerase chain reaction RNA Ribonucleic Acid RP HPLC Reverse phase high pressure liquid chromatography RPM Rotations per minute RT Reverse transcriptase SA M S adenosyl methionine SCX Strong cation exchange SDM Site directed mutagenesis


16 SDS Sodium dodecyl sulfate SILAC Stable Isotope Labelling with Amino acids in Cell culture SIM Single ion monitoring Spr Spectinomycin resistance SRM Selective reaction monito ring Ta Thermoplasma acidophilum TBS Tris buffered saline TBST Tris buffered saline with Tween 20 TEA Triethylamine TOF Time of flight Tris N tris(hydroxymethyl) aminomethane Ub Ubiquitin UV Ultraviolet XIC Extracted ion chromatogram V Volt Vh Volt hour


17 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 POSTTRANSLATIONAL MODIFICATION AND THE 20S PROTEASOME SYSTEM OF THE H ALOARCHAEON Haloferax volcanii By Matthew Adam Humbard August 2009 Chair: Julie A. Maupin Furlow Major: Microbiology and Cell Science While much of the study of proteasome systems in all organisms, eukaryotes and prokaryotes, focuses on substrate recognition ubiquitin like modifiers, or protein targeting, the 20S core particle and regulatory ATPases are subject to a wide variety of modifications that include, but are not limited to, phosphorylation and acetylation. Despite the large number of modificat ions known, the roles these modifications play within various proteasomes remain unclear, as they remain relatively unstudied. This study focused on identifying and studying specifically the sites of modification at the residue level, including mapping and altering these sites in order to better understand how proteasomes themselves are assembled and controlled. Through the use of proteomic approaches, we have identified post translational modification on all three subunits of 20S core particles in Halofe rax volcanii as well as on one of the proteasome activating nucleotidases, PanA. In addition to mapping phosphorylation sites to the alpha1, alpha2, beta subunits of the 20S core particle, and PanA, both alpha type subunits are also N terminally acetylate d. A novel post translational modification, methylesterification, was also identified on aspartic and glutamic acid residues of the alpha1 protein. This is the first report of methylation of a proteasomal subunit. Phenotypic characterization of variant protein s unable to undergo N terminal acetylation revealed that the N terminus of alpha1 plays a role in the stability of the protein, and that this can alter the hypoosmotic response, growth rate and yield.


18 Further investigation into N terminal acetyla tion in Hfx. volcanii revealed this is a generalized pathway and nearly 30 % of soluble proteins are modified in this manner. Additionally, variant alpha1 proteins that were unable to undergo phosphorylation severely inhibited growth of Hfx. volcanii cell s and altered their pigmentation. A candidate serine/threonine kinase from the atypical Rio kinase family, was shown to catalyze autophosphorylation in vitro and phosphorylat e r ecombinantly purified alpha1 proteins in vitro Altogether, this study demons trates that the proteins that make up the proteasomal system in Hfx. volcanii are heavily modified post translationally, there is a generalized protein N terminal acetylation pathway at work in the haloarchaeon, and a Rio type I kinase autophosphorylate s a s well as phosphorylate s alpha1 in vitro


19 CHAPTER 1 LITERATURE REVIEW Introduction This literature review is designed to present the most current primary scientific literature concerning proteasomes and post translational modification in the three domains of life This review will highlight on the assembly, function, and the role post translational modification plays on the regulation of proteasome systems. It will also look at the mechanisms employed by different organisms with respect to N terminal maturation, phosphorylation, methylation a nd ubiquitinlike modification. Proteasome Structure and Function Proteasomes are large, energy dependent, proteases that act as the central house keeping protease responsible for protein turnover in the removal of misfolded or damaged proteins within the cell (Chen et al. 2004) Proteasomes are conserved in a rchaea, e ukaryotes, and in the Actinobacter ia class of bacteria (De Mot et al. 1999; Volker and Lupas, 2002; Wolf and Hilt, 2004; De Mot, 2007) This function, post translationally controlling the levels of proteins, places the proteasome in a unique regulatory role. Several biological processes are controlled through protein degradation by proteasome s Some of these processes include DNA replication, cell division, metabolism, and antigen presentation by major histocompatibility complex I (MHC I) presenting cells of the immune system (Josefsberg et al. 2000; Clarke, 2002; Goldberg et al. 2002; Kang et al. 2002; Kirkland et al. 2007; Kirkland et al. 2008a) Disregulation of the proteasome system has been implicated in the progression of several diseases in humans including Alzheimer s, Amyotrophic Lateral Sclerosis (ALS ) Parkinsons and several different kinds of cancer (Kabashi and Durham, 2006; Hung et al. 2006; van Leeuwen et al. 2006; Olanow and McNaught, 2006; Zavrski et al. 2007; Hayslip et al. 2007; Stapnes et al. 2007)


20 Proteasomes are also essential for cellular growth in both e ukaryotic organisms (Fujiwara et al. 1990; Georgatsou et al. 1992; Lee et al. 1992; Nishimura et al. 1993b; Saville and Belote, 1993; Gerards et al. 1994) as well as the haloarchaeon Haloferax volcanii (Zhou et al. 2008) Proteasomes in the G ram positive a ctinobacter Mycobacterium tuberculosis have been shown to be necessary for virulence, as a part of nitric oxide stress response, but are not essenti al for growth (Darwin et al. 2003) 20S C ore P article S tructure and S ubunit A rrangement The 20S core particle of proteasomes ha s a conserved quaternary structure from species to species. The 20S proteasome consists of a stack of four different seven membered rings made up type superfamily. The rings are stacked with two heptameric rings subunits on the inside type subunits (Figure 11. Left panel.) (Lowe et al. 1995; Tamura et al. 1995; Groll et al. 1997; Zuhl et al. 1997b; Nagy et al. 1998) Eukaryotic organisms such as Saccharomyces cerevisiae encode at least seven different type subunits The E ukaryotic proteasome s are made up of heteroheptameric rings, each ring is made up of seven different subunits ) Each half proteasome contains 14 unique subunits, all with a defined space in the structure (Groll et al. 1997) In higher eukaryotes, a mixed population of proteasomes may exist through the use of i, i, and i ). Proteasomes that contain these specialized subunits are called immunoproteasomes (Akiyama et al. 1994; Hisamatsu et al. 1996; Foss et al., 1998; Rivett et al. 2001) and have reduced activity for cleavage after acidic amino acids (Gaczynska et al. 1996) families. The a ctinobacter Rhodococcus sp. encodes two subunit s These subunits form heteroheptameric rings much like the yeast (Tamura et


21 al., 1995; Zuhl et al. 1997a; De Mot et al. 1998) Archaeal organisms usually encode one type type subunits as is the case with Thermoplasma acidophilum (Lowe et al. 1995; Seemuller et al. 1995) The haloarchaeon Haloferax volcanii is somewhat unique in this respect, encoding type subunits type subunit type subunits form homoheptameric rings, unlike the yeast and Rhodococcus sp. This allows for several different kinds of proteasomes in a Hfx. volcanii cell. purified from Hfx. volcanii cells (Wilson et al. 1999; Kaczow ka and MaupinFurlow, 2003; Reuter et al. 2004; Zhou et al. 2008) ( Figure 11) Analysis of the crystal structures of both archaeal and eukaryotic 20S core particles shows that the interior consists of three chambers, the proteolytic chamber found betwe en the two inner rings and two ante rings (Figure 1 1. Right panel.) (Lowe et al. 1995; Groll et al. 1997; Dorn et al. 1999; Groll et al. 2003) The two antechamber s are formed by an aperture ring, where all substrates must pass. The ring side, excludes folded proteins from entering the catalytic chamber and requires the assistance of ATPases for the recruitm ent and translocation of substrates. The central chamber of proteasomes houses the catalytic activity. The N subunits are exposed after a processing event in this chamber (Arendt and Hochstrasser, 1997) While prokaryotic proteasomes typically lack the subunit complexity seen in eukaryotic proteasomes, usually encoding t subunits in eukaryotic 20S proteasome s (Zwickl et al. 1992; Heinem eyer et al. 1994) The enzymatic activity of eukaryotic proteasome s immunoproteasome (Tanaka and Kasahara, 1998) The r subunits are


22 subunits (Arendt and Hochstrasser, 1999) Proteasomes can display a variety of proteolytic activities including t ryptic, chymotryptic, and posta cidic cleavage (Lee et al. 1992; Nishimura et al. 1993b; Gerards et al. 1994) The result of proteolysis of proteins are short peptides, 3 to 24 amino acids in length. The fate of these peptides remains unclear. Conflicting hypotheses exist concerning whether the peptides passively diffuse away from proteasomes or are actively removed (Kohler et al. 2001; Hutschenreiter et al. 2004) The resulting peptides can be used by the cell to regenerate amino acid pools with downstream peptidases or as signaling molecules (Uebel and Tampe, 1999) Assembly of 20S C ore P article Maturation of 20S proteasomes is a series of sequential folding, assembly and processing events. Some assembly steps or intermediates ar e common to all three domains and others are not. Differences in subunit complexity are reflected in the complexity of assembly pathways for 20S core particle s in the three domains of life. Each domain, A rchaea E ukar ya and B acteria has different requi rements and unique challenges to properly assemble 20S proteasome s In the case of some archaeal proteasomes, functional complexes can be assembled entirely in vitro or in recombinant systems In the case of the archaeon Methanosarcina thermophila, when e quimolar amounts of purified 7 subunits with the propeptide intact) are mixed approximately 50 % are processed. Electron microscopy revealed 20S proteasomes assembled properly (MaupinFurlow et al. 1998) 7 ring. The archaeal proteasomes from Thermoplasma acidophilum are also made up of only one Co units of T acidophilum proteasomes in E coli yields fully assembled and active 20S proteasomes (Zw ickl et al. 1994) as is also the case with Methanosarcina thermophila (Maupin Furlow et al. 1998) Additionally, expressing


23 Aeropyrun pernix precursor subunit from Archaeoglobus fulgidus results in a proteolytic active chimeric 20S proteasome (Groll et al. 2003) This spontaneous formation of functional 20S pr oteasomes indicates that there is no need for proteasome specific chaperones for proper assembly and the ability to form chimeric proteasomes comprised of recombinantly expressed subunits from different organisms suggests that even phylogenetically distant archaea share an assembly pathway for 20S proteasomes. Not all archaeal proteasomes can be reconstituted in vitro In the case of haloarchaea, functional proteasomes have never been reconstituted in vitro for haloarchaea, however assembly intermediates c E. coli and (7)) can properly fold (Wilson et al. 1999) A 7 intermediate can be d H aloferax volcanii proteasomes. W s from T. acidophilium are expressed in E. coli they assemble into heptameric rings 7) and di heptameric structure s 77). N terminal substitution and d T acidophilium revealed that a helix from residues 22 33 is important in forming the stable intermediate heptameric ring. A deletion of amino acids 2 34) is unable to form rings or complete 20S proteasomes. L ikewise Glu 25Pro is unable to form rings or proteasomes (Zwickl et al. 1994) Archaeoglobus fulgidus has the same rigid structure whether it is incorporated into the 20S proteasome or not. ring is believed to be the earliest a ssembly intermediate in the archaeal proteasome assembly pathway and is thought to serve as scaffolding to recruit pre subunits to form a half proteasome. The presence of half proteasomes can be seen when A fulgidus threonine 12 to glycine ( Thr 12G ly ) is used in place of the wild subunit. 20S


24 proteasome s did not form properly with this mutation The crystal structure of the A fulgidus T hr 12G ly proteasome revealed two half proteasomes had a 4.5 gap between them. The gap had regions of disorder that did not resolve in the crystal structure (Groll et al. 2003) This observation suggests that the half proteasome is an assembly intermediate and dimerization of the half proteasomes is followed by t he propeptide subunits. Bacterial proteasomes can be found in the Actinobacter class proteobacteria (Tamura et al. 1995; Zuhl et al. 1997a; Zuhl et al. 1997b; De Mot et al. 1998; Nagy et al. 1998; De Mot, 2007) The first of these bacterial proteasomes to be studied in detail was from Rhodococcus sp. strain N186/21 (Tamura et al. 1995) Rhodococcus Expression of e (Zuhl et al. 1997b) Similar to the archaeal example discussed above, chimeric proteasomes with subunits from different Actinobacter such as Frankia, could spontaneously assemble into functional 20S proteasomes (Pouch et al. 2000) type subunits were expressed alone, no ring structure w as observed. This Lys 33A la from Frankia sp. was co form properly, although half proteasomes were also observed (Mayr et al. 1998) Lys33Ala subunit is unable to cleave off the propeptide, so the propeptide is still present in assembled 20S proteasomes. The half proteasome is a short lived intermediate in the bacterial proteasome assembly pathway but not the heptameric rings. There is spe culation that the Rhodococcus proteasome most likely assembles and


25 the heterodimers oligomerize to form a half proteasome, consisting of 7 The assembly intermediates of Rhodococcus erythropolis were measured by real time mass spectrometry confirming that the bacterial proteasome does not pass through the ring as an intermediate (Sharon et al. 2007) be detected but the majority of subunits are either assembled into whole proteasomes or are in the short lived intermediate half proteasome s The half proteasomes come together spontaneously and stimulate the cleavage of the propeptides after dimerization. E ukaryotic proteasome s ha ve maximized the diversity of subunits and as a consequence ha ve the most complicated and specific assembly pathway. Mammalian proteasome s pass through several intermediates, originally ident ified as 13S, 15S, and 16S, before their biochemical composition could be determined (Schmidtke et al. 1997) T he first step in eukaryotic proteasome assembly is similar to the first step in archaeal proteasome assembly, the ring. In eukaryotic organisms, as opposed to archaeal organisms, this means the formation of a heteroheptameric ring containing seven diff Each of the seven different subunits has a defined place in the structure. Observations of the individual subunits in vitro of S. cerevisiae can dimerize and form double ring like structures (Yao et al. 1999b) Similarly of S. cerevisiae mixed together results in the formation of a random ring like structure with variable subunit positioning in the rings (Gerards et al. 1997) Since these structures can form rand omly, eukaryotes have a devoted set of proteasome specific chaperones that aid in the formation of incorrect ring arrangements. Proteasome biogenesis factors (Pba or PAC 1 4) are a set of ring formation


26 (Rosenzweig and Glickman, 2008b) Pba1 and Pba2 bind to open rings that contain up to 5 The remaining two subunit inserted last. ring structure (Velichutina et al. 2004) Once ring as a template. ring type subunits are incorporated in a specific and sequential order. ring is bound with Pba1 and Pba2; Pba3 and Pba4 bind to the ring to nit. (Ramos et al. 2004) displacing Pba3 and Pba4 from the growing structure. At this point, the half proteasome is comple Dimerization of the half This is achieved through the long C (Ramos et al. 2004) and a protein complex called Blm10 (PA200) responsible for proteasome activation (Ortega et al. 2005) E ukaryotic proteasome s are completed when the two half proteasomes come together, the propeptides on type subunits are cleaved and Pba1, Pba2, and Ump1 are degraded by the proteasome. proteasome structure is conserved between yeast and mammals (Hirano et al. 2008) The chaperones responsible for this assembly pathway are also widely di stributed throughout eukaryotes (Hirano et al. 2008) subunit P rocessing type subunits of 20S proteasomes belong to the Ntn hydrolase family Ntn hydrolases are typically synthesized as inactive precursors and undergo an autocatalytic internal


27 cleavage exposing an N terminal threonine, serine, or cysteine. Threonine (Thr1) serves typ e subunits in 20S proteasomes. A substitution of serine for the active site threonine (T hr 1S er ) in Methanosarcina thermophila 20S proteasomes reduc ed both the chymotrypsin and post glutamyl peptidase activity by 30 and 70 %, respectively (Maupin Furlow et al. 1998) Likewise, Thermolplasma acidophilium 20S proteasome r1Ser) has reduced proteolytic activity against proteins (Kisselev et al. 2000) subunits and some inactive (Thomson and Rivett, 1996) are removed in an autocatalytic event as the final step of proteasome assembly and maturation. The prope support ing protecting the cells from hydrolase activity (Khan and James, 1998) preventing Nacetylation of the active site (Arendt and Hochstrasser, 1999; Jager et al. 1999) and aiding in proteasome assembly (Zuhl et al. 1997a) The propeptides of active subunits in 20S proteasomes can vary in length fr om 4 amino acids to over 70, as is the case interferon (Lee et al. 1990) Some of the p type subunits play a role in proteasome assembly. The Rhodococcus 64) still is able to form functional proteasom es in the Rhodococcus sp. However, the assembly efficiency is greatly reduced. Providing the propeptide in trans improved the in vitro assembly of the 20S proteasome (Zuhl et al. 1997b) Not all propeptides are required for proper assembly as is the case with Thermoplasma acidophilum 8) subunit from T acidophilum


28 subunit in E. coli and still assemble into active 20S proteasomes, indicating that the propeptide does not have a role in assembly of the Thermoplasm a proteasome (Zwickl et al. 1994) Propeptides are essential for growth and prevent N terminal acetylation of active site residues in yeast. chymotrypsinlike activity and viability of Saccharomyces cerevisiae cells (Arendt and Hochstrasser, 1999) The chymotrypsin(Chen and Hochstrasser, 1996; Arendt and Hochstrasser, 1997) The and propeptides are dispensable for cell growth indicating that they are not necessary for mature 20S proteasome formation. Even though th he mutants expressing the ( 19 or ) and ( 29 or ) subunits ha ve a lower growth rate, but viable. Cells expressing both defective subunits ha ve an increased growth defect. 20S proteasomes containing these leader peptide lacking subunits ha ve drastically reduced peptidase activity. had no peptidylglutamyl peptide hydrolyzing (PGPH ) had a 60 % reduction in trypsin like activity. The activity is restored in a nat1 mutant ( nat1 encodes Ard1), an acetyltransferase gene The acetylation of the N subunits results in the inactivation of the activity by blocking the nucleophile required to catalyze the reaction (Arendt and Hochs trasser, 1999) may be active. In Rhodococcus sp. in the crenarchaeota Aeropyru m pernix one is active and the other is not (Koonin et al. 2001) In eukaryotic


29 Inactive subunits may or may not undergo the propeptide cleavage. The active subun proteasome assembly (Seemuller et al. 1996) are conserved in yeast (Chen and Hochstrasser, 1995) slime mold (Schauer et al. 1993) rat (Lilley et al. 1990) and human (Lee et al. 1990) Structure and Folding of subunit s 20S proteasome assembly and maturation continuously exclude s the proteolytic active site from the cytoplasm first by blocking the N terminal threonine active site with a propeptide and second by sequestering the act ive site within the structure after maturation. Conserved aminoacid residues in the N type subunits of proteasomes create structural elements that keep proteasomes in an autoinhibited state by obstruct ing entry of substrates into the 20S core particle. The Tyrosine a spartic a cid a type subunits in all three domains. The YDR motif makes up the end of a N helix that extends into the aperture ring and is an essen tial component of N terminal activities of 20S core particles independent of ATPase action (Groll et al. 2000; Benaroudj et al., 2003; Bajorek and Glickman, 2004) subunits, gated channel may be a general m echanism for regulating the activity of the proteasome (Groll et al. 2000) The rate limiting step in protein degradation by proteasomes is translocation of the ring to the active site contained within the central chamber and formed by t rings. Eukaryotic 20S core particles have no protease and relatively low peptidase activity in the absence of the 19S cap or chemical pretreatment with SDS or heat treatment


30 (Coux et al. 1996) Peptidase activity of the yeast 20S proteasome is blocked by the N termina l A deletion of the N helix 9) subunit abolishes the N terminal gating of the 20S core particle A single substitution of Asp 9A la increases peptidase activity (Groll et al. 2000) One difference between the crystal structure of eukaryotic and prokaryotic 20S core particle s rings. The structure s of the 20S core particle from Thermoplasma acidophilum (Lowe et al., 1995) and Archaeglobus fulgidus (Groll et al. 2003) reveal a common axial channel of 13 that connects the interior of the core particle with the surroundings. The X ray structure of the yeast 20S particle did not contain this channel (Groll et al. 1997) These differences in structures might reflect differences between crystal forms and not actual differences within the cell. The N terminal 11 amino acids of the A. fulgidus less ordered in the 20S proteasome than the 16S half proteasome precursor (G roll et al. 2003) Asp 9Asn, Asp9 is present in the YDR motif of A. fulgidus in A. fulgidus does not have an obvious change in peptidase activity (Groll et al. 2003) 12 in T. acidophilum (a deletion of the N terminal helix including the YDR motif) abolished the need for ATPase activity of PAN in the degradation of aciddenatured GFPssrA or casein (Benaroudj et al., 2003) The cons ring gating sterics, is present in all known a type subunits to date. Although the mechanism may not be exactly the same, a ring aperture Furthermore, cryoelectron microscopy of the M ycobacterium tuberculosis 20S core particle reveals closed ends that subunit These 8 N terminal residues diminish peptidase activity of 20S core particles as well (Hu et al. 2008)


31 Proteasome Activation Proteasomes, like all energy dependent proteases with known structures, e xist in cells in an autoinhibited state, sometimes referred to as the latent state. As discussed previously, the outer rings of the 20S core particles act to prevent diffusion of random proteins into the proteolytic core. External forces or proteins have to act on core particles and substrates to stimulate the ticles. This can be achieved in vitro with the addition of heat or denaturing agents such as SDS or urea that denature the substrate proteins or disrupt the axial gating of 20S core particles, or in vivo with the assistance of different protein complexes This section will discuss the various structures and proteins that have been shown to influence 20S proteasome activity in different organisms. 19S R egulatory P article ring s of eukaryotic 20S proteasomes to form 26S or 30S proteasomes in a reversible and ATP dependent process The 26S proteasome is a 20S core particle with one 19S cap bound, the 30S proteasome is when both ends are capped with 19S caps. 20S core particles associated with 19S caps are commonly referred to as 26S proteasomes, despite the number of 19S caps in complex. The 19S regulatory particle is responsible for the recognition, binding, unfolding, and transl ocation of substrates into the proteolytic chamber of the proteasome. Unfolding and translocation of substrates are ATP dependent processes (Kohler et al. 2001; Navon and Goldberg, 2001; Takeuchi and Tamura, 2004) In yeast, t he 19S complex consists of at least 17 unique gene products that make up two different substr uctures, the lid and the base. The base structure is made up of 6 r egulatory p article t riple A (Rpt) ATPases, Rpt1 to Rpt6, and three r egulatory p article n onATPase (Rpn) subunits, Rpn1, Rpn2, and Rpn10. The base structure is the ATP hydrolyzing portion of the structure and is responsible for the mechanical unfolding and translocation of substrates into the


32 20S core particle. The lid is attached to the base structure through the Rpn10 subunit of the base. The functions of the lid portion of the structur e include recognition and binding of substrate proteins (such as polyubiquitination proteins). The lid structure is made up of Rpn3, Rpn 59, Rpn11 and Rpn12. Although a complete crystal structure of 19S regulatory particles, as well as 26S proteasome s, has not been resolved for any eukaryotic organism, extensive cryoelectron microscopy and proteinprotein interaction studies in yeast, Caenorhabditis elegans Drosophila melanogaster and humans have been conducted. The 19S regulatory particle s activate t he 20S core particle s by changing the conformation of N terminal extensions type subunits making up the axial gate to the proteasome (Kohler et al. 2001) The base subunits bind to the rings in the 20S core particle. This causes structural changes in the 20S core particle. 19S RP by the C terminal tails of Rpt2, Rpt5 and Rpt1 (Smith et al. 200 7; da Fonseca and Morris, 2008) Specifically, the Rpt2 subunit has been shown to physically change the structure of the axial channel of the 20S proteasome by opening it and allowing substrates to enter (Kohler et al. 2001) Rpt1 and Rpt2 of yeast can form hetero oligomers when recombinantly expressed in E. coli and Rpt4 can form homooligomers (Takeuchi and Tamura, 2004) These complexes are enough to hydrolyze ATP and unfold substrates to be exposed to the protease activity of the archaeal 20S core particle in vitro 11S R egulator / PA28 The 11S regulator PA28 or PA26 in Trypanos oma brucei (Yao et al. 1999a) stimulates peptidase activity of 20S proteasomes in an ATP independent manner but does not stimulate proteolysis of folded proteins (Dubiel et al. 1992; Ma et al. 1992) It is a multiprotein complex made up of two different subunits, PA28PA28 (Mott et al. 1994; Ahn et al. 1995;


33 Kohda et al. 1998) and possibly (Realini et al. 1994) When recombinantly produced, (Song et al. 1997) An amino (Song et al. 1996; Zhang et al. 1999) This heteroheptameric structure is the most likely physiological structure since a ll three subunits can activate 20S proteasome s by themselves but the level of activation is greatly increased when are combined (Kuehn and Dahlmann, 1997; Song et al. 1997) A crystal structure of the Trypanosome PA26 activator and the yeast 20S proteasome in com plex was determined (Whitby et al. 2000) as well as the PA26 bound to an archaeal 20S proteasome (Forster et al. 2005) The structure ring was altered in the combined structure s In the yeast : Trypanosome structure, t retained the confirmation previously seen in the 20S core particle structure. The remaining four s in their N termini from the 20S structure alone. that normally establish the gating mechanism of 20S proteasomes adopted a new conformation binding to the interior of PA26, thereby ope ning the aperture to the proteolytic chamber. The archaeal 20S proteasome and Trypanosome PA26 structure revealed that only one side Lys 66. Amino acid substitu tions in position Lys 66 to alanine Lys 66A la Lys 66S er ) abolished PA26 interaction and stimulation. The idea that this would only interact with one amino acid side chain is supported by the observation that PA28 activity can be abolished w ith a single substitution in its C terminal domain (Ma et al. 1993; Song et al. 1997; Li et al. 2000)


34 Although the exact biological role of the PA28 activator is debated, the fact that the interferon could indicate that it plays a role in modulating the activity of the immunoproteasome. The three specific subunits of the immunoproteasome, interferon (Ahn et al. 1995; Groettrup et al. 1995) (Rechsteiner et al. 2000) It has also been suggested, since 11S regulators can bind to 26S proteasomes, they may facilitate the diffusion of peptides out of the proteolytic cham ber. This function could potentially be the physiological role of the activator in the immunoproteasome. The length of the product peptide generated by 20S proteasome s is determined by the amount of time the product spends in the proteolytic chamber (Tanaka and Kasahara, 1998) If the 11S regulator opens the opposite axial gate, larger products (peptides 8 10 amino acids in length), compatible with MHC I presentation could diffuse out of the core particle and be used for antigen presentation (Tanaka and Kasahara, 1998) PA200 (Blm10) PA200 ( Blm10 ) is a 200 kDa protein that binds to the outer ring of the 20S core particle similar to the 19S regulatory particle (Ortega et al. 2005) It is wide spread in mammalian tissues but also found in lower eukaryotes such as yeast. PA200 is a large protein made up of solenoid structure (Schmidt et al. 2005) PA200 in yeast was originally thought to only aid in the assembly pathway or mature 20S proteasomes by bringing together two half proteasomes (Fehlker et al. 2003) Negativestain electron microscopy showed that PA200 interacts with the mature core particle forming a cap over the 20S proteasome channel (Schmidt et al. 2005; Iwanczyk et al. 2006) Additional work showed that PA200 binds to mature and enzymatically active 20S core particles and opens the axial channel subunits enhancing peptidase activity but not protea se activity (Iwanczyk et al.


35 2006) Current purposed role s and physiological function of PA200 is to regulate substrate translocation or prod uct release of 20S proteasomes Th ese hypothes e s are supported by the ability of PA200 to distinguish between open and close gate conformations of the mature 20S core particle. Since PA200 preferentially binds open state core particles, PA200 binding to one may facilitate gate opening at the trans end of the core particle. 20S core particles with two PA200s bound have reduced peptidase activity and hybrid RP CP PA200 complexes have a lower rate of protein degradation (Lehmann et al. 2008) These two observations support the model where PA200 binding affects the trans axial gate. So far, t his section has discussed three examples of protein complexes 19S RP, PA28, and PA200, that are able to simulate protease activity of 20S core particles. All three protein complexes ring of core particles alter the structure of th e axial gate formed ring. Although this may not be the only way to affect proteolytic activity of 20S core activation in these three examples. Other strate gies may include unfolding of proteins so they ring structure Proteasome A ctivating N ucleotidases (PAN) Similar to the Rpt subunits within the base of the 19S regulatory parti cle of eukaryotic 26S proteasome s archaeal species encode for ATPase subunits thought to regulate 20S proteasome function (Zwickl et al. 1999; Wilson et al. 2000) Some of t hese archaeal regulatory particles are referred to as PAN ( P roteasome A ctiv ating N ucleotidases) and share sequence homology to the ATPase subunits of the 19S RP in eukaryotes (Zwickl et al. 1999) PAN complexes can even interact with eukaryotic proteasomes (Smith et al. 2005) The conserved C terminal motif hydrophobic X tyrosine (HbXY) where X is any amino acid, has been shown to be essential for


36 PAN proteasome function (Smith et al. 2007; Zhou et al. 2008) It is believed that this C terminal tail is required for functional association of PAN complexes with the 20S core particle. Some archaea encode more than one PAN protein. Two PAN pro teins, PanA and PanB, have been identified in the haloarchaeon H fx. volcanii (Reu ter et al. 2004) and two in Methanosarcina mazei (Medalia et al. 2006) Another homologue from M ethanococcus jannaschii has been shown to increase the activity of proteasomes from Methanosarcina thermophila and Thermoplasma acidophilum (Zwickl et al. 1999; Wilson et al. 2000) The PAN proteins, PanA and PanB, from H fx volcanii have been shown to form heterohexamers as well as homododecamers (Reuter et al. unpublished) and the M. jannaschii PAN assembles into dodecamers that can associate with 20S proteasomes (Wilson et al. 2000) PAN proteins can bind and unfold proteins in vitro without additional components. This unfolding event is ATP dependent (Benaroudj and Gol dberg, 2000; Navon and Goldberg, 2001) Following the unfolding event, proteins bound by PAN are translocated into the catalytic chamber of the 20S proteasome to be degraded (Smith et al. 2005) PAN is hypothesize d to be involved in substrate select ion and delivery to 20S core particles. Binding to substrates by PAN stimulates ATP hydrolysis, unfoldase activi ty, and translocation of the denatured peptide into the proteolytic chamber of the proteasome (Benaroudj et al. 2003) Substrates of the PAN complexes are fed into the proteolytic chamber C to N terminus after being mechanically unfolded on the surface of the ring (Navon and Goldberg, 2001) Not all archaea encode PAN proteins. Archaea that are apparently lacking PAN, may encode different AAA ATPases thought to have similar function, such as VAT or Cdc48 as is the case for Thermoplasma acidophilum (Gerega et al. 2005) Targeting of Proteins for Degradation The ability to quickly turnover specific proteins as well as damaged or misfolded proteins is an e ssential function in all cells. Bacteria, a rchaea, and e ukary a have evolved specific


37 pathways that target certain proteins for destruction. Generalized pathways of protein degradation exist for damaged or misfolded proteins, but directed and targeted app roaches that include a myriad of post translational tagging or m odifications are also employed. This section will review four different targeting mechanisms employed in different organisms that use a co or post translational tag to signal degradation. Ss rA tagging A targeting mechanism used by bacterial cells involves tagging proteins that have stalled at the ribosome due to lack of charged tRNA, a transcription error, or as a strategy to regulate cellular metabolism through proteolysis (Abo et al. 2000) This involves addition of a short 11 amino acid tag to the C terminus of protein that targets it for degradation (Komine et al. 1994; Gottesman et al. 1998; Gillet and Felden, 2001) This tag is called SsrA for s m all s table R NA A encoded protein. The ssrA molecule is a tmRNA or a t ransfer m essenger RNA and acts as both a transfer RNA and a messenger RNA. The s srA is charged with an alanine (A) and enters a stalled ribosome at the A site. The original, stalled, piece of mRNA is displaced by the tmRNA that codes for an additional 10 amino acids. Th e result is a n eleven amino acid C terminal tag (A ANDENYALAA) (Karzai et al. 2000) SsrA tagged proteins are recognized by either adapter molecules, such as SspB (Wah et al. 2002) or proteases, such as ClpAP, ClpXP, Ft s H, and Lon (Gottesman et al. 1998; Smith et al. 1999; Farrell et al. 2005; Herman et al. 1998) The adapter molecule SspB is not required for degra dation of SsrA tagged substrates in E. coli but it enhances the rate of degradation by the ClpXP complex (Flynn et al. 2001; Wah et al., 2002) Mutations in the ssrA sequence resulting in amino acid substitutions alanine 10 to aspartic acid ( A la 10Asp ) and A la 11Asp greatly reduce the efficacy of the targeting (Gottesman et al. 1998) These substitutions interfere with the SsrA / ClpXP interaction. The ClpXP complex binds residues 1, 2, and 8 10 in the SsrA tag. The adapter molecule SspB binds


38 residues 1 4 and residue 7 (Flynn et al. 2001) Even though the S srA tagging system seems unique to the bacteria, S srA tagged proteins can be degraded by 20S proteasomes and PAN complexes from archaeal species (Benaroudj and Goldberg, 2000; Navon and Goldberg, 2001; Benaroudj et al. 2003; Medalia et al. 2006) G FPS srA is a common substrate used for proteolysis assays for archaeal proteasomes (Benaroudj and Goldberg, 2000; Benaro udj et al. 2003; Reuter and MaupinFurlow, 2004; Medalia et al. 2006; Benaroudj and Goldberg, 2000) Since PAN proteins can thread substrates in a C to N terminus direction into the proteasome (Navon and Goldberg, 2001) perhaps there is a conserved C terminal degradation signal in the archaea. N end R ule The N end rule is a protein targeting pathway that connects the N terminal amino acid of a protein to th e in vivo stability, or half life, of that protein with respect to proteolysis (Gonda et al., 1989; Varshavsky, 1996) Although currentl y the N end rule has not been demonstrated in any archaeal organism, the pathway is conserved between bacteria and eukaryotes (Mogk et al. 2007) The machinery involved in the N end rule pathway may vary from domain to domain but the recognition of the N terminal motifs is highly conserved. The N end rule pathway involves a series of post translational modifications at the N terminal amino acid of a protein, eventually leading to the degradation of the protein by either the proteasome / ubiquitin pathway in eukaryotes or the ClpAP / ClpS system in bacteria (Mogk et al. 2007) With respect to the N end rule, N termina l a mino acids can be described as primary, secondary, or tertiary destabilizing residues. These classifications are based on the number of post translational modifications the residues undergo before being targeted to proteolytic machinery. The p rimary d estabilizing residues in mammals are phenylalanine, tryptophan, tyrosine, leucine, arginine, lysine, histidine and isoleucine (Varshavsky, 1996) They do not


39 require any additi onal post translational modification before entering the proteasome degradation pathway. In the case of eukaryotic organisms, the primary destabilizing residue tagged substrates enter the proteasomeubiquitin pathway by interacting with E3 ubiquitin ligas es called N recognins (Tasaki et al. 2005; Meinnel et al. 2006; Xia et al. 2008) In bacteria, there are fewer primary destabilizing residues. Even though there are fewer, the primary destabilizing residues in bacteria are conserved ( Phe Trp Tyr and Leu ) (Varshavsky, 1996) Proteins with a primary destabilizing residue at the N terminus are targeted to the ATP dependent proteolytic system of ClpAP and may use the adapte r ClpS (Erbse et al. 2006; Hou et al. 2008) Secondary stabilizing residues, lysine and arginine in E. coli and aspartic and glutamic acid in Vibrio vulnificus and eukaryotic cells, require a primary destabilizing residue be attached to the primary amine at the N terminus of the protein. In the E. coli example, this is carried out by the L/FK,Rtransferase (Aat) and results in a leucine (primary destabilizing residue) as the N terminal residue with the secondary, lysine or arginine in this example, in the N terminal penultimate position of the protein (Abramochkin and Shrader, 1995) The L/FK,Rtransferase can also transfer phenylalanine methionine, and tryptophan to the N terminus of a protein less efficiently but the dominant physiological role is to transfer a leucine (Abramochkin and Shrader, 1995) In V. vulnificus there is an additional transferase, LD,Etransferase (Bpt) that catalyzes the formation of the peptide bond between the N terminal acidic residues, a spartic or glutamic acid, with leucine (Graciet et al. 2006) Aspartic acid and glutamic acid are the only secondary destabilizing residues in eukaryotic system s and in these systems arg inine is conjugated to the N terminus of aspartic acids or glutamic acids by R transferases (Ate1) (Tasak i and Kwon, 2007)


40 Tertiary destabilizing amino acids are only found in eukaryotes to date and require two post translational modifications before entering the proteasome degradation pathway. The tertiary destabilizing residues are aspar a gine, glutamine and cysteine (cysteine is only a tertiary destabilizing residue in mammals) (Varshavsky, 1996) Both aspar agine and glutamine undergo enzymatic deamidation to aspartic and glutamic acid, respectively by the enzyme Ntan1 (Baker and Varshavsky, 1995) Aspartic acid and glutamic acid are secondary destabilizing residues and are substrates for R transferases (Ate1). Cysteine is modified through a reaction with nitric oxide to form oxidized cysteine (Cys S O3H). This oxidized cysteine is a substrate f or the Ate1 R transferase (Varshavsky, 2008) Ubiquitin C onjugation S ystem and P olyubiquitin C hains The ubiquitin conjugation system is the primary targeting mec hanism for protein degradation in eukaryotic cells. Ubiquitin is a conserved 76 amino acid protein and is covalently attached through a series of enzymatic reactions to proteins at lysine residues or the N terminus and targets them for degradation by 26S proteasome s in eukaryotic organisms (Hershko and Ciechanover, 1998) The addition of ubiquitin to proteins is a multi step process that is carried out by a series of enzymes. The chemistry that activates and transfers the ubiquitin protein to a target protein appears to h ave evolved from the thiamine biosynthesis and molybdopterin synthesis pathways (Hochstra sser, 2000; Hochstrasser, 2009) Step one is carried out by the Ubactivating enzyme, E1. The C terminal glycine residue of ubiquitin is activated by forming an intermediate ubiquitin adenylate through the hydrolysis of ATP and the release of pyrophosph ate. A cysteine residue on E1 substitutes for the adenylate resulting in a thioester linkage and the release of adenosine monophosphate (AMP). The activated ubiquitin protein is then transferred to a nother cysteine residue in a n E2 enzyme (ubiquitin carr ier proteins or ubiquitinconjugating enzymes) resulting in a new thioester bond. The ubiquitinligated E2


41 enzyme physically interacts with an E3 enzyme that is bound to a substrate. One of two different events can occur. The E3 enzyme can present a sub strate directly to the E2 and the substrate is ubiquitinated by the E2 or ubiquitin can be transferred to the E3 and onto a free primary amine in the bound protein. In either case, the ubiquitin is linked to either amine of a lysine residue amine of the N terminus resulting in an atypical isopeptide bond (distinct amide) This process may be repeated ligating ubiquitin to ubiquitin until a polyubiquitin chain consisting of four or more ubiquitin molecules is formed (Figure 1 2) In some cases, another enzyme designated E4 (accessory factor) is needed to elongate the ubiquitin chain (Koegl et al. 1999) The second ubiquitin in the chain is connected to the first at a lysine residu e in the primary sequence of ubiquitin, usually Lys48. There are examples of polyubiquitin chains being assembled on Lys6, Lys 11, Lys 27, Lys29, Lys33, Lys 63, but these events seem to be more unique and not always associated with protein degradation (Li and Ye, 2008) The classic signal for degradation by 26S proteasomes is a polyubiquitin chain consisting of at least four ubiquitin molecules connected through Lys48. The four ubiquitin chain signal s 19S regulatory particle s to bind and unfold the substrate. The complexity of this process is reflected in th e enzyme complexity. While a typical organism may have only one or two different E1 enzymes, there can be dozens of different E2 and hundreds of E3 ligases encoded in a single genome (Hershko and Ciechanover, 1998; Huang et al. 2004; Li and Ye, 2008) The E3 enzymes seem to provide the selectivity of substrates with each E3 enzyme having a na rrow range of possible substrates. Several 19S regulatory particle s ubunits can bind ubiquitin, increas ing the concentration of ubiquitinated substrates at 26S proteasomes. Rpt5 subunits of base structures in 19 RP are involved in ubiquitin recognition (Lam et al. 2002) There is some debate how important the Rpt5 interaction with ubiquitin is, since there are several other candidates that may be more


42 available for initial interactions with substrates. The nonATPase subunits Rpn10 and Rpn1 can bind and interact with ubiquitinated proteins (Young et al. 1998; Elsasser et al. 2002) More recently, Rpn13 is thought to be the subunit in the lid portion of the regulatory particle that is involved in initial interaction with ubiquitin tagged substrates (Husnjak et al. 2008; Schreiner et al., 2008) Ubiquitin itself is not degraded in this process. Eukaryotic organisms synthesize deubiquitinating enzym es (DUBs) or ubiquitin specific proteases, that remove the ubiquitin from substrates while they are at the 19S cap. Deubiquitinating enzymes s erve multiple roles in the cell including processing ubiquitin to an active form, proof reading of polyubiquitin chains, and regenerating ubiquitin by removing polyubiquitin chains from proteins as they are translocated into 20S core particles (Amerik and Hochstrasser, 2004) Deubiquitinating enzymes play an important regulatory role with ubiquitin as they initiate ubiquitin immediately after it is translated by removing the N terminal leader sequence and regenerates it so it is not degraded by the proteasome. Pupylation The existence of an ubiquitin like molecule in prokaryotes has been speculated in the past (Durner and Boger, 1995; Maupin Furlow and Ferry, 1995; Bienkowska et al. 2003; Spreter et al., 2005) The discovery of Pup ( p rokaryotic u biquitinlike p rotein) demonstrates that Mycobacterium uses a similar protein tag to modify proteins possibly target them for degradation by the 20S proteas ome The pup gene is in an operon with the proteasome 20S core particle genes prcB and prcA Actinobacter class of bacteria The C terminal sequence, Lys Gly Gly Gln is 100 % conserved throughout the Actinobacter and is similar to the C terminus of ubiquitin, Arg Gly Gly The 6.9 kDa connector protein ca lled Pup has been shown to be covalently linked to proteins and target them for degradation by the 20S


43 proteasome (Iyer et al. 2008; Pearce et al. 2008) Pup is covalently attached to the amine of a lysine side chain of a substrate after the C terminal glutamine is deaminated to a glutamic acid. This de amination is carried out by Dop, a deaminase encoded in M. tuberculosis Dop binds ATP as a cofactor but does not hydrolyze it in the process of deamination (Striebel et al. 2009) The free carboxylic acid is phosphorylated in an ATP dependent manner and the phosphate group is displaced by the target protein (Iyer et al. 2008) This process is catalyzed by a carboxylate amine ligase called PafA. PafA is required for the conjugation of P up to proteasome substrates in Mycobacterium (Pearce et al. 2008) (Figure 1 2) There are dozens of speculated targets for puplyation (Mukherjee and Orth, 2008) and subsequent degradation by the 20S proteasome in Mycobacterium but there are also other questions surrounding this mechanism. For example, i t is unclear if being modified by one Pup protein is sufficient for degradation or if polyPup chains, as is the case with ubiquitin, must form for efficient degradation. Also, it is unclear whether there are Pup specific proteases, depupylating enzymes, that remove Pup proteins or chains from substrates before they are degraded. Pupylation is a n exciting new post translational modification found in the Actinobacter that could be associated with protein turnover. Co and Post translational Modif ication During or after translation, polypeptides undergo a series of maturation events that may include proteolytic processing, folding, localization, and chemical modification before they are mature, functional proteins There are over 200 known protein modifications that can occur across the three domains of life ( some of the modifications discussed in this section can be seen in Figure 1 3) These modifications can be post translational or co translational, stable or reversible. Protein modification is responsible for increased protein diversity across all proteomes. Considering post translational modifications, proteomes may be two or three orders


44 of magnitude more complex than the corresponding genome would predict (Walsh et al. 2005) The majority of proteins that undergo a modification often times are subjected to multiple modifications (Yang, 2005) Some of these modifications include disulfide bond formation, glycosylation, lipidat ion, biotinylation, phosphorylation, methylation, acetylation, and nitrosylation. Nearly every amino acid can undergo a side chain modification with the exceptions of leucine, isoleucine, valine, alanine, and phenylalanine. Some stable modifications are required for proper f olding or stability of a protein O ther transient modifications are used to modulate the function of proteins This section will discuss common post translational modifications that occur in the different domains of life emphasizing the mechanisms of mod ification and the enzymes that catalyze the reactions. N terminal Processing and Maturation All RNAencoded protein synthesis begins at the N terminus. The N terminal residues of growing polypeptide chains are the first portions of proteins exposed to the enzymatic activities of the cytoplasms of organisms. Residue specific alterations to the N terminus of proteins can occur co translationally and post translationally. Modifications such as removal of initiator methionine deformylation of formyl methion amino group by acetylation, or propeptide cleavage are examples. The removal of the initiator methionine and amino of the polypeptide are two modifications that a ffect the majorit y of proteins in eukaryotic cells (Brown and Roberts, 1976) Def ormyla tion of m ethionine Formylated methionine is the initiating residue in bacterial translation. Neither eukaryotes (with the exception of protein synthesis that takes place in the mitochondria or chloroplast) or archaea use formyl methio nine as an initiating residue (Ramesh and RajBhandary, 2001) Deformylation is the co translational removal of the formyl group on methionine and takes place


45 on nearly all polypeptides in bacterial cells (Fry and Lamborg, 1967; Adams, 1968; Giglione and Meinnel, 2001; Giglione et al. 2004) Peptide deformylases catalyze the cleavage of the N formyl group from nascent N formyl methionine polypeptides in bacteria and organelles. Peptide deformylases are related to amino peptidases and belong to the superfamily of HEXXH containing metalloproteases. All the metalloproteases of this large family use two histidine residues of the HEXXH motif as m etal ligands. Peptide deformylases constitute a new subfamily in which cysteine s are the third metal ligand (Meinnel et al. 1996) Methionine r emoval Initiating m ethionine removal by methionine aminopeptidase s is an essential co translational process that occurs in all organisms and compartments within the cell where protein translation takes place across all three domains of life (Bradshaw et al. 1998; Giglione et al. 2004) This process is carried out by a devoted set of peptidases called methionine aminopeptidases (MAP). MAP activity is the major source of amino acid diversity at the N terminus of proteins and 80 % of all proteins undergo this modification in the cell (Waller, 1963; Brown, 1970; Matheson et al. 1975) There are two families of methionine aminopeptidases, MAP1 (type 1) and MAP2 (type 2). The different MAP families have diffe rent phylogenetic distributions Archaea, such as Pyrococcus furiosus and Haloferax volcanii have one MAP2 while bacteria, such as Escherichia coli have one MAP1 (Lowther and Matthews, 2000) Higher e ukaryotic organisms can have both MAP1 and MAP2 in the cytoplasm and MAP1 in organelles. Although these two families of MAP enzymes have only low levels of sequence identi ty, they have similar three dimensional crystal structures (Lowther and Matthews, 2000) Whether or not a protein is a M AP substrate is dictated by the nature of the N terminal (second) penultimate amino acid. The radius of gyration of the second amino acid defines whether or not the initiating methionine can be removed (Hirel et al. 1989) When small ami no


46 acids, such as glycine, alanine, proline, serine, threonine, valine and cysteine, are found in the penultimate position of a protein, the methionine is efficiently removed from the N terminus. Other amino acids, asparagine, aspartic acid, leucine and i soleucine, can also have their initiator methionine removed but at a lower efficiency (Hirel et al. 1989) N terminal acetylation Nacetylation is a common co translational modification in e ukaryot ic organisms with 50 90 % of soluble pr oteins acetylated on N terminal serine, alanine, glycine or threonine residues after the removal of the initiating methionine (Brown and Roberts, 1976; Driessen et al. 1985; Polevoda and Sherman, 2003b) In contrast, bacterial N terminal acetylation is rare and thought to be post translational (Waller, 1963; Tanaka et al. 1989) Recent reports in the haloarchaea have demonstrated that N terminal acetylation may be more common in archaea than in bacteria with an estimated 20 30 % of all soluble proteins being putative substrates (Falb et al. 2006; Humbard et al. 2006; Aivaliotis et al. 2007; Mackay et al. 2007; Kirkland et al. 2008b) Both bacteria and eukaryotic organisms synthesize multiple N terminal acetyltransferases (NAT) In Escherichia coli there are three N terminal acetyltransferases; RimI, RimJ, and RimL (Yoshikawa et al. 1987; Tanaka e t al. 1989) There are three major N terminal acetyltransferases found in Saccharomyces cerevisiae (Polevoda and Sherman, 2003a) They are designated NatA, NatB, and NatC and act on specific substrates. NatA. NatA is the primary NAT in yeast cells with multiple substrates ident ified in vivo and over 2500 predicted protein targets. NatA substrates appear to be degenerative, encompassing a wide range of sequences, especially those with N terminal residues of serine or alanine. Approximately 90 % of proteins with a serine threon ine, glycine, or alanine in the N terminal position are acetylated in yeast (Driessen et al. 1985; Pol evoda and Sherman, 2003b) and a third, auxiliary subunit Nat5p (San) (Gautschi et al. 2003) NatA activity requires two


47 subunits, Ard1p and Nat1p (Mullen et a l. 1989; Park and Szostak, 1992) The probable role of Nat1p is substrate binding and positioning of NatA at the ribosome and Ard1p is the catalytic subunit. The nat1 and ard1 mutants were unable to acetylate proteins in vivo including those with serin e or alanine as the N terminal amino acid. The mutants were tested against 24 proteins known to be acetylated in vivo by twodimensional gel analysis (Polevoda et al. 1999; Polevoda and Sherman, 2003b) The mutants shared similar growth phenotypes including temperature sensitivity, slowe r growth on nonfermentable sugars, failure to enter G0, defects in sporulation, and sensitivity to salt and detergents (SDS) (Mullen et al. 1989) Even though mutants that affect NatA activity have a pleiotropic phenotype in yeast, the activity is not essential. Interestingly, the NatA activity is essential in Trypanosoma brucei during the mammalian and insect stage of development (Ingram et al., 2000) The exact mechanism that makes this activity essential in T. brucei is not currently known. It is speculated that it is required for proper chromatin remodeling or telomeric silencing. Nat5p is not essential or required for the acetylation of the 24 model NatA substrates Knockouts of the nat5 gene in yeast does not share the phenotypes of the ard1 or nat1 mutants (Polevoda and Sherman, 2003a) It is possible that Nat5p acetylates a subset of not yet identified targets. Recent studies have identified possible su bstrates for Nat5p containing NatA complexes in humans (Arnesen et al. 2006) NatB. NatB acetyltransferase contains the catalytic subunit Nat3p a nd the auxiliary subunit Mdm20p. Both are required for NatB activity (Polevoda et al. 2003) NatB substrates have common N terminal sequences of Met Met Met Asn Met Asp or Met Glu All Met Asp and Met Glu eukaryotic pr oteins that have been analyzed have been shown to be N terminally acetylated. NatB acetylates two essential proteins, actin and tropomyosin in yeast. In addition, it has been shown to acetylate the small subunit of ribonucleotide reductase Rnr4p,


48 ribosom al proteins S21 and S28 (Arnold et al. 1999) 26S proteasomal subunit s Pre1p Rpt3p, and Rpn11p (Kimura et al. 2003) Out of all three of the N terminal acetyltransferase activities in yeast, mutations in either nat3p or mdm20 have the most severe phenotypes. It was originally believed to be essential for cell viability (Kulkarni and Sherman, 1994) Further examination revealed that cells were defective in acetylation of t wo essential proteins, actin and tropomyosin. This defect led to the temperature and osmotic sensitivity, inability to utilize non fermentable carbon sources, and reduced mating, and increased sensitivity to DNA damaging agents (Polevoda et al. 2003) Acetylation of tropomyosin is required for association with actin filaments and lack of actin acetylation decreases sliding velocity and disrupts cytoskeleton function (Jeong et al. 2002) The disruption of the cytoskeletal network leads to the pleiotropic phenotype of nat3p and mdm20 mutations. NatC. The catalytic subunit of NatC is Mak3p and encoded by the mak 3 gene NatC is made up of three subunits Mak3p (catalytic), Mak10p and Mak31p (Polevoda and Sherman, 2001) All three subunits are required for the activity of NatC. Knockout strains for the genes responsible for the NatC activity have slower growth rates at higher temperatures when grown on nonfer mentable sugars. The substrates for NatC in the cel l are peptides beginning with an N terminal methionine and followed by an isoleucine, leucine, tryptophan, or phenylalanine (Met Ile, Met Leu, Met Trp, and Met Phe). The activity of NatC is required for the acetylation of the viral major coat protein, gag, with an Ac Met Leu Arg Phe N terminus as well as two 26S proteasomal subunits Pup2p and Pre5p (Kimura et al. 2003) NatC activity is clearly not essential, yet the activity is conserved through eukaryotes and similar substrates are acetylated by the enzyme complex in different species (Polevoda and Sherman, 2003a)


49 RimI, RimJ, and Rim L Only four proteins from E. coli are known to be Nacetylated. They are the ribosomal s ubunit L7 (L12 is the unacetylated form) (Isono and Isono, 1981) S5 (Cumberlidge and Isono, 1979) S18 (Isono and Isono, 1980) and the elongation factor Tu (Arai et al. 1980) Three N terminal acetyltransferases have been identified in E. coli RimI, RimJ and RimL. These three acetyltransferases are responsible for the Nacetylation of L12, S5 and S 18 respectively. The bacterial acetyltransferases apparently do not form a complex with other proteins as the NAT genes of eukaryotic organisms, but act alone. Purified RimL can catalyze the acetylation of the primary N term inal amino of L12 without an accessory proteins (Miao et al., 2007) Since RimL can act in vitro on folded protein substrates, it is believed that the modification of bacterial proteins by N terminal acetylation is posttranslation al rather than co translational. Additionally, the presence of both L7 and L12 (the acetylated and unacetylated forms of L7 protein) on mature ribosomes also indicates that the acet ylation is post translational. Acylamino acid h ydrolysis Acylamino acid releasing enzymes (AAREs) are able to remove acetylat ed amino acids from the N terminal end of peptides and proteins (Radhakrishna and Wold, 1989; Krishna and Wold, 1992; Sokolik et al. 1994) The AARE s have been found in both eukar yotes and archaea but not in bacteria (Ishikawa et al. 1998; Mori and Ishikawa, 2005; Perrier et al. 2005) The phylogenetic distribution follows the trend of generalized protein acetylation pathways. In vitro studies reveal these enzymes can cleave Acetyl Ala, Acetyl Thr, Acetyl Met, Acetyl Gly, and AcetylSer from the N terminus of proteins and peptides (Sokolik et al. 1994) There are two additional peptide hydrolases that can remove acetylated amino acids from the N terminus of peptides, acetyl Met and acetyl Cys hydrolases that are involved in actin processing (Sheff and Rubenstein, 1992a; Sheff and Rubenstein, 1992b) It is unclear whether these enz ymes are


50 related to AARE in structure or function. The exact roles of AARE are currently not known in the cell but they could be involved in recycling amino acids for future synthesis. Phosphorylation Protein phosphorylation has been referred to as the fl agship of reversible post translational modification and is common in all three domains of life (Johnson and Barford, 1993) The vast majority, if not all, cellular process es are affected by phosphorylation of proteins. Ph osphorylation of proteins regulates DNA replication, transcription, translation, metabolism, transport of proteins and metabolites, cell division, protein degradation, and serves as a relay chemical in signal transduction pathways in both prokaryotes and e ukaryotes (Kennelly, 2002; Kennelly, 2003; Eichler and Adams, 2005) Phosphorylation can occur on the side chains of amino acids serine, threonine, tyrosine, aspartic acid, arginine, lysine, cysteine, glutamic acid, and histidine. In the case of ser thr, tyr, and asp, phosphorylation events create a phosphoester bond with the hydroxyl group. Histidine is different. Histidine residues are phosphorylated on a nitrogen atom in the imidazole ring resulting in phosphoramidite bonds (P N). There are two nitrogen atoms that can serve as the acceptor of the phosphate group, atom 1 and 3, resulting in the formation of 1phosphohistidine or 3phosphohistidine A phosphate group can be donated from phosphate of adenosine triphosphate (ATP), phosphate of adenosine diphosphate (ADP), phosphoenolpyru va te (PEP) acetyl phosphate (AcP) polyphosphate or a nother phos phorylated protein. Kinases catalyze the addition of a phosphate group to a substrate. Kinases are one of the l argest group of known post translational enzymes with 5 18 putative protein kinases identified in the human genome alone (Venter et al. 2001; Manning et al. 2002; Johnson and Hunter, 2005) The co mplexity and large number of protein kinases implies a large number of substrates, ranging in the thousands. It is estimated that one third of all intracellular protein s in eukaryotic


51 organisms exist in different phosphorylation states resulting in approximately 20,000 distinct phosphoproteins within the cell. In eukaryotes, kinases are classified by structural motifs in the primary amino acid sequence or by substrate. There is a kinase superfamily of enzymes and 13 atypical protein kinase (aPK) subfamil ies that do not fit into a typical eukaryotic protein kinase (ePK). Typical eukaryotic protein kinases contain a catalytic domain of 250 300 amino acids that bind nucleotides, bind the peptide substrate, and carry out the phosphoryl transfer reaction. Kinases that carry out diverse functions in the cell still have this conserved catalytic domain. Recent genomic data has revealed the atypical protein kinase families that share little to no homology to ePK (LaRonde LeBlanc and W lodawer, 2005b) Histidine / Aspartic Acid Kinases. Histidine and aspartic acid phosphorylation are part of the signal transduction cascade known as two component systems. Two component signal transduction systems are widespread in bacteria and archaea but appear to be restricted to plants, yeast, fungi and protozoa in eukaryotes (Wolanin et al. 2002) The histidine kinase in a twocomponent system can be embedded in the membrane of the cell. In response to external stimuli, the kinase uses ATP to autophosphorylate on a histidine residue creating a phosphoramidate bond (P N), before transferring the phosphate to an aspartic acid residue on a second protein, known as a response regulator (Klumpp and Krieglstein, 2002) The phosphorylated response regulator often times is a transcription factor and directly affects transcription within the bacterial or archaeal cell. Although twocomponent systems have not been found in animals, there are example s of histidine phosphor ylation. Phosphorylation on histidine residues has been demonstrated for histone H4 in rats (Chen et al. 1977) p36 (Motojima and Goto, 1993) and p38 (Hegde and Das, 1987) There is also an atypical protein kinase family that shares sequence similarity to histidine


52 kinases, the branched ketoacid dehydrogenase kinase (Popov et al. 1992) The role of histidine phosphorylation in mammalian cells is currently unknown, although it is speculated to be an important signaling mechanism in liver regeneration cell signaling, differentiation, and cell motility (Klumpp and Krieglstein, 2009) Serine / Threonine Kinases. Serine / t hreonine kinases transfer phosphate groups to create phosphoserine and phosphothreonine through the formation of phosphoester bonds. Phosphorylation of serine and threonine are the most common f orms of phosphorylation with about 90 % of the phosphorylation events occurring on the hydroxyl side chain of serine and 9 % on threonine Every organism has examples of serine/threonine kinases in their genome with nearly 500 in the human genome alone (Venter et al. 2001) In addition to the typical eukaryotic protein kinase superfamily, there are 10 additional serine/threonine kinase families that contain kinases that control transcription (Le Douarin et al. 1996) ribosome biogenesis (LaRonde LeBlanc and Wlodawer, 2005b) stress response (Abraham, 2004) metabolism (Popov et al. 1997) heat shock (Shemetov et al. 2008) apoptosis (Izquierdo and Valcarcel, 2007) translation (Roberts et al. 2006) and signal transduction (Radziwill et al. 2003) A typical serine/threonine kinase will have two major domains, the N terminal domain helix and a C helices. There is a hinge region between the two domains. This allows for movement of the two domains in relation to one another in response to binding of substrates. There are several sub domains associated with kinase structure, including a nucleotide binding domain (typically GXGXXG), a cataly tic loop (with Asp / Asn residues for phosphate transfer), a metal binding loop (DFG l oop), and in some cases an activation loop (APE) (Hanks and Hunter, 1995) Phosphorylation of the


53 activation loop is a common mechanism to negatively regulate kinase activity (Johnson et al. 1996) Tyrosine Kinases. Tyrosine phosphorylation is the least common type of phosphorylation that takes place on proteins. Less than 1 % of all protein phosphorylation events result in phosphotyrosine (~0.05 0.5 % ) (Sefton et al. 1980; Hunter, 1998; Machida et al. 2003) The archetype of an eukaryotic tyrosine kinase is the membrane receptor that autophosphorylates or phosphorylates a binding partner a t the beginning of a signal transduction cascade. Examples of bacterial and archaeal tyrosine phosphorylation have also been detected, although it is unclear if the role of tyrosine phosphorylation is conserved across all domains. Tyrosine phosphorylation of a cellular receptor in eukaryotic cells is closely associated with dimerization and the subsequent transfer of the phosphate to another kinase closely physically associated with the receptor kinase (Hunter, 1998) Glycosylation Protein glycosylation describes a large number of post translational modifications that regulate protein targeting and enzymatic func tion. It is estimated that nearly 50 % of all eukaryotic proteins are glycosylated (Apweiler et al. 1999; Morelle et al. 2009) Eukaryotic glycosylation begins in the lumen of the endoplasmic reticulum and finishes in the Golgi apparatus. Glycosylation was originally believed to be restricted to the Eukarya. It is now known that bacteria and archaea can undergo both N linked and O linked glycosylation. O linked g lycosylation O gylcosyl is a modification that takes place on serine and threonine residues in eukaryotic proteins O ften the modification consists of the addition of monosaccharide s, usually N acetylglucosamine (GlcNAc). The addition of the monosaccharide is typically catalyzed by a devoted O GlcNAc transferase located in the Golgi apparatus and subsequently removed by a


54 devoted hydrolase (Moloney et al. 2000a) There are few examples of polysaccharide additions in O linked glycosylation, such as with the Notch protein (Moloney et al. 2000b) The Notch protein is important in eukaryotic cells as an extra cellular signaling protein and can be modified by chains of two or three different monosaccharides. These different glycosylation events play important roles in the maturation and function of the Notch signaling pathway. Both archaeal and bacterial S layer proteins can undergo O linked modification as well. An O linked glycosylation pathway has been detected in the bacteria Campylobacter jejuni and C. coli (Szymanski et al. 2003) Even though only a few targets have been identifed, the pat hway appears to be similar to the eukaryotic pathway (Szymanski et al. 2003) Both Halobacterium salinarum and Haloferax volcanii have S layer proteins that are modified on threonine residues with a disaccharide galactose glucose (Mescher and Strominger, 1976; Sumper et al. 1990) and Sulfolobus acidocaldarius cytochrome B558/566 contains an O linked mannose subunit (Hettmann et al. 1998) Little is known about the mechanism and enzymology of O linked glycosylation in archaea or its relation to eukaryotic or bacterial pathways. N linked glycosylation In eukaryotic cells, N linked glycosylation is both more common and more complex than O linked glycosylation (Mechref and N ovotny, 2002) The glycan chain is assembled in the endoplasmic reticulum and transferred to an asparagine residue within the sequence motif Ser/Thr X Asn by a oligosaccharyltransferase (Trombetta and Parodi, 2001) The glycan chain is made up of seven different monosaccharides. This heptasaccharide structure is found on all eukaryotic N linked glycosylation substrates. The transfer of the saccharide chain to the asparagine resid ue is through a polyisoprenol based lipid carrier dolichol pyrophosphate (Parodi and Martin Barrientos, 1977) Additional modifications can take place on the glycan chain after


55 it has been attached to the target protein including the addition and subtraction of monosaccharides (Walsh et al. 2005) It is believed that the process of N linked glycosylation that takes place in eukaryotes evolve d from the archaeal system (Burda and Aebi, 1999) This is supported by the observations that antibiotics that interfere with N linked glycosylation in eukaryotes also interfere with N linked glycosylation in archaea. Antibiotics such as bacitracin and tunimycin both inhibit the dolichol carriers in eukaryotes and inhibit N linked gl ycosylation in the archaeon Halobacterium sali narum (Wieland et al. 1980) Interestingly, the same antibiotics do not interfere with N linked glycosylation of the S layer protein of another haloarchaeon Haloferax volcanii (Eichler, 2001) This difference is likely due to variations in the attachment pathwa ys between the two organisms. Other Modifications There are literally hundreds of covalent modifications that can take place on different residues within a protein. The incredible number and diversity of post translational modifications makes it impossible to exhaustively cover in this section. The following examples are of common post translational modifications that eit her occur in archaeal organisms, are related to protein stability or are involved in protei n targeting that have not been discussed previously. Methylation Even though several of proteins are post translationally methylated, protein met hylation remains a rather under studied modification. A protein can be methylated on the amine groups of alanin e, arginine, glutamic acid, histidine, lysine and proline, the hydroxyl groups of glutamic acid and aspartic acid, or the thiol group of cysteine (Nochumson et al. 1978; Paik and Kim, 1986) In all of the examples listed previously, the methyl group is donated from S -


56 adenosyl methionine (SAM). O linked methylation is reversible (Paik et al. 1975) N link ed methylation is irreversible as is the case with the trimethylation of the N terminus of the E. coli protein L11 (Dognin and WittmannLiebold, 1980) Both bacteria and archaea methylate proteins involved in chemotaxis as a result of external stimuli. Halobacterium salinarum uses methylation to regulate separate taxis responses for phototaxis and chemotaxis (Hildebrand and Schimz, 1990; Hou et al. 1998) Som e N methylation events (irreversible methylation of side chain amino group of lysine) are thought to increase the thermal stability of a protein and lessen the tendency to aggregate (Febbraio et al. 2004) DNAbinding and ribosomal proteins are also methylated. H istone proteins are also heavily methylated (Cheung et al. 2000) This pattern of methylation of histone proteins is conserved in archaeal organisms. T he Sul7 family of histone proteins found in Sulfolobus species are methylated on lysine residues (Baumann et al. 1994; Knapp et al. 1996; Wardleworth et al. 2002) Another mechanism of methylation is the i soaspartate methylation pathway. This pathway is involved in protein repair and over expression increases heat s hock response in Escherichia coli (Kindrachuk et al. 2003) Isoaspartyl residues form spontaneously through deamidation of asparagine or isomerization of aspartic acid. The carbonyl group of the amino acid attacks the amine of the amino acid backbone forming a five membered Lsuccinimidyl ring. This ring spontaneously opens to either aspartic acid or the atypical Lisoasparyl residue. The rate of formation of the isoasparyl residue is three times the rate for the formation of aspartic acid (Johnson et al. 1987) Methylation of isoaspartate by peroxisome proliferatoractivated receptor interacting protein with methyltransferase domain ( PIMT ) increases the rate at which the isoasparyl residue returns to the succinimide ring form. The ring rehydrolyzes to either


57 aspartate or isoaspartate. If the ring resolves to isoaspartate, the process repeats (Vigneswara et al., 2006) Although this process may seem inefficient, after several rounds of methylation, the damaged protein can regain function (Johnson et al. 1987) Acylation There are several different kinds of acylation that can take place on a protein. The most common forms of acylation are acetylation of amino group of lysines and amino groups of N termini myristoylation of N terminal glycines, and palmi toylation of cysteine residues. Having already discussed N terminal acetylation, the following section will focus on the acyl modification lipidation Lipidation Lipid modification of proteins include s protein isoprenylation, palmitoylation, glycosylphophatidylinositol lipid anchoring, N myristoylation and several other modifications Isoprenylation has been observed in the haloarchaea Halobacterium salinarum and Haloferax volcanii (Sagami et al. 1994; Konrad and Eichler, 2002) N myristoylation is an irreversible cotranslational modification where a myristate (14 carbon) is covalently linked to an N terminal glycine residue of a protein (Johnson et al. 1994) The myristate molecule is attached to the N terminal glycine through an amide bond after the initiating methionine is removed by a methionine aminopeptidase (Utsumi et al. 2001; Maurer Stroh et al. 2002) The enzyme that catalyzes the addition of the myristate is a N myristoyl transferase (NMT). NMT transfers the myristate group from the intermediate compound myristoyl CoA, with release of CoA in the process. Proteins modified by N myristoylation are typ ically found in the membrane. The hydrophobicity of the myristate group helps to localize the proteins to the membrane and can aid in hyrdrophobic protein protein interactions (Farazi et al. 2001) Palmitoylation of proteins is the addition of a C16 chain from a fatty acylCoA donor to a cysteine residue, rather than an N -


58 terminal glycine. This modification is referred to as S palmitoylation (Bijlmakers and Marsh, 2003) While it is not widespread in eukaryotic organisms, the modification plays an important role for the proper localization and function of Ras GTPase signaling (Resh, 1999) Post translational M odification of P roteasomal P roteins Subunits of 20S core particles and associated proteins are subject to several f orms of post translational modification. In addition to the propeptide processing that some undergo, subunits from 20S and 26S proteasomes undergo Nacetylat ion phosphorylat ion, S glutathionat ion (Demasi et al. 2003) O linked glycosylation (Zachara and Hart, 2004; Zhang et al., 2003a) and N myristoyl at ion (Lee and Shaw, 2007) Even though the functions of some of the previously mentioned modifications are unknown, the large number of modifications indicates the proteasome system is heavily regulated (summarized in Table 11) All seven type subunits and are N terminally acetylated (Kimura et al. 2000) Additionally, 20S cor e particle type subunits from haloarchaea Halobacterium salinarum and Natronomonas pharaonis are also N terminally acetylated (Aivaliotis et al. 2007; Falb et al. 2006) including type subunits for the haloarchaeon Haloferax volcanii (Humbard et al. 2006) The crystal structures of 20S core particles rings show that the N helix type subunits poi nts into the aperture, creating a steric gate. 20S proteasomes purified from a nat1 yeast strain had a two fold increase in chymotrypsinlike peptidase activity (Kimura et al. 2000) Analysis of the N purified from this nat1 strain revealed that 5 out of the seven were no longer modified. These data suggest acetylation 20S c ore particles is important in establishing or maintainin g the regulatory gate ring type type subunits of 20S core particles in yeast t en out of the seventeen subunits of 19S regulatory caps in yeast are also N-


59 acetylated: Rpt3, Rpt4, Rpt5, Rpt6, Rpn2, Rpn3, Rpn5, Rpn6, Rpn8, and Rpn11 (Kimura et al. 2003) Phosphorylation of subunits from 20S core particles and 19S regulatory particles is well established. Four out of the six Rpt subunits are phosphor ylated in the 19S regulatory particle, Rpt2, Rpt3, Rpt4, and Rpt6, as well as one Rpn subunit, Rpn8. The exact roles of many of the phosphorylation events is unclear at this time. It has been speculated that the phosphorylation of subunits in response to interferon may influence the shift to the immunoproteasome (Bose et al., 2001; Rivett et al. 2001; Bose et al. 2004) Phosphorylation of the 19S regulatory particle Rpt6 has b een shown to stimulate 19S association with the 20S to form the 26S proteasome 3. Dephosphorylation stimulates the disassembly (Satoh et al. 2001) Rpt2 subunits from 19S regulatory particles are N myristoylated in Saccharomyces cerevisiae after the initiating methionine is removed (Kimura et al. 2003) Rpt4 subunits from several organisms are predicted by primary amino acid sequence to be N myristoylated. The organisms predicted to have m yristoylated N termini are Caenorhabditis elegans Gallus gallus Drosophila melanogaster Homo sapiens Mus musculus Schizosaccharomyces pombe and Saccharomyces cerevisiae (Maurer Stroh et al. 2002) N myristoylation has been shown to aid in the translocation of proteasomes between nuclear envelope and the endoplasmic reticulum. This translocation event may even be coupled to a phosphorylation of a serine residue on Rpt4 found near the N terminus The exact role of this modification in the 26S proteasome is unknown, but N myristoylated is known to affect proteinprotein and protein membrane interactions (Johnson et al. 1994; Farazi et al. 2001)


60 Other modifications of proteasomal subunits have been seen such as O N acetylglucosamine (O GlcNAc) and S glutathionylation. Recent reports demonstrate a number of subunits from both Drosophila (Sumegi et al. 2003) and mammalian proteasomes (Zhang et al., 2003a) are glycos ylated with O GlcNAc moieties. This modification is quite widespread in Drosophila with 5 out of 19 subunits of the 19S cap and 9 out of 14 of the subunits of the core par ticle being reversibly modified in this way (Sumegi et al. 2003) The modification of mammalian proteasomes by O GlcNAc negatively regulates proteasome activity and blocks the degradation of the transc ription factor Sp1 (Zhang et al. 2003a) The changes in different levels of glycosylation of the proteasome correspond with periods of low nutrition. The lower the nutrition, the less glycosylation, the more Sp1 can be degraded by the proteasome (Zhang et al. 2003a) An additional post translational mechanism of negatively regulating the proteasome is the S glutathionylation of yeast 20S proteasomes in response to oxidative stress (Demasi et al. 2001; Demasi and Davies, 2003; Ishii et al. 2005) While the demonstration of this modification is solely in vitro it makes sense to temporarily and reversibly inhibit the activity of the 20S proteasome in yeast immediately following an oxidative stress. A temporary decrease in proteasome activity would prevent the degradation of redox signaling factors such as AP 1. Also, preemptively modifying the proteasome subunit cysteines wi th S glutathion would prevent the formation of irreversible oxidation of thiol groups to Cys SO2H or Cys SO3H, allowing the proteasome to eventually be reactivated in the absence of oxidative stress (Demasi et al. 2003) Mass Spectrometry T andem mass spectrometry has emerged in the past decade as a reliable and sensitive method for identificat ion and analysis of small molecules such as peptides and post translational modifications. Tandem mass spectrometry typically utilizes site specific proteases, such as Trypsin or AspN, to fragment proteins into peptides of predictable size (Olsen et al. 2004) The


61 resulting peptides may or may not be separated by highpressure liquid chromatography before analysis by a mass spectro meter The work flow for tandem mass spectrometry is a series of steps where the sample interacts with an ion source, a mass analyzer, a collision chamber, a second mass analyzer, and a detector. Different mass spectrometers have different types of ion s ources, mass analyzers, collision chambers, and de te ctors Peptides are ionized and enter a detector that measures the masses of the most abundant peptides. After the masses are measured, the peptides are selectively moved into a collision chamber where they are fragmented again through a process called collision induced dissociation (CID). The masses of the resulting fragments are measured in another mass analyzer. The output of the second mass analyzer is called an MS/MS or MS2. Different strategies for ionization may include matrix assisted laser desorption ionization (MALDI) or electron spray ionization ( ESI) (Bakhtiar and Nelson, 2000) Different mass analyzers can be used in combination with the different ionization strategies such as quadrupoles (Q) or time of flight tubes (TOF). In tandem mass spectrometry least two mass analyzers are needed A mass spectrometer can have the same mass analyzers as is the case for a MALDI TOF/TOF or an ESI Q qQ or have different mass analyzers such as ESI Q T OF. In combinati on with other proteomic techniques such as two dimensional gel electrophoresis or strong cation exchange chromatography (SCX) a large number of proteins can be separated and identified by mass spectrometry Recent advances in proteomics and specifically mass spectrometry have allowed scientists to obt ain accurate quantifiable readings of protein levels in different samples. This section will review and highlight the role mass spectrometry has played in the identification of phosphorylation sites as well as review the current state of the art of quanti tative mass spectrometry techniques.


62 Phosphorylation S ite I dentification by Tandem Mass Spectrometry A dvances in tandem mass spectrometry (MS/MS or MS2) have made the highthroughput identification of multiple phosphorylation sites on hundreds of proteins simultaneously possible (Resing and Ahn, 1997; Sickmann and Meyer, 2001) Prior to tandem mass spectrometry, scientists relied on the use of radioactive phosphates, two dimensional gel electrophoresis or N terminal sequencing to determine sites of phosphorylation. While not only being technically difficult, they often required the use of radioactive phosphate and therefore limited the types of analysis that were available. Recent advances in immobilized metal affinity chromatography (IMAC) (Posewitz and Tempst, 1999; Zhou et al. 2000; Ficarro et al. 2002; Raska et al. 2002; Nuhse et al. 2004) dyes and stains (Jacob and Turck, 2008) and use of phosphospecific antibodies (Zhang et al. 2002) have allowed for the highthroughput identification of hundreds of phosphorylation sites. The incorporation of phosphopeptide selective techniques with tandem MS/MS has created an exponential increase in the total number of phosphorylation sites identified. In 2003, nearly 500 novel phosphorylation sites were identifi ed on proteins. That is greater than the sum of all the phosphosite s identified in the primary literature from 1990 to 2001. Of those nearly 500 phosphosites, almost 70 % of them were identified by MS/MS experiments (Loyet et al. 2005) Label based methods for the detection of phosphopeptides have been used to map the positio elimination reaction phosphate elimination of phosphoserine and methyldehydr oalanine (Knight et al. 2003) These resulting alkene groups can be used as nucleophiles for the attachment of various chemicals that are easily detected such as fluorophores (McLachlin and Chait, 2003) Specifically, the fluorescence affinity tag ( or FAT)


63 that consists of cysteamine linked to a fluorescent rhodamine tag, has been used to selectiv ely modify and tag phosphoproteins for offline purification prior to MS/MS analysis (Stevens, Jr. et al., 2005) All of these phosphospecific tools have given rise to increased understanding of global levels of phosphorylation in vivo and further fuels understanding of post translational modification. Quantitative Mass Spectrometry Proteomic researchers not only want to identify what proteins or what modifications are present in an organism but a lso to rank proteins in order of abundance or relative quantity to one another. The ability to quantitatively measure the difference between biological states is fundamental to a detailed understanding of cellular biology. Early attempts at quantifying proteomic data involved the use of protein score to rank abundance (Allet et al. 2004) A protein is scored by the number of times a peptide from that protein is identified in a mass spectrometry run, t he higher the score the more abundant. This initial strategy proved ineffective i n providing repeatable quantitative data (Ishihama et al. 2005) New strategies have been developed in the past decade that provide comparative and absolute quantification of proteins in complex mixtures. There are two basic strategies of quantitative mass spectrometry, using unmodified proteins and comparing two samples under the same conditions (labelfree) or using tags, chemical or isotopes, to take direct measurements of protein amounts (labeling based methods) Label free m ethodology There are several practical situations w here direct comparison of two different samples is required in the absence of isotopic labeling or chemical tagging To address this need, there are two different quantitative techniques tha t do not involve the use of any kind of label The first


64 involves counting and comparing the number of spectra of a protein, and the second compar es the intensity of the mass spectrometric signal of precursor ions be longing to a specific protein. The firs t strategy is called s pectral counting and is a quantitative method that involves comparing the number of MS/MS spectra assigned to each protein. Examples of this technique using idealized proteins spiked into yeast extract demonstrates that spectral coun t ing is correlative to protein quantity over two orders of magnitude (Liu et al. 2004) The correlation is strong enough to compare proteomic measurements with estimated protein copy numbers in yeast cells (Ghaemmaghami et al. 2003) There are some obvious disadvantages and limitations to spectral counting. While spectral counting can provide a general id ea of the relative abundance of a protein between two samples, it cannot be used to compare two different proteins in the same preparation. Also, spectral counting is correlated less with protein abundance as the mixture becomes more complex. Since many proteomics researchers use the bench mark of two peptides per protein, spectral counting usually under estimates the differences in protein levels. Additional statistical tools and techniques have been added to this pr actice to make it more accurate (Gilchrist et al. 2006; Washburn et al. 2001) Since there is a direct correlation between the numbers of peptides identified in a single run to the abundance of that protein, a common way to estimate the dominant proteins in a complex mixture is called protein abundance indices (PAI). PAI can use any observed parameter in a mass spectrometry run, but combines multiple readings from all observed peptides The relationship between protein quantity and peptide identifications was observed to be logarithmic. Correlating protein quantity to peptide nu mbers is referred to as exponentially modified PAI (emPAI) (Ishihama et al. 2005) The second strategy involves the use of peak intensity of the first MS run to estimate relative protein quantity. This strategy is often referred to as extracted ion chromatogram (XIC)


65 counting (Bondarenko et al. 2002) Extr acted ion chromatogram based methods are more reliable and sensitive than spectral counting alone. The principle is similar in that the intensity of a signal is still measured but rather than measuring the number of times the mass spectrometer records a peptide hit, the nature of the chromatography peak is analyzed in order to make a comparison. The area under the curve for a specific peptide can be linearly related to the relative quantity of that protein. Two peptides from different chromatographic pr eparations can be directly and accurately compared. There is no way to determine the MS detector response to a random peptide due to difference in ionization properties but a strategy that utilizes the peak areas of three of the most abundant peptides of a protein has proven to be quantitative. This process combines XIC with PAI and is termed xPAI (Rappsilber et al. 2002) By averaging the signal of multiple peptides from different proteins in the same mass spectrometry run, a rough idea of the relative quantity of those two proteins can be estimated. This has proven to be more accurate than just spectral counting alone when comparing two dissimilar proteins in the same sample. Thorough comparisons of these different approaches have been performed (Old et al. 2005; Anderson and Hunter, 2006; Ono et al. 2006; Bantsch eff et al. 2007) The overall conclusions of these studies reveal that the two techniques spectral counting and XIC, can be used to make different kinds of comparisons. S pectral counting, with or without emPAI, is more accurate at comparing proteins f rom two different runs than XIC based methods, and peak area intensities (XIC based methods) are more accurate at estimating the difference s between two proteins in the same sample than spectral counting alone (Old et al. 2005) Together, spectral counting and XIC based methods give researchers valuable insight into relative protein quantities between samples and within the same sample.


66 Label based m ethod ology Additional methodologies for the quantitative analysis of protein samples have been developed that use tags consisting of either a chemical that modifies peptides or stable isotopes to alter the molecular weight of peptides. Stable isotope labeling refers to a set of strategies that involves the incorporation or labeling of proteins of peptides with heavy isotopes of carbon (13C), nitrogen (15N), oxygen (18O) or hydrogen (deuterium, 2H). There are four basic strategies for the incorporation of he avy stable isotopes into mass spectrometry samples, (i) adding an isotopically labeled peptides that mirrors a physiological one, (ii) incorporation of isotopes during enzymatic digestion of protein, (iii) reacting proteins or peptides with isotopically l abeled markers, or (iv) having the isotopes incorporated by living cells metabolically. Examples of each of these strate gies will be discussed further. The addition of a synthetic peptide to a sample that was synthesized with an isotopically labeled amino acid (15N alanine for example) is a way to measure the absolute quantity of peptide in the sample. This strategy is called Absolute quantification (AQUA) (Des iderio and Kai, 1983; Gerber et al. 2003; Kirkpatrick et al. 2005) AQUA is widely accepted as the most analytical mass spectrometry technique for the measurement of peptide quantity in a sample. A synthetic peptide labeled with stable isotopes of known sequence is spiked into a sample. The quantity of the AQUA peptide is known. Since the AQUA peptide has the exact same sequence as a peptide previously identified in the sample, the ionization and detection should be the same for both. This removes t he variability normally associated with comparing two unique peptides. This technique not only allows for the relative comparison of peptide quantities in the sample, but since the exact amount of AQUA peptide added to the sample is known, the exact amount of peptide in the sample can be measured (Desiderio and Kai, 1983) AQUA has several disadvantages, most notably the synthesis of the synthetic peptide can be tedious and expensive.


67 Furthermore, e ach AQUA experiment requires new peptides to be synthesized and different experiments and peptides cannot be directly compared. Even with these drawbacks, AQUA remains the most standard method for quantification of mass spectrometry data. The secon d, albeit unpopular, strategy to incorporate a label is to perform the protease digestion in the presence of H2 18O. During a trypsin digestion, 18O is incorporated into the peptide during cleavage if the digestion is performed in the presence of H2 18O (Mirgorodskaya et al., 2000; Yao et al. 2001) A problem with this technique is the variability in the amount of 18O that is incorporated into the peptide. One or both of the oxygen atoms may be exchanged for the solvent 18O at the C termina l end of the peptide. In addition, some proteases will only incorporate one 18O into the peptide such as Lys N. This will result in only a 2 Da shift in molecular weight and such a small shift in weight is not adequate for quantitative measurement. Chemi cal tagging approaches are popular ways to quantify different proteomic samples. Different reagents can be reacted with the sulfhydryl, amine, and carboxyl groups of amino acids. Isotope coded affinity tag (ICAT) was one of the first commercially availab le tags (Gygi et al. 1999) The ICAT reagent consists of a cysteine reactive group and a polyether linker region with 8 deuteriums (heavy) or 8 hydrogens (light) and a biotin molecule for the recovery of the modified peptides. Two different samples will be reacted with either the light or the heavy ICAT reagent. The modified proteins are mixed and digested with a protease such as trypsin. The labeled peptides are isolated in an avidin column and analyzed by MS The fact that ICAT reagents only react with sulfhydryl groups is both an advantage and a disadvantage. Cysteine is a relatively infrequent amino acid so a complex mixture is significantly simplified after the avidin chromatography. A simplified mixture aids in producing accurate quantifiable data.


68 However, s ome proteins contain only one or no cysteines. For these proteins, quantifiable data will not be generated in an ICAT experiment (Wu et al. 2006) Another tagging strategy derivitizes amine groups of N termini and amine groups of lysine residues. This tagging method is called i sobaric tag for relative and absolute quanti fic ation (iTRAQ) (Ross et al. 2004) The iTRAQ labeling system is a set of reagents that use N hydroxysuccinimide ( NHS ) chemistry to react to the amines but each of the different (up to 8) iTRAQ reagents give a unique reporter ion in the MS/MS sc an (114 Da 121 Da). In an ICAT experiment, protein pools were labeled, iTRAQ reagents are typically added to pools of peptides since modification of lysine residues prevents cleavage by trypsin. Since each of the tags are a different mass and give a si milar fragmentation spectrum, the samples can be mixed to reduce variability from run to run (Wu et al. 2006) The last strategy tha t will be discussed with be in vivo incorporation of stable isotopes. There are two basic approaches. The first involves growing cells in media that contains light nitrogen (14N) or heavy nitrogen (15N). T w o different cultures are grown, one in the heavy and one in the light nitrogen. After both are grown, the cultures are mixed and processed for downstream MS analysis. Both the heavy and the light peptides are present in each run so the ratio of the two peptides is directly compared (Conrads et al. 2001) Mass shifts due to this technique can be difficult to predict since the labeling can be in both the aminobackbone of the protein as well as the amino acid side chains. The s econd approach for in vivo labeling of proteins is called stable isotope labeling with amino acids in cell culture (SILAC) (Ong et al. 2002) SILAC uses 13C lysine, 2H leucine, 13C tyrosine, or 13C / 15N arginine as the heavy isotopes. SILAC is more often used than the 14N / 15N method pr eviously discussed because the mass defects are easier to predict making data analysis less difficult. The workflow is very


69 similar. The heavy amino acids are added to cultures that are actively growing. After the growth (and labeling) is completed, the samples are mixed and processed as one sample for mass spectrometry. The different masses can be directly compared in the mass spectrometry run. The most common strategy for SILAC is the use of a heavy arginine (either 13C or 15N) since every tryptic fragment would contain a predictable mass defect due to the presence of the C terminal arginine. Project Rational and Design The objective of this study is to identify post translational modifications of the subunits of 20S proteasomes and PAN proteins fro m Haloferax volcanii Due to the ease of genetic manipulation as well as the relative simplicity of the proteasome system, Hfx. volcanii is an ideal model organism to study archaea and proteasomes. Since little was known concerning post translational modifications of proteins in archaea or proteasome subunits, the first goal was to establish how the different subunits were modified. Once the nature of the modification was known, the location on each of the proteins was identified Once the modification and site of the modification was known, mutations were introduced into the genes in order to perturb the primary amino acid sequence surrounding the modification, and blocking or disrupting the modification. Once successful, cell expressing variant protei ns were assayed for a variety of phenotypes including temperature tolerance and osmotic stress. P roteins were purified and assayed for activity and presence of the modifications Finally, bioinformatic techniques identified candidate genes responsible for the modifications. Candidate enzymes were tested for their ability to modify the wild type or variant proteins in vitro


70 Figure 11. Crystal s tructures of 20S proteasomes from Thermoplasma acidophilium (PBD:1PMA) (Lowe et al. 1995) configuration with a central channel joining three inner cavitie s. The central cavity is lined by the catalytic sites, which mediate the hydrolysis of peptide bonds. The axial pores on each end of the cylinder restrict access of substrate. Graphic rendered with Pymol.


71 Figure 12. Ubiquitination and pupylation pathway s Ubiquitin is activated by E1 and transferred to an E2 enzyme. Depending on the E2 enzyme, the ubiquitin will either be transferred to an E3 and then a substrate, or directly transferred to a substrate bound by a RING protein. A polyubiquitin chain will form on the substrate, targeting it for degradation by the proteasome. The Pup protein is deaminated by Dop and phosphorylated, possibly by PafA. The phosphorylated Pup is covalently attached to a substrate protein that may be degraded by the pr oteasome.


72 N-terminal acetylation NH2CH C N H CH C N H CH Gly O Gln O Met CH3N H N H N H Gly O Gln O Met O N-terminal acetylation NH2CH C N H CH C N H CH Gly O Gln O Met CH3N H N H N H Gly O Gln O Met O Phosphoserine Phosphothreonine Phosphotyrosine Phosphoaspartate 1-phosphohistidine 3-phosphohistidine Phosphorylation of Amino Acids O H O NH N OH OH N NH PO OO O P OO O O O P OOO O CH3P OOO O P OO O N N P O OO OH CH3 Phosphoserine Phosphothreonine Phosphotyrosine Phosphoaspartate 1-phosphohistidine 3-phosphohistidine Phosphorylation of Amino Acids O H O NH N OH OH N NH PO OO O P OO O O O P OOO O CH3P OOO O P OO O N N P O OO OH CH3 Lysine N-acetyl-Lys N-acetylation HN NH3+ HN O NH O CH3O Lysine N-acetyl-Lys N-acetylation HN NH3+ HN O NH O CH3O Lysine N-alkyl-Lys -N-alkylation HN NH3+ HN O NH CH3O Lysine N-alkyl-Lys -N-alkylation HN NH3+ HN O NH CH3O UDP-Glucose O-linked GlycosylationThreonine Glc-O-Thr CH3OH CH3O O OH OH OH OH UDP-Glucose O-linked GlycosylationThreonine Glc-O-Thr CH3OH CH3O O OH OH OH OH N-terminus N-myristoly-Gly-protein N-myristoylation 11NH2O N H O O CH3 N-terminus N-myristoly-Gly-protein N-myristoylation 11NH2O N H O O CH3 Figure 1-3. Modified amino aci d structures. Modificati ons, N-terminal acetylation, phosphorylation, N-acetylation, -N-alkylation (methylation), O-linked glycosylation, and N-mystri stoylation, are in red.


73 Table 1 1. List of known post translational modifications of proteasome subunits. Subunit Organism Modification Enzyme Reference 7 Ca ndida albicans ~P CK2 (Pardo et al. 1998) 6 Candida albicans ~P CK2, rhCK2 (Fernandez et al. 2002) 3 Candida albicans S248~P CK2 (Fernandez et al. 2002) 5 Candida albicans ~P CK2 (Fernandez et al. 2002) 7 Carassius auratus ~P (Horiguchi et a l. 2005; Tokumoto et al. 2000) 2 Carassius auratus Y120~P, A1 N acetylalanine (Horiguchi et al. 2005; Tokumoto et al. 2000) 1 Drosophila melanogaster Y103~P (Frentzel et al. 1992; Haass et al. 1989) 1 Homo sapiens N acetylmethionine (DeMartino et al. 1991; Kristensen et al. 1995; Kristensen et al. 1994; Tamura et al. 1991) 3 Homo sapiens ~P S249, N acetylserine CKII (Claverol et al. 2002; Kristensen et al. 1995; Kristensen et al. 1994; Tamura et al. 1991; Beausoleil et al. 2004) 4 Homo sapiens ~P CKII (Kristensen et al. 1995; Kristensen et al. 1994; Tamura et al. 1991) 2 Homo sapiens Y120~P N acetylalanine (Kristensen et al. 1995; Kristensen et al. 1994; Tamura et al. 1991) 5 Homo sapiens S56~P (DeMartino et al. 1991; Beausoleil et al. 2004) 7 Homo sapiens S250~P, N acetylated 1 Mus musculus N acetylmethionine (Elenich et al. 1999) 3 Mus musculus N acetylserine S249~P ( Elenich et al. 1999) 2 Mus musculus N acetylalanine, Y120~P (Seelig et al. 1993) 5 Mus musculus S56~P (Elenich et al. 1999) 1 Oryza sativa S~P CKII (Umeda et al. 1997a; Umeda et al. 1997b) 1 Rattus norvegicus N acetylmethionine (Tokunaga et al. 1990) 3 Rattus norvegicus N acety lserine S249~P, S250~P, S243~P (Castano et al. 1996; Tokunaga et al. 1990) 4 Rattus norvegicus ~P (Castano et al. 1996) 5 Rattus norvegicus S56~P 6 Rattus norvegicus alkylation 2 Rattus norvegicus N acetylalanine, Y120~P (Tokunaga et al. 1990) 7 Rattus norvegicus ~P 3 Rattus norvegicus alkylation (Nishimura et al. 1993a) 2 Saccharomyces cerevisiae acetylated NAT1 (Emori et al. 1991; Groll et al. 1997; Iwafune et al. 2002; Kimura et al. 2000)


74 Subunit Organism Modification Enzyme Reference 7 Saccharomyces cerevisiae acetylated, S258~P, S263~P, S264~P, ~P CK II, NAT1 (Emori et al. 1991; Groll et al. 1997; Iwafune et al. 2004; Iwafune e t al. 2002; Pardo et al. 1998; Kimura et al. 2000) 3 Saccharomyces cerevisiae N Myristoylation, acetylated NAT1 (Emori et al. 1991; Groll et al. 1997; Kimura et al. 2000) 6 Saccharomyces cerevisiae acetylated MAK3 (Heinemeyer et al. 1994; Kimura et al. 2000) 5 Saccharomyces cerevisiae acetylated MAK3 (Chen and Hochstrasser, 1995; Groll et al. 1997; Kimura et al. 2000) 1 Saccharomyces cerevisiae acetylated NAT1 (Kimura et al. 2000) 4 Saccharomyces cerevisiae acetylated NAT1 (Chen and Hochstrasser, 1995; Groll et al. 1997; Iwafune et al. 2002; Kimura et al. 2000) 4 Saccharomyces cerevisiae acetylated NAT3 (Groll et al. 1997; Kimura et al. 2000) 3 Saccharomyces cerevisiae acetylated NAT1 (Groll et al. 1997; Kimura et al. 2000) Xenopus laevis ~P (Tokumoto et al. 1999; Wakata et al. 2004) 2 Xenopus laevis N acetylalanine, Y120~P (Tokumoto et al. 1999; Wakata et al. 2004)


75 CHAPTER 2 MATERIALS AND METHOD S Chemicals, Strains and Media Materials Organic and inorga nic analytical grade chemicals were from Fisher Scientific (Atlanta, GA) and Sigma Chemical Co. (St. Louis, MO ) unless otherwise indicated Restriction endonucleases and DNA modifying enzymes were from New England BioLabs (Beverly, M A ). Desalted oligonuc leotides were from Integrated DNA Technologies (Coralville, IA ). Hi Lo DNA molecular weight standards were from Minnesota Molecular, Inc. (Minneapolis, MN) Agarose, real time polymerase chain reaction ( RT PCR) materials and Precision Plus protein molec ular mass were purchased from Bio Rad (Hercules, CA) Radioactive chemicals were purchased from Perkin Elmer (Waltham, M A ) and StrepTactin Superflow resin was purchased from Qiagen (Valencia, CA ). Site directed mutagenesis materials were from Invitroge n (Carlsbad, CA). Polyvinyl difluoride membranes for Western blots were from Amersham Biosciences ( Piscataway, NJ). Strains, Media, and Plasmids Strains and plasmids used in each of the studies are listed in T ables contained within each chapter. Escherichia coli ( Life Technologies ) was used for routine cloning, and GM2163 ( New England Biolabs ) was used for purification of plasmid DNA for transformation of Hfx. volcanii as previously described (Cline and Doolittle, 1992) Hfx. volcanii strains were grown in ATCC 974 medium at 42 or in minimal medi um ( lactate and glycerol ) adapted from Allers et. al. (Allers et al., 2004) for radiolabeling studies consisting of 2.5 M NaCl, 85 mM MgCl26H2O, 85 mM MgSO47H2O, 55 mM KCl, 5 mM CaCl22H2O, 5 mM NH4Cl, 4.5 mM NaBr, 1.5 mM NaHCO3, 10 mM KPO4 pH 7.5, 30 mM Tris pH 7.5, 0.25 %


76 (v/v) dllactic acid, 15 mM succinic acid, 0.025 % (v/v) glycerol. Media were s upplemented with 2 nM MnCl24H2O, 1.5 nM ZnSO47H2O, 8 nM FeSO47H2O, 2 nM CuSO45H2 ml leucine, ml methionine, ml tryptophan, ml glycine, d ml Lpantothenic acid, ml ml thymidine and hypoxantine, 800 ng ml thiamine 100 ng ml dbiotin. E. coli st rains were grown in Luria Bertani broth at 37 ml ml of novobiocin as needed. For salt stress tests, the concentration of NaCl was reduced to 1, 1.125, 1.25, 1.375 and 1.5 M from the typi cal 2.25 M NaCl of the normal ATCC 974 medium. Inoculum for the salt stress test was generated by growing cells in normal salt ATCC 974 medium. Cells were streaked from 80 C glycerol stocks onto fresh plates, grown twice to log phase in 2 ml medi um and used as an inoculum for the salt stress test Each subculture was inoculated to an initial OD600 of 0.01 to 0.02. Experiments were performed in triplicate and t he mean S.D. was calculated. Casamino acid medium used in the construction of acetyltra nsferase knockouts consisted of 2.4 M NaCl, 80 mM MgCl26H2O, 175 mM MgSO47H2O, 50 mM KCl, 120 mM Tris buffer (pH 7.5), 3 mM CaCl2, and 0.5 % (w/v) casamino acids. Media were mluracil and / ml 5fluroorotic acid as needed. DNA Techniques Cloning Genes were amplified by PCR using DNA polymerases including Thermalase (Invitrogen), Accuprime, or Phusion (New England Biolabs). The fidelity of all cloned PCRamplified products was confirmed by DNA sequencing using the dideoxy termination method with Perkin Elmer/Applied Biosystems and LICOR automated DNA sequencers (DNA Sequencing Facilities, Interdisciplinary Center for Biotechnology Research and Department of Microbiology and Cell Science, University of Florida). Produc ts and vectors were cut with


77 restriction enzymes Nde I, Blp I, Kpn I, Bbv CI, BamHI, Eco RI or Xba I according to M anufacturers specifications (New England Biolabs) as needed. Digested vector DNAs were treated with shrimp alkaline phosphatase (Roche Scientific) for one hour at 37 cloning, Vent polymerase was used to fill in Blp I overhangs The resulting product s w ere treated with T4 polynucleotide kinase (PNK) as per M anufacturers instructions (New England Biolands) Ligation reactions were preformed using T4 DNA ligase at 16 24 hours (New England Biolabs). For cloning products encoding a n in frame 3` Strep_II tag, PCR products and pJAM816 (Humbard et al. 2009) were cut with Nde I and Kpn I Digested pJAM816 was treated with shrimp alkaline phos phatase (Roche Scientific) for one hour at 37 Products were ligated using T4 DNA ligase as previously described. DNA Electrophoresis S izes of PCR products and plasmids were analyzed by electrophoresis using 0.8 2 % (w/v) agarose gels in TAE buffer ( 40 mM Tris acetate, 2 mM EDTA, pH 8.5) with Hi Lo DNA molecular weight markers as standards (Minnesota Molecular, Minneapolis, MN ). Gels were photographed after staining with ethidium bromide at 0.5 gml with a Mini visionary imaging system (FOTODYNE, Hartland, WS ). Plasmid Isolation and Transformation Plasmids were isolated by Qiagen M iniprep kit according to M anufacturers protocols (Qiagen Inc., Valencia, CA). When applicable, plasmids were purified from agarose slices by QIAquick gel extraction kit (Qiagen). Hfx. volcanii strains DS70, H26, and different mutants were transformed with plasmid DNA isolated f rom E. coli GM2163 ( dcmdam-) according to Cline et. al. (Cline and Doolittle, 1992)


78 Site directed M utagenesis Site directed mutagenesis was performed using the QuikChange Site Directed Mutagenesis kit (Stratagene) as per M anufacturers instructions wi th the following modifications. Pf u DNA polymerase (Stratagene) was used for generation of all mutations with exception of psmC mutations in which Phusion DNA polymerase (New England Biolabs) was used. A n elongation time of 7 minutes and 30 seconds was used for the gener ation of 11 kb products. Restriction enzyme digests were carried out with D pnI ( Stratagen e or New England Biolabs). Oligonucleotides used in site directed mutagenesis rea ctions are listed in Table A 2 and were not HPLC purified. Generation of N terminal A cetyltransferase K nockouts Generation of N terminal acetyltransferase gene knockouts (HVO_1954 and HVO_2709) followed the previously described protocols of Allers et. al. (Allers et al. 2004) Primers used in the gene deletions are listed in Table A 4. The PC R product s of the chromosomal regions of both genes ( 500 base pairs) were treated with T4 polynucleotide kinase ( PNK) and blunt end cloned into the Eco RI site of plasmid pTA131. Inverse PCR was used to remove the coding region of the genes (start to sto p codons). The inverse PCR products were self ligated and transformed into E. coli Inverse PCR clones were purified and transformed into Hfx. volcanii strain H26 (uracil auxotroph) Transformations were plated on CAmedi um without uracil. Cells that s uccessfully incorporated the plasmid into the chromosome survive d the uracil auxotrophy. Transformants were challenged in uracil containing medi um with 5 f luoroorotic acid (5 FOA). The plasmid recombine s out of the chromosome either restoring the wild ty pe copy of the gene or deleting the target gene. 5 FOA resistant colonies are checked for the presence or absence of the target gene by PCR of the chromosomal region using primers 700 bp target gene.


79 Genome A nalysis NCBI Local BlastP (Altschul et al. 1997) with BioEdit sequence editor software v7.0.4.1 (Hall, 1999) was used to compare the theoretical Hfx. volcanii DS2 proteome (, April 2007 version ) (unpublished). Phylogenetic and molecular evolutionary analyses of the primary sequences of proteins were conduc ted using MEGA v3.1 (Kumar et al. 2004) Pairwise and multiple sequence alignment was performed using Clustal W (Thompson et al. 1994) Evolutionary distances were estimated from the protein sequences using the p distance substitution model. Consensus tree inference was by neighbor joining with bootstrap phylogeny test (1000 replicates; 64238 seed) and pairwise gap deletion. Regions of high variability and low complexity were filtere d out of the search using Gblocks server (Castresana, 2000) RNA Techniques RNA Isolation and RTPCR Total RNA was isolated using the R N easy Mini kit for bac teria (Qiagen) as previously reported (Reuter et al. 2004) with the following modifications One milliliter of cells (OD600 = 0.8) was harvested by centrifugation and lysed in 12O. Lysed cells were treated with Dnase I (Sigma) according to M anufacturers protocol. RNA samples applied to the R N easy Mini RNA isolation column were centrifuged for an additional 2 minutes before elution to further dry out the membrane. Quality of RNA was determined by 0.8 % (w/v) agarose gel electrophoresis in 1 TAE buffer after heating the RNA samples to 50 minutes. Total RNA concentration was determined from A260nm. Total RNA was used to generate cDNA using t he iScript cDNA synthesis kit ( Bio Rad ).


80 qRTPRC Quantitative real time PCR was performed using the iQ SYBR Green Supermix ( Bio Rad ) and iCycler MyiQ real time PCR detection system ( Bio Rad ). qRT PCR was performed as both a dilution series of the cDNA lib rary and as triplicate samples of a dilution in the linear range of detection for both the psmA and ribL transcripts. A standard curve using a PCR product specific for the psmA gene was performed to measure the linear range of detection of the iCycler. P rimers specific for the ribL a ribosomal protein gene, coding region were used as a control. The primers for the psmA gene were previously reported (Reuter et al. 2004) The ratio of ribL from the two samples was calculated and the resulting scalar was used to correct the transcript levels of the psmA gene from the different sampl es. Protein Techniques Protein Expression in Escherichia coli Hfx. volcanii proteins were expressed in E. coli BL21 (DE3) with pSJS1240 (expressing rare tRNA genes for protein expression). P lasmids and strains used for synthesis of 20S proteasome subunits are listed in Table 31 or Table 51. Strains were grown in Luria Bertani broth at 37 and 200 rpm ml1 of ampicillin 50 ml1 of kanamycin, 50 ml1 spectinomycin, or 50 ml1 of chloramphenicol as needed. Cells were grown to mid log phase (OD600 = 0.5 0.8) for 1 hour and induced with isopropyl D thiogalactopyranoside for 3 5 hours. Protein Expression in Haloferax volcanii 20S proteasome proteins and protein kinases were expressed in Hfx. volcanii strains from plasmids listed in T ables 3 1, 41, and 51. Cultures were grown in ATCC 974 medium at 42 and 200 rpm in Fernbach flasks to late stationary phase (OD600 = 2.5 3) Media were supplemented with or ml1 of novobiocin or 4 ml1 mevinolin as needed.


81 Protein Quantification Protein concentrations were determined by Bradford method (Bradford, 1976) with bovine serum al bumin as the standard (Bio Rad) with the following modifications. Reaction volume for B radford reagent was added to a 1:10 to 1:50 dilution of protein sample and incubated for 1 10 minutes. The assay was linear between 100 and 800 ml1 of protein. Protein Separation and Chromatography Cells were harvest ed by centrifugation at 6000 g for 30 minutes and lysed by passing through a French pressure cell ( 3500 PSI) in preparation for downstream chromatography. Cell lysates and chromatography fractions were monitored for purity by staining with Coomassie blue R 250 or SYPRO Ruby (Invitrogen) after separation by reducing 12 % SDS PAGE according to Laemmli (Laemmli et al. 1970) Molecular mass standards for SDS PAGE included phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) ( Bio Rad ). Nickel affinity c hromatography Cells were harvested by centrifugation (10,000 g, 20 minutes 4 C) and resuspended in 60 ml of 20 mM Tris buffer (pH 7.2) supplemented with 2 M NaCl (buffer A) and 5 mM imidazole. Cells were passed through a chilled Frenc h pressure cell at 20,000 lbin2. Cell debris was removed by centrifugation (16,000 g, 20 minutes 4 C). The filtrate obtained with a 0.45 pore size filter (Nalge Nunc International, Rochester, NY) was applied to a 5 ml Ni2+Sepharose column (HiTr ap chelating; Amersham Biosciences) equilibrated with buffer A and washed with a step gradient consisting of 5 and 60 mM imidazole in buffer A. Fractions containing His tagged proteins were eluted in b uffer A with 500 mM imidazole.


82 Strep t actin c hromatogr aphy Strep_II tagged (Trp Ser His Pro Gln Phe Glu Lys) proteins were purified by application of cell lysate onto a 0.2ml Strep T actin column (Qiagen) equilibrated with 20 mM Tris buffer (pH 7.5) with 2 M NaCl (buffer L). The column was washed with 10 ml of buffer L and proteins were eluted in 20 mM Tris buffer (pH 7.5) with 2 M NaCl and 1.5 mM desthiobiotin (buffer E). Fractions containing protein were pooled and dialyzed against buffer A using D Tube Dialyzer (Novagen). Column resin was regenerated by washing 3 times with 5 column volumes of 100 mM Tris buffer pH 8.0 with 150 mM NaCl 1 mM EDTA and 1 mM HABA (hydroxyazop henyl benzoic acid) (buffer R). Gel f iltration c hromatography Protein s amples (0.5 ml per run) were applied to a Superose 6 HR 10/30 gel filtration column (Amersham Biosciences) equilibrated in 20 mM Tris buffer (pH 7.2) with 2 M NaCl at a flow rate of 0.1 0.3 ml min.1. Molecular mass standards for gel filtration included cytochrome C ( 12 kDa ), carbonic anhydrase ( 29 kDa ), serum al bumin (66 kDa), alcohol dehydrogenase (150 amylase (200 kDa), apoferritin (443 kDa), thyroglobulin (669 kDa), and Blue Dextran (2000 kDa) (Sigma). Elution of protein was monitored by UV absorbance at 280 nm. Peptide Synthesis The synthetic phosphopeptide RGEDMSMQALpSTL (where pS represents fluorenyl methyoxycalbomyl amino acid chemistry on an ABI 432A peptide synthesizer. P eptide purity and molecular weight were measured after synthesis with MALDI TOF. The NH2MQGQAQQQAYDKbiotin was synthesized in the same way for N terminal acetyltransferase assays. L alanine N FMOC (U-13C3 and 15N) was purchased from Cambridge Isotope Laboratories Incorporated (Andover, MA) and used for the synthesis of two AQUA


83 peptides Ac etyl MQGQ A QQQA YDR and QGQ A QQQA YDR, for quantitative mass spectrometry standard controls ( heavy isotope amino acids are underlined and bold) Two D imensional Gel Electrophoresis Two dimensional gel ele ctrophoresis was preformed as previously described (Karadzic and MaupinFurlow, 2005; Kirkland et al. 2006) with the following modifications, 2 ml of cells at OD600 = 1, were centrifuged at 10,000 g at 4 and lysed in dH2O. The lysed cells were treated with D N ase I for 30 minutes at room temperature. Proteins were precipitated from cell lysate by mixing 100 % cold acetone with the cell lysate. The resulting protein pellet w as washed 5 times with 100 % cold acetone and resuspended in with 10 % glycerol in 20 mM Tris buffer (pH 7.2) T otal protein loaded onto an 11 cm ReadyStrip IPG Strip ( Bio Rad ) with a p I range of 3.9 5.1. Isoelectric focusing was performed using a Protean IEF Cell (Bio Rad ) for 40,000 Vh. Proteins were separated on the second dimension using Criterion Precast 12.5 % SDS PAGE gels (11 cm with IPG +1 well) ( Bio Rad ) at 16 Proteins were stained in gel with SYPRO Ruby (Invi trogen) according to manufacturer Gels were imaged on a Typhoon (Amersham) and spots were excised manually and identified by mass spectrometry. Immunoblotting For immunoblotting, purified proteins and cell lysates were separated by denaturing (SDS) and mercaptoethanol) PAGE gels (Laemmli et al. 1970) After separation, proteins were transferred to Hyb ond P membranes (Amersham Bioscience, Piscataway, NJ) using 10 mM 2N morpholinoethanesulfonic acid (MES) buffer at pH 6.0 with 10 % (v/v) methanol at either 20 V for 16 hours or 200 V for 90 minutes. Transferred membranes were blocked with 10 % (w/v) mil k solution in Tris buffered saline (TBS) for 1 16 hours with rocking. Primary antibod ies PanA generated in rabbit (Kaczowka and Maupin -


84 Furlow, 2003) were suspended in 5 % (w/v) milk in Tris buffered saline wit h 0.05 % Tween 20 (TBST) at the following dilutions: 1:2000 PanA, and 1:5000 Primary antibody solution was incubated with the membrane for 60 90 minutes rocking. Primary antibody solution was washed 2 with TBS for 15 minutes Th e secondary antibody was goat anti rabbit IgG Alkaline phosphatse (AP) Blot s were developed in AP buffer ( 100 mM Tris buffer (pH 9.5) 100 mM NaCl, and 5 mM MgCl2) with nitro blue tetrazolium (NBT) and 5bromo4chloro3 indolyl phosphate ptoluidine (BCIP) and imaged using a Versa Doc 1000 with Quantity One v. 4 Software ( Bio Rad ). L abeling Hfx. volcanii C ells with O rthophosphate in M inimal M edia Cells grown to log phase in 1 3 ml (13 100 mm2 tubes, 42 were used to inocul ate 100 ml cultures of lactate minimal medi um in 500 ml flasks by a 1:100 dilution. These larger cultures are grown to OD600nm = 0.6 0.8 (log phase) (42 The cells were washed with phosphate free medium and concentrated to 10 % their origi nal volume. This cell suspension was incubated at 42 C in a standing water ml1 32P (Perkin Elmer NEX053H ) for up to 20 hours. Cells were washed with phosphate free lactate minimal medium three times and stored at 20C until further analysis Pulse Chase For pulse chase, H fx volcanii cells were grown in 200 ml of minimal (Hv Min) medium in 500 ml flasks to an OD600 of 0.4 to 0.6 (200 rpm, 42 C). Cells were collected by centrifugation at 10,000 g for 15 minutes at 40 C and resus pended in 80 ml of prewarmed (42 C) HvMin medium. Cells were incubated at 42 C for 1 hour with shaking in 500 ml flasks (200 rpm). Radioactively labeled methionine and cysteine (500 Ci of 35S Met and 35S Cys mixture) (EXPRE35S35S Protein Labeling Mix, Perkin Elmer) were added to the culture and incubated for an additional 20 minutes (42 C, 200 rpm). Samples were divided into 8 10 ml fractions in 15


85 ml conical tubes. Translation was arrested immediately for one tube (0 min utes ) by addition of 1 ml of stop solution (22 % [w/v] sodium azide, 100 g/ml puromycin) and incubat ed on ice. The remaining 7 samples were incubated with 1 ml of chase solution (8 mg/ml cold methionine a nd cysteine, 100 g/ml puromycin, 1 mg/ml protease inhibitor cocktail) at 42 C and 200 rpm. Each sample was arrested at appropriate time intervals (1, 3, 5, 10, 30 and 60 minutes and 12 hours) by the addition of 1 ml of stop solution and chilling on ic e. Each 10 ml sample was collected by centrifugation at 8,000 g at room temperature for 10 minutes. Pellets with approximately 1 ml of medium were resuspended and re pelleted in 2 ml microcentrifuge tubes at 14,000 g at room temperature for 5 minutes Cell pellets were frozen at 80 C for a maximum of 48 hour pri or to immunoprecipitation (IP). Immunoprecipitation (IP) For IP, protein A Sepharose beads (Pharmacia) [10 mg per ml phosphate buffered saline (PBS) with 0.01 % (w/v) sodium azide] were aliq uoted into 1.8 ml Eppendorf tubes (75 l per tube), washed once with cold PBS and resuspended in 1 ml cold PBS. The beads were charged by addition of 10 12 l of polyclonal antiserum for 4 12 hours at 4 C with continuous agitation and washed 5 with cold PBS to remove unbound antibody. Cell pellets from pulse chase labeling (described above) were prepared for IP by resuspension in 150 l of denaturing lysis buffer (1 % [w/v] SDS, 50 mM Tris Cl, pH 7.4, 5 mM EDTA, 10 mM DTT, 1 mM PMSF, 300 mM NaCl), boiling for 10 minutes, and addition of 1.35 ml nondenaturing lysis buffer (1 % [v/v] Triton X 100, 50 mM Tris Cl, pH 7.4, 5 mM EDTA, 0.02 % [w/v] sodium azide, 10 mM iodoacetamide, 1mM PMSF, 300 mM NaCl). Lysate was incubated with ~500 units of Benzonas e (2 l) (Sigma Aldrich) at room temperature for 30 minutes with occasional mixing. Samples were clarified by centrifugation at 10,000 g for 5 minutes, and clarified lysate was added to the charged beads. The lysate/bead mixture was incubated at 4 C with r ocking for 3


86 hours. Beads were washed 5 with IP wash buffer (0.1 % [v/v] Triton X 100, 50 mM Tris Cl, pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.02 % [w/v] sodium azide, 0.1 % [w/v] SDS and 0.1 % [w/v] deoxycholine) and one final time with cold PBS. SDS reducing dye (20 l of 100 mM Tris Cl, pH 6.8, 10 % [v/v] beta mercaptoethanol, 2 % [w/v] SDS, 10 % [v/v] glycerol, 0.6 mg/ml bromophenol blue) was added. Samples were boiled for 10 minutes and centrifuged at 14,000 g for 5 minutes to remove the beads. Proteins in the supernatant were separated by 12 % SDS PAGE (200 V for 45 minutes), and their migration was compared to Precision Plus Protein dual color standards ( Bio Rad ). Gels were incubated in fixing solution (10 % methanol, 7 % acetic acid) for 30 minutes and an intensifier solution (1M salicylic acid) for 1 hour. Gels were washed with H2O, dried under vacuum for 1.5 2 hours, and exposed to radiographic film for a minimum of 10 days at 80 C. Band intensity was quantified using a Versa Doc 1000 with Quantity On e v. 4 Software ( Bio Rad ). Molecular Modeling Crystal structure files were downloaded from RCSB and the protein data bank. Three dimensional analysis of structure was done on Rasmol (Sayle and Milner White, 1995) Pymol, and VMD (Humphrey et al. 1996) structure viewers. Homology based structure determination was done by using SWISS MODEL (Peitsch, 1996) Enzyme Assays Peptide Hydrolyzing Activity Chymotrypsinlike peptide hydrolyzing activity of 20S proteasomes was assayed in buffer E with 20 mM N Suc Leu Leu Val Tyr 7amido 4methylcoumarin (LLVY AMC). Pepti de substrate was solubilized in 100 % final). Release of 7 amino 4methylcoumarin was monitored by determining the increase in


87 Fluorocolorimeter (Spectro nic Instruments) or with Bio Tek Syntergy HT 96well plate reader (Bio Tek Instruments) Assays were performed at 37 C and 65 C. Assay conditions of pH 7.5, 2 M NaCl and 65 C were previously determined optimal for Hfx. volcanii 20S proteasomes (Wilson et al. 1999) Kinase Assays K inases were purified to electrophoretic purity by Strep Tactin chromatography as described above. Purified kinase s were dialyzed using a D tube dialysis tube (Novagen) into 20 mM Tris buffer (pH 7.2) with 2 M NaCl (or 2 M KCl) at 4 Kinase activity was measured by incubating with different concentrations of Mn2+ and Mg2+ (0 250 mM) for one hour in the presence of 200 -32P] adenosine 5 t riphosphate (Perkin Elmer) in a reaction volume o Kinase reactions were inactived by addition of reducing SDS PAGE buffer and separated by 12.5 % SDS PAGE gels. Gels were stained with Coomassie brilliant blue G and dried to W hatman paper. X ray film was exposed to t he dried gel for 1 10 days. Mass Spectrometry Sample Preparation Protein fractions from Ni2+Sepharose or Strep Tactin chromatography were either separated by reducing 12 % SDS PAGE or provided as insolution samples for mass spectrometry Proteins were stained in gel with B io Safe Coomassie (Bio Rad). Protein bands corresponding to the molecular weights of the proteins of interest (such as the subunits of 20S proteasomes, 20 30 kDa) were excised from the gel and destained with 100 mM NH4HCO3 in 50 % (vol/vol) acetonitrile (4 C, overnight). Protein samples were reduced, alkylated ingel or in solution, and digested with sequencing grade trypsin, AspN, chymotrypin, or GluC (Promega).


88 Three Dimensional Ion Trap (LCQ Deca) The subset of data used to analyze the N terminal proteome was generated on a Thermo LCQ Deca quadrupole ion trap mass spectrometer (LCQ DECA MS) in line with a 5 cm 75 inner diameter Pep M axTM capillary column (LC Packings). Sequest Analysis software was used to interface with the LCQ Deca and run protoc ols. All runs had a 30 or 60 minute s gradient from 5 50 % acetylnitrile with a flow rate of 10 min ute1. Parent ion scans (MS) were followed by three or four data dependent MS/MS scans. Hybrid ESI Q ToF (ABI QSTAR) Capillary reverse phase highperformance liquid chromatography (HPLC) separation of the protein digests was performed using a PepMa xTM C18 column (75 m inside diameter, 15 cm length; LC Packings, San Francisco, CA) in combination with an Ultimate capillary HPLC system (LC Packings, S an Francisco, CA) operated at a flo w rate of 200 nl min ute1. A gradient (60 minute s ) from 5 to 50 % (v/v) acetonitrile in 0.1 % (v/v) acetic acid was used. Tandem mass spectrometry (MS/MS) analysis was performed using a hybrid quadrupole time of flight (QTOF) instrument (QSTAR) equipped with a nanoelectrospray source (Applied Biosystems, Foster City, CA) and operated with Analyst QS 1.1 data acquisition software. Triple Quadrupole (ABI QTRAP) Quantitative mass spectrometry techniques were performed using a triplequadrupole tandem mass sp ectrometry analyzer (QTRAP) equipped with a nanoelectrospray source (Applied Biosystems). Capillary reverse phase highperformance liquid chromatography (HPLC) separation of the protein digests was performed using a PepMapTM C18 column (75 m inside diame ter, 15 cm length; LC Packings, San Francisco, CA) in combination with an Ultimate capillary HPLC system (LC Packings, San Francisco, CA) operated at a flow rate of 200 nl min ute1. A gradient (60 minute ) from 5 to 50 % (v/v) acetonitrile in 0.1 % (v/v) a cetic acid


89 was used. A control peptide (amino acids 13 unmodified) was included in all samples to minimize variability. Peptides were targeted for precursor ion scanning using a multiple reaction monitoring pr ogram (MRM) developed in the MIDAS workflow software system. Relative quantities of different peptides were measured by integration of the respective peaks on the initial MS scan using the integrati on function on Analyst QS 1.2. Database Searching and Sof tware Peptides were identified using MASCOT algorithms (Perkins et al. 1999) that searched a custom, non redundant database based on the hypothetical proteome of translated open reading frames from the H fx volcanii genome ( Eisen, professional communicatio n April 2007, ). Probability based MOWSE scores were estimated by comparison of search results against estimated random match population and are reported as ~10 log10(p), where p is the ab solute probability. Individual ion scores greater than 32 indicates identity or extensive homology (p<0.05). Carbamidomethylation was used as a fixed modification due to sample preparation. Variable modifications that were searched included deamidation of asparagines and glutamine, N terminal acetylation, oxidation (single and double) of methionine, and phosphorylation of serine, threonine, or tyrosine.


90 CHAPTER 3 N TERMINAL ACETYLATION OF ALPHA TYPE SUBUNITS OF 20S PROTEASOMES IN H ALOFERAX VOLCANII Intr oduction Proteolysis is important in regulation and protein quality control. Energydependent proteases are crucial to early stages of protein degradation. In eukaryotes and archaea, proteasomes are the central energy dependent proteases of the cytosol and are essential for growth (Rosenzweig and Glickman, 2008a; Zhou et al. 2008) The catalytic component of type subunits hydrolysis are forme d by the N type subunits and are sequestered within the central channel of the barrellike structure. Energy dependent triple A ATPases, including regulatory particle triple A ATPases (Rpt) in eukaryotes and proteasomeactivatin g nucleotidases (PAN) in archaea, mediate the unfolding and translocation of substrate proteins rings for degradation within the 20S core (Smith et al. 2007; Rosenzweig and Glickman, 2008a) One major difference between eukaryotic and prokaryotic 20S core particles is in the subunit N termini, the site of substrate entry appears open at the ends of the 20S cylinders of Thermoplasma acidophilum (Lowe et al. 1995) Archaeglobus fulgidus (Groll et al. 2003) and Mycobacterium tuberculosis (Hu et al. 2008) In contrast, X ray structures of the 20S core particles of yeast (Groll et al. 1997) and bovine (Unno et al. 2002a; Unno et al. 2002b) do not contain this opening. Instead the extreme N pore in a gate like structure.


91 Evidence suggests that all 20S proteasomes are gated, and the major differences observed ring gat e in crystal structures are not physiological. For example, the N terminal 11 amino acids of the A. fulgidus the 20S proteasome structure, are more ordered in the 16S half proteasome precursor (Groll et al., 2003) Furthermore, cryoelectron microscopy of the M. tuberculosis 20S core particle subunit and which diminish peptidolytic activity. Consistent with this, deletion of the N 12) of the T. acidophilum subunit abolishes the need for an ATPase ( i.e ., PAN) in the degradation of aciddenatured GFP SsrA or casein (Benaroudj et al. 2003) In addition, the conserve ring gating, is present in all type subunits to date (Groll et al. 2003) Thus, although the mechanism may not be exactly the same, prokaryotic proteasomes are thought to gate th ring aperture. A gated channel formed by the N rings may be a general mechanism for regulating the activity of proteasomes. The rate limiting step in proteasome mediated protein r ings to the active sites contained within rings (Kohler et al. 2001) This mechanism is suppo rted by the finding that eukaryotic 20S core particles have no peptidolytic activity in the absence of Rpt proteins or mild chaotropic agents such as SDS or heat treatment (Coux et al. 1996) Furthermore, peptidase activity of the yeast 20S proteasome is blocked by the N 9) or single substitution (D9A) of N (Groll et al. 2000) An additional gating mechanism could be employed by post translational modifications of the N type subunits of 20S proteasomes are modified by


92 Nacetylation in several eukaryotes and haloarchaea including Haloferax volcanii (Tokunaga et al., 1990; Kimura et al. 2000; Kimura et al. 2003; Falb et al. 2006; Humbard et al. 2006) In yeast, N acetyltransferase 1 (NAT1 ) is responsible for the Natype nat1 mutant have twofold higher chymotrypsinlike peptidase activity in the absence of SDS compared to wild type suggesting that Ngate (Kimura et al. 2000) In Hfx. volcanii both acetylated and instead cleaved by an app arent methionine aminopeptidase. A large scale proteomic survey reveals Nacetylation is common to other proteasomal type proteins of the haloarchaea (Falb et al. 2006) In this previous survey, the ratios of Nacetylated and cleaved type proteins were quantified by spectral counting and estimated to be around 3:1 and 4:3 for Halobacterium salinarum and Natronomonas pharaonis respectively (Falb et al. 2006) So far, this existence of two unique forms termini in the cell simultaneously has only bee n observed in the haloarchaea. In this chapter demonstrated to be Nacetylated on their initiator by an apparent methionine aminopeptidase. Q uantitative MS/MS was used to precisely determine the ratio of the NHfx. volcanii Site directed mutagenesis was also used to examine how the Nacetylated state. Residues that perturbed this state had profound phenotypic consequences heat and hypoosmotic stress.


93 Results Isoforms of 20S Proteasome Subunits Many subunits of 26S and 20S proteasomes from eukaryotes have isoforms that are generated by post translational modification and can be separated by 2DE. In order to investigate this in archaea, an H fx volcanii strain which synthesizes a C terminal histidine His) of 20S proteasomes (Kaczowka and Maupin Furlow, 2003) was used to enrich for proteasomal complexes by nickel affinity chromatography. Based on previous work (Kaczowka and Maupin Furlow, 2003) samples enriched for proteasomes us ing this method and strain are proteolytically active and are composed primarily of 20S pr oteasomes and heptamers heptameric rings are also observed. Therefore, this general approach was used to enrich for proteasomes from log phase cells for further analysis. Samples were separated by 2 DE using an immobilized pH gradient from 3.9 to 5.1 prior to reducing 12 % SDS PAGE (Fig ure 31). subunits of 20S protea somes (37.5, 34.5 and 30 kDa, respectively, based on SDS PAGE) (Wilson et al. 1999) were digested in gel by trypsin and the corresponding peptide mixture analyzed by tandem mass spectrometry. All of these spots wer 20S proteasomal proteins of H fx volcanii as indicated in F igure 31. Based on these results, all three of the H fx volcanii chains (Fig ure 3 1). These proteaso me fractions were composed H fx volcanii cells (Wilso n et al. 1999; Kaczowka and MaupinFurlow, 2003) Approximately identified (Fig ure 31; a to h). terminal histidine tag, this


94 specific isoforms detected in the chain. In detected (Fig ure 3 1; i to m). Based on SDS PAGE, four of these isoforms (indicated as i, j, k and m) were similar in migration to the matur ersus previous study (Wilson et al. 1999) which used Edman degradation to determine the N terminal r49 (residue number according to the deduced type proteasomal proteins, which undergo autocatalytic processing to remove an N terminal propeptide and expose the active site T hr during 20S proteasome assembly (Seemuller et al. 1996; MaupinFurlow et al. 1998) The propeptide of the H fx volcanii pr ecursor is rather large and accounts for 5,427 Da of the molecular mass of the polypeptide. This difference in proteins by SDS PAGE. Thus, the three most basic a nonhich copurifies during the proteasome enrichment procedure and migrates too (Fig ure 31, n and o). and type isoforms detected was significantly greater than the three polypeptides deduced from the isoforms suggested that the proteasomal polypeptides are modified in the cell by mechanisms which alter their pI.


95 It should be noted that the uppermost band of 66kDa directly associated with the nicke l Sepharose column, based on its purification from not only the His tag expression strain (DS70/pJAM204) but also parent strain DS70. This protein was recently identified as PitA (Bab Dinitz et al. 2006) and contains an N terminal chlorite dimutase related domain and C terminal antibiotic biosynthesis monooxygenase related domain linked by a central histidine region which most likely accounts for its divalent metal af finity. In contrast to the PitA protein, His expression strain. Analysis of Proteasome Isoforms by Mass Spectrometry To enhance the number of peptide fragments de tected by mass spectrometry and increase the likelihood of detecting post or co translational modifications of the proteasome isoforms, the proteasome enriched fractions were separated by reducing 1D (vs. 2D) SDS sed from the gel as bands, reduced, alkylated, and cleaved with either trypsin, sequentially with trypsin and chymotrypsin or with GluC endoprotease. This approach enhanced the protein sequence coverage when compared to analysis of protein spots from 2D gels (data not shown), most likely due to the higher amount of protein isolated from the 1D gels. The peptide fragments resulting from the protease digests were separated by reversed phase HPLC and analyzed by ESI QTOF MS. Using this approach, the trypsi His purified sample resulted in 37.9 % % % coverage of ( Table 3 2 and 33) with Mascot total protein scores of 2162, 338, and 161, respectively (coverage and scores based on complete polype ptide deduced from GenBank nos. AF126262, AF126261 and AF126260) (Wilson et al. 1999) In combination with a trypsin and % and 56 %


96 Mascot total protein scores fo were 763, 422 and 328, respectively. Summaries of these Mascot search results are presented in Table 32 and 3 3. The Glu C endoprotease was used to enhance co verage of the N terminus of 2. terminally Acetylated acetylated and unmodifed forms. The ion corresponding to the N with the acetylation had an individual MOWSE score of 76 and an Expect of 5.5e7. It contained 12 amino acids, from 1 to 12 (MQGQAQQQAYDR), and ha d a monoisotopic mass of 1480.67 Da. It eluted from RP HPLC between 20.01 minutes and 20.35 minutes as a doubly charged ion (741.343, 2+). The peptide contained a n oxidation on methionine 1 and an N terminal acetylation site. The MS/MS fragmentation of the ion produced a complete yion series as well as several key b ions including b(1) with a mass of 190.05 Da ( Figure 3 2). This mass correspond ed to a methionine with an oxidation to methionine sulfoxide (147.0354) plus an acetylation (42 Da). Additional b and a ions support ed this oxidation / acetylation mass. There were several internal ion fragmentations resulting in major peaks in the spectrum. The major peaks are labeled and listed in inset table in Figure 32. It should be noted that the N terminally acetylated ion was obtained in multiple experiments each time with multiple ions. In addition, an unmodified N te This suggest ed either were acetylated or the acetyl group was removed during collision induced dissociation (CID). This second possibility is unlikely since the unmodified fragment had a unique elution time from HPLC. If the acetyl group was lost in CID, the peptide should be chemically indistinguishable from the modified one prior to CID. It is far more likely that the unmodified and modified subunit exists in a cell simultaneously. Since the Ni2+-


97 Sepharose chromatography fractions of cells expressing included both 20S proteasomes, we were unable to determine whether both the unmodified and present in functional 20S proteasomes. I s N ot A cetylated detected which spanned residues 2 to 12 (QGQAQQQAYDR). It was a doubly charged ion (m/z 646.8) with a Mascot individual ion score of 43 and an E value of 1 e 3. The MS/MS fragmentation spectrum contained a complete y ion series as well as four unique b ions supporting an N terminal cleavage event. This result suggest ed proteins in the cell are acetylated, and a subset may instead be cleaved by methionine aminopeptidase. Thus, N A cetylated F P rotein s I s D ominant In order t o better understand the relative ratios of these forms, quantitative mass His6 proteins purified from Hfx. volcanii DS70 (pJAM204) cells was performed. Initially, spectral counting was combined with a comparative integration of peak area of each of the isotopic envelopes of the two N terminal peptides. The ratio of the Ac1 to N2 Table 34 ), heavily favoring the acetylated (Ac1) to the unacetylated methionine removed form (N2). To confirm the integration intensity of the MS scan from the Q ToF instrument, different instrumentation, a triple quadrupole, was used with a multiple reaction monitoring (MRM) initiated detection and sequencing workflow (MIDAS). Using this software, the mass spectrometer only detects the ions that are defined by a set of parameters. The ions defined were for the two forms of the N terminal peptide (733.79, 2+ for Ac1 and 647.17,2+ for N2) and for additional selectivity, product ions from the second MS scan (MS/MS) were used to identify the correct species. The resulting filtering identified two peptides that matched those


98 defined mass / charge ratios and product ions. The calculated ratio of these two ions was 100:1 (Fig ure 3 5). Amino A cid S ubstitutions in the P enultimate P A ffect A cetylation and C leavage of the I nitiator M ethionine R esidue As a result of N terminal processing and protein maturation by methionine aminopeptidase (MAP) and Nacetyltransferase (NAT) enzymes, at least four different forms of a protein are possi ble. These include proteins that retain their initiator methionine residue in either an Nacetylated (Ac1) or unmodified form (N1) as well as those which undergo MAP cleavage resulting in a cleaved methionine (N2) which can also be Nacetylated (Ac2). Unlike bacteria, formyl methionine has not been identified as an initiating residue in archaea (Ramesh and RajBhandary, 2001) These final N terminal states of a protein ar e thought to be governed by th e nature of the first three amino acids, the initiating methionine, the penultimate and the antepenultimate amino acid (Polevoda and Sherman, 2003b) Taking this into consideration, a directed mutagenesis of the psmA Q2P, Q2S, Q2T, Q2V and a deletion of the N R12) resulting in fusion of the initiating methi onine Hfx. volcanii strain with the wild type psmA gene deleted from the chromosom e (GZ130) and all of the variants had a C variants were subjected to tryptic dig est and mass spectrometry analysis by ESI Q ToF (ABI counting (Table 3 5 was acetylated on the second amino acid, alanine, after removal of the initiator methionine (Ac2)


99 100 % R12 (G13) was not modified by either acetylation or methionine (Ac1) similar to wild cleaved and uncleaved, acetylated and unacetylated forms of the protein (N1, Ac1, N2, Ac2) with the ratios of these different forms varying among different chromatography preparat ions (Table 3 5). Co purifying Non proteasomal Proteins In addition to the 60kDa nickel binding protein PitA already discussed above, t hree additional proteins were identified by tandem spectrometric analysis of the proteasomeenriched samples. These inc luded HVO_1577, HVO_2108, and HVO_2645 (Table 3 6) of calculated molecular masses 19377.70, 20752.38, and 32702.80 Da. Whether these proteins copurify with His or purify by nickel affinity chromatography alone remains to be determined. Proteins of molecular masses in the range of 15 to 40 kDa were n ot detected by SYPRO Ruby stained SDS PAGE gels of proteins purified by nickel affinity chromatography from parent strain DS70. However, mass spectrometry can be more sensitive at protein detection than gel stains due to the diversity of protein chemical properties. The high content of His residues in HVO_2108 and HVO_2645 as well as the prediction that these proteins bind metal (Table 3 6) is consistent with the likelihood that these proteins His protein. It is less clear regarding HVO_1577 which is not predicted to bind metal. This latter protein contains a DUF293 domain and is a distant homolog of t he heat shock transcriptional repressor HrcA, recently shown to bind and regulate a bacterial GroEL (Wilson et al. 2005) Thus, it is tempting to speculate that HVO_1577 may associate with proteasomes.

PAGE 100

100 20S P roteasomes R apidly I solated by T wo step A ffinity P urification from Hfx. volcanii To reduce contaminant background and to further purify 20S proteasomes for kinetic studies, a n efficient, two step affinity purification protocol was developed to rapidly isolate 20S proteasomes from H fx. volcanii It involved fusing a StrepII ta g to the C subunit and a hexahistidine (His6) tag to the C were produced from a synthetic operon [ psmB strepII StrepII) followed by psmA his6 His6)] using the strong Halobacterium cutirubrum rRNA P2 promoter and T7 phage transcriptional terminator on a plasmid provided in trans To examine this approach, cell lysates of Hfx. volcanii DS70 expressing the synthetic operon were first subject to nickel affinity chromatography to isolate His6 proteins and complexes. The second column, StrepTactin (a His6 proteins. The purity of the resulting fractions was determined by tandem mass spectrometry (MS/MS) (Table 37) in addition to SDS PAGE and Superose 6 gel filtration (Figure 3 4 by reducing SDS PAGE revealed only 20S proteasome specific prote in bands including: a (Fig ure 3 4). The appearance of only one major peak in the gel filtration chromatogram at the molecular weight of 20S proteasomes (600 kDa ) further supports the conclusion that the purification of 20S proteasomes fr om Hfx. volcanii cells under native conditions and is similar to a previously reported technique for purifying 20S proteasomes from yeast cells (Wang et al. 2007)

PAGE 101

101 20S P roteasomes H ave D ifferent R atios of M P roteins t han Whole cell L ysates Measuring the relative levels of acetylation in purified 20S proteasomes containing the % o 20S proteasomes were in the Ac1 form (acetylated on the initiator methionine residue) (Table 3 5 12 and wildtype did not display any difference in acetylation patter ns whether or not they were successfully incorporated into 20S proteasomes. In methionine removed (N2), and acetylated on the second amino acid (Ac2) forms. When 20S associated in non 20S proteasome complexes. Thus, although modification of t he penultimate selected for incorporation into 20S proteasomes or 20S proteasome structural elements are required for proper acetylation of the N CP P eptidase A ctivity was E 212 M odifications The influence of the N on the chymotrypsinlike activity of composition purified from Hfx. volcanii DS2 ( i.e ., Tris HCl buffer at pH 7.5 with 2 M NaCl at 65 (Wilson et al. 1999) Assays were also performed at 37 12 variant showed a 3to 5fold increase in activity compared to wild type at both temperatures examined (Table 35 ). The N ( 212), and this change is likely to allow the passive diffusion of cleavable peptides into the

PAGE 102

102 proteolytic core. Interestingly, the Q2A variant which has only modest changes in its N terminus including the removal of the initiator methionine and acetylation of the exposed N terminal alanine was also enhanced (nearly 2 fold at 65 has a more open gate conformation tha n wild variants displayed activity comparable to wild type at both temperatures examined, consistent with the Nacetylated state of these CPs which is similar to wild type. V ariants (Q2D, Q2P and Q2T) A ccum ulate d as H eptameric R ings in H aloferax volcanii detected in multiple N terminal forms (N1, Ac1 and/or N2), accumulated to much higher levels type or ot Hfx. volcanii (Fig ure 36 A). Analysis of cell lysate by 2 D gels revealed a 30 kDa isoform chain predominated in the Hfx. volcanii GZ130 cells (grown at 37 C) expressing ure 39). Thi 3 8 expressing cells. Additional differences in cell lysate separated by 1D gels were also observed includin g an ~70 kDa protein wild type (Fig ure 36 protein excess of the 2D gels (Fig ure 39). To inve proteins enriched by Ni2+Sepharose chromatography were compared by Superose 6 gel filtration chromatography (Figure 36B ). For cells expressing wild appeared in excess (Q2D, Q2T and Q2P) was of molecular mass consistent with heptameric rings (170 kDa), with the levels of the 600kDa CPs sim ilar to wild type (Fig ure 3 6B

PAGE 103

103 be trapped in an assembly or degradation intermediate of heptameric rings. qRT PCR analysis revealed no dramatic dif ference in the levels of psmA specific transcript among the various strains (e.g ure 36C ), suggesting the enhanced transcriptional event suc h as protein half life or translation efficiency. Complementation of the H ypoosmotic S tress P henotype of psmA M utant Hfx. volcanii requires high concentrations of salt to grow (1.7 to 2.5 M NaCl) (Mullakhanbhai and Larsen, 1975) Recently characterized strains of Hfx. volcanii that are lacking in proteasomal subunit genes, most notably psmA hypoosmotic environments, low salt containing media (Zhou et al. 2008) For the psmA mutants, this increased sensitivity to low sa lt media can be rescued by the addition of a psmA his6 gene in trans (plasmid pJAM204) (Zhou et al. 2008) In order to understand how the rying concentrations of NaCl (Fig ure 3 7). As previously reported, while the psmA strain (GZ130) is fully complemented to wild type by providing psmA his6 gene in trans the negative control (pJAM202c vector alone) failed to rescue the hypoosmolarity phe notype caused by deletion of the psmA 12 variants had dominant negative phenotypes with even greater growth inhibition when expressed in trans than the psmA knockout (GZ130) alone (Figure 37) type cells (Figure 3 7).

PAGE 104

104 Cells E D is play a M ore T hermotolerant P henotype Curiosity about the possibility of a temperature sensitive phenotype of cells producing proteasomal CPs with an N i.e. R12 and Q2A) led us to measure the temperature sensitivity of Hfx. vol canii GZ130 ( psmA proteins in trans. Strains expressing the N significant temperature sensitive phenotype compared to strains expressing wild However, to our surp density at 50 C than those expressing wildpsmA mutant strain backgrounds, revealing that this growth phenotype at 50 C associated with the variants was dominant over the genomic encoded wild type at 50 C (Fig ure 38times the final d ensity of those with wildure 38A). The growth rate of the psmA cells expressing wild from plasmids was further analyzed at temperatures ranging from 37 to 50 C (Fig ure 38B). At all temperatures measured, cells expressi rate than cells expressing wild C of the psmA strain expressing psmA his6 in trans (GZ130pJAM204) was comparable to H26, the parent strain of this study (data not shown). Likewise, the Arrhenius plot for cells expressing wild Hfx. volcanii DS70 (Robinson et al. 2005) It is surprising that such a dramatic increase in overall growth rate, cell yield and compared to wildtype, since the difference between the two strains expressin g these variants at the genetic level is only two nucleotide substitutions in the penultimate codon of the 5 end of

PAGE 105

105 the psmA gene resulting in a single amino acid substitution. While the thermostability of extremophilic proteins is relatively common, inc luding optimal activity of the Hfx. volcanii CP at 6575 (Wilson et al. 1999) increasing the growth rate, growth yield and stress tolerance of the entire organism by making a subtle change to a structural gene is less common. There are a few examples of accomplishing this by over expressing heat shock proteins, Hsp70 (Nollen et al., 1999; Nakamoto et al. 2000) ShsP (Takeuchi, 2006) or by uncoupling the regulation of heat shock inducible transcription factors (hsf) (Wagstaff et al. 1998) in various organisms. Oddly, the connection between proteasomal activity and acquired stress tolerance appears inversely proportional with the more active, poten tially ungated and unregulated, proteasomal CPs ( i.e ., Q2A and 12 variants) rendering the Hfx. volcanii cells less tolerant of stress than those with gated CPs. Interestingly, this type of inverse relationship correlates with previous studies in eukary otic cells including the observed increase in thermotolerance of yeast after inhibition of proteasomal CP activity, apparently due to an induced heat shock response (Bush et al., 1997; Lee and Goldberg, 1998) In our study, the increased stress tolerance of Hfx. volcanii does not appear to be a product of enhanced CP peptidase activity. Both wildtype temperatures (37 and 65 (Table 35) and their thermostab ility was comparable, based on preincubation at 75 to 80 Although 2locked into a constitutive heat shock phenotype when grown at 37 C, it did reveal unusually ure 3 6). Based stimulate the prote in folding activity of chaperones such as those of the triple A ATPase family

PAGE 106

106 or more directly act as a chaperone to stabilize proteins needed for cell proliferation and/or stress response. Chaperone activities have been found to be closely associated wit h energydependent protease pathways such as the proteasome, Clp and FtsH systems (Gottesman et al. 1997; Suzuki et al. 1997; Yano et al. 2005) It is also possible that the Q2T strain is in an induced heat shock state based on the observed increase in the levels of a 7 0kDa protein and decrease in the levels of a 37 kDa protein compared to wildtype by 1D PAGE (Fig ure 36A); however, these differences could not be resolved by 2 D PAGE to facilitate rapid protein identification. terminally Acetylated acetylat ed also include this modification. In order to enhance the probability of observing peptides containing the N was digested with Glu C endoprotease. The rationale for using GluC (vs. trypsin) was based on the abundance of basic residues in the N e.g. Arg3 and Lys6). Trypsin was predicted to cleave carboxyl to these basic residues and gen erate short N terminal peptide ions that would not be readily detected by LC MS/MS. The Glu (m/z 776.6) which contained the first 26 amino acids of the deduced protein sequence ( Acetyl MNRNDKQAYDRGTSLFSPDGRIYQVE). This peptide had a Mascot individual ion score of 24 and an E value of 0.1. The MS/MS fragmentation spectrum of this N terminal peptide (Fig ure 3 3) contained primarily b type ions including a 185.1 Da ion which corresponded t o an acetylated methionine and several y type ions. The peptide contained an acetyl group on the N terminus, and the fourth amino acid (Asn4) was deamidated. The low Mascot ion score and corresponding high expect value were likely a consequence of the le ngth and amino acid composition of the peptide. However, the ability to manually assign all the peaks in the spectrum and the consistency of the data support

PAGE 107

107 the validity of this identification as an acetylated N all of the N Discussion and Conclusions This study demonstrates that both type proteasomal subunits in Hfx. volcanii are Nacetylated are n ot acetylated but rather missing the initiator methionine To elaborate on the Ns t he acetylated (Ac1) and unacetylated (N1) form he acetylated form was shown to be in 100fold excess of th e unacetylated form in whole cells and purified 20S proteasomes or CPs. Amino acid substitutions in the N terminal penultimate position and removal of the N acetyltransferase and methionine aminopeptidase (MAP) activities of Hfx. volcanii Deletion of the N helix ( R12) and substitution of the penultimate glutamine for an alanine residue (Q2A) had the most profound influence on these activities. The N terminal deletion ( R12) rende unavailable for modification by Nform. Likewise, although the Q2A modification did not impair Nthe site of this modification to the N2 position based This latter result suggests that the M A bond of the Q2A variant is a much better substrate for MAP cleavage than the MQ bond of wildMAP enzymes (Wiltschi et al. 2009) In contrast to R12 and Q2A, the Q2D, Q2P, and these latter site directed mutations did result in an increase in the population of unacetylated N1 and/or N2 proteins within the CP did not change.

PAGE 108

108 N terminal variants had a collection of phenotypes and acetylated or unacetylated state. For i.e R12) or im paired in the position of Nacetylation ( i.e., Q2A in the Ac2 vs. Ac1 form) had enhanced levels of peptidase activity suggesting the gate of the CP was in a more open state than wild type allowing better access of substrate to the proteolytic active sites harbored within the central chamber. Interestingly, the Hfx. volcanii strains producing these open CPs displayed reduced growth in low salt medium suggesting that gating of the CP (likely to protect the cell from uncontrolled proteolysis) is important for cell viability under these stressful conditions. All of the other site directed mutations examined did not impact the level or residue position of the Nsuggesting the CP gate is not artificially opened by these mutations. Although these latter strains produced apparently gated CPs, a number of them ( i.e ., Of these, two strains were further examined (i.e growth rates and improved tol erance to heat and hypoosmotic stress compared to wildtype. produced with wilderation and survival of cells. Before systematic surveys of Nacetylated proteins in the archaea had occurred, it was unclear whether the acetyl groups that modified the N termini of the proteasomal proteins, as detected in this study, are common or rare events in Hfx. volc anii C otranslational Nacetylation is common in eukaryotes with 50 to 90 % of all proteins acetylated on N terminal

PAGE 109

109 serine, alanine, glycine, or threonine residues after the removal of the initiating methionine (Brown and Roberts, 1976; Driessen et al. 1985; Polevoda and Sherman, 2003b) In contrast, N terminal acetylation is presumed to be post t ranslational and rare in prokaryotes based primarily on studies of E. coli (Waller, 1963; Tanaka et al. 1989) Additionally, w hether Nacetylation occurs co and/or post translationally in Hfx. volcanii or other arc haea remains to be determined. acetylated by NatA (NAT1 catalyt ic subunit) or NatC (MAK3 catalytic subunit) during translation (Kimura et al. 2000) Thus, similar to eukaryotic cells, it is possible that Hfx. volcanii uses a co translational mech anism to acetylate the N helix for this modification. If so, incomplete Nacetylation during translation of the Q2T, Q2D and Q2P variants could explain cetylated and some are not. If the protein. Alternatively, the intermediates in which the co translationally added Nacetyl group was removed and the otein has yet to be degraded. If so, modifying the N terminal penultimate protein. Although an archaeal N end rule has yet to be described, N terminal residues, i ncluding those in the penultimate position, are known to substantially influence protein stability in bacteria (Tobias et al. 1991) and eukaryotes (Varshavsky, 2000) Alternatively, the Nis post translational and requires CP structure including the N helix

PAGE 110

110 but not the initiator methionine. If so, it would be interesting to understand how the NAT al CP complex, since the site of N the CP cylinder. Whether the acetylation is specific to mature H fx volcanii 20S proteasomes or lower molecular mass complexes and what the function of thi s modification is remain to be determined. In S. cerevisiae type and type subunits t erminal acetylation (Kimura et al. 2000) This yeast 20S proteasome purifies in a latent state that is activated by mild chaotropic agents such as SDS and heat. Presumably, these age nts selectively denature the N type subunits which form tightly closed constrictions on each end of the cylinder (Groll et al. 1997) resulting in an opening of the channel of the 20S proteasome cavity. Although the role(s) of proteasomal acetylation remains to be determined, 20S proteas omes purified in the latent state from a S. cerevisiae nat1 slightly more active in catalyzing chymotrypsin like peptidase activity than those purified from the parental strain (Kimura et al. 2000) Thus, the deletion of six of the nine N ring) are speculated to change the higher order structure, possibly opening the central channel in the absence of mild chaotropic agents (Kimura et al. 2000) and/or regulatory ATPases. Early studies suggested that archaeal 20S proteasomes are not gated. T his was based on the disordered configuration of the N Thermoplasma acidophilum 20S proteasome purified from recombinant E. coli (Lowe et al.

PAGE 111

111 1995) and the ability of archaeal 20S proteasomes to hydrolyze short peptides in the absence of chaotrophic agents (Dahlmann et al. 1992; MaupinFurlow et al. 1998) However, the activities of most if not all archaeal 20S proteasomes are stimulated by h eat and/or SDS (Dahlmann et al. 1992; MaupinFurlow et al. 1998) and deletion of re sidues 2 12 of the recombinant T. acidophilum capacity of 20S proteasomes to degrade proteins with little tertiary structure (e.g casein) (Benaroudj et al. 2003; Smith et al. 2005) Based on this, the N termini type subunits do appear to function as a gate that prevents entry into 20S proteasomes of polypeptides seven residues or larger. Thus, regulated acetylation of the H fx volcanii control the configuration of the 20S ga te and ultimately modulate substrate entry and/or the association of regulatory ATPases. ty pe subunits from 20S proteasome enriched fractions in the haloarchaeon Haloferax volcanii are acetylated. The acetylated (Ac1) and unacetylated (N2) form of t protein were quantified and the acetylated form was shown to be in 100fold excess. Using terminal acetyltransferase and methionine aminopeptidase activity revealed that there is a more generalized acetylation pathway in Hfx. volcanii ariants had a collection of phenotypes. Some of the 12, while other improved stress response to heat and hypoted in whole cells as a mixture of acetylated and unacetylated forms, only acetylated proteins were found in complex with assembly 20S proteasomes. Taken together, this

PAGE 112

112 ld play a role in proper proteasome assembly.

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113 Table 3 1. Strains and plasmids used in this study Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli F recA1 endA1 hsdR17 (r k m k + ) supE44 thi 1 gyrA relA1 Life Technologies GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL 136 dam13::Tn9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs Haloferax volcanii DS70 Wild type isolate DS2 cured of plasmid pHV2 (Wendoloski et al. 2001) H26 DS70 pyrE2 (Allers et al. 2004) GZ130 psmA (Zhou et al. 2008) Plasmid pBAP5010 Ap r Nv r ; 11 kb shuttle expression vector derived from pHV2; includes P2 rrn upstream of citrate synthase gene (Jolley et al. 1997) pJAM202 Ap r Nv r ; pBAP5010 containing P2 rrn psm B h is 6 H is 6 expressed in Hfx. volcanii (Kaczowka and MaupinFurlow, 2003) pJAM202c Apr; Nvr; pJAM202derived control plasmid (Zhou et al. 2008) pJAM204 Ap r Nv r ; pBAP5010 containing P2 rrn psmA h is 6 ; H is 6 expressed in Hfx. volcanii (Kaczowka and MaupinFurlow, 2003) pJAM809 Ap r Nv r ; p JAM202 containing P2 rrn hvo 1862 s trep II ( KpnI site i nserted upstream of S trepII coding sequence) Maupin Furlow, unpublished pJAM816 Ap r Nv r ; pJAM809 containing psmB strepII StrepII expressed in Hfx. volcanii This study pJAM2510 Ap r Nv r ; p BAP5010 containing P2 rrn psmA Q2P h is 6 ; Q2P H is 6 expr essed in Hfx. volcanii This study pJAM2511 Ap r Nv r ; pBAP5010 containing P2 rrn psmA 12 h is 6 ; 12H is 6 expressed in Hfx. volcanii This study pJAM2512 Ap r Nv r ; pBAP5010 containing P2 rrn psmA Q2S h is 6 ; Q2S H is 6 expressed in Hfx. volcanii T his study pJAM2513 Ap r Nv r ; pBAP5010 containing P2 rrn psmA Q2T h is 6 ; Q2T H is 6 expressed in Hfx. volcanii This study pJAM2514 Ap r Nv r ; pBAP5010 containing P2 rrn psmA Q2D h is 6 ; Q2D H is 6 expressed in Hfx. volcanii This study pJAM2515 Ap r Nv r ; pBAP5010 containing P2 rrn psmA Q2A h is 6 ; Q2A H is 6 expressed in Hfx. volcanii This study pJAM2545 Ap r Nv r ; 797 bp PCR product blunt end ligated into a This stud y

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114 Strain or Plasmid Genotype; oligonucleotide used for amplification Source Blp I cut and Vent treated pJAM816 StrepII and H is 6 expressed in Hfx. volcanii pJAM2546 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and Q2A H is 6 expressed in Hfx. volcanii This study pJAM2547 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and Q2 D H is 6 expressed in Hfx. volcanii This study pJAM2548 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and Q2 P H is 6 expressed in Hfx. volcanii This study p JAM2549 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and Q2 S H is 6 expressed in Hfx. volcanii This study pJAM2550 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJ AM816 StrepII and Q2 T H is 6 expressed in Hfx. volcanii This study pJAM2551 Ap r Nv r ; 797 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and Q2 V H is 6 expressed in Hfx. v olcanii This study pJAM2552 Ap r Nv r ; 764 bp PCR product blunt end ligated into a Blp I cut and Vent treated pJAM816 StrepII and 12 H is 6 expressed in Hfx. volcanii This study

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115 Table 3 1 2 T). Protei na Res. no.b Mascot Ion Score Sequence 1 T 45.2 % 1 12 0.04 76 MQGQAQQQAYDR; N acetyl; Oxidation (M) 13 22 0.01 48 GITIFSPDGR 23 30 0.03 44 LYQVEYAR 35 43 0.01 32 RGTASIGVR 44 55 0.03 32 TPEGVVLAADKR 56 68 0.03 38 SRSPLMEPTSVEK; O xidation (M) 72 88 0.06 58 ADDHIGIASAGHVADAR 89 95 0.01 48 QLIDFAR 105 116 0.02 34 YGEPIGIETLTK 150 163 0.01 36 LYETDPSGTPYEWK 2 T 47.8 % 12 22 0.02 59 GTSLFSPDGR 23 29 0.03 44 IYQVEYAR 43 53 0.03 61 TADGVVLAALR 54 6 7 0.04 34 STPSELMEAESIEK 104 115 0.03 49 YGEPIGVETLTK 116 148 0.13 42 TITDNIQESTQSGGTRPYGASLLIGGVENGSGR ; Deamidation (NQ) 149 162 0.00 45 LFATDPSGTPQEWK T 37.9 % 21 36 0.03 55 SNVFGPELGEFSNADR 57 68 0.02 45 TEEGVVLATDMR 69 7 8 0.01 47 ASMGYMVSSK; Oxidation (M) 83 109 0.05 65 VEEIHPTGALTIAGSVSAAQSLISSLR 119 137 0.04 57 RGEDMSMQALSTLVGNFLR; 2 Oxidation (M) 236 243 0.02 44 HQNFEGLE

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116 Table 3 1 2 ial trypsin and chymotrypsin digest ( TC). Proteina Res. no.b Mascot Ion Score Sequence 1 TC 32.1 % 13 22 0.03 48 GITIFSPDGR 58 68 0.06 33 SPLMEPTSVEK 72 88 0.01 43 ADDHIGIASAGHVADAR 89 95 0.04 43 QLIDFAR 105 116 0.10 37 YGEPIGI ETLTK 126 149 0.06 62 TQVGGARPFGVALLIGGVENGTPR 135 149 0.06 46 GVALLIGGVENGTPR 2 TC 34 % 43 53 0.05 59 TADGVVLAALR 54 67 0.05 48 STPSELMEAESIEK 71 87 0.05 74 LDDALGAATAGHVADAR 104 115 0.06 49 YGEPIGVETLTK 116 133 0.0 5 69 TITDNIQESTSGGTRPY 134 148 0.04 56 GASLLIGGVENGSGR TC 49.4 % 57 68 0.07 68 TEEGVVLATDMR 69 78 0.05 38 ASMGYMVSSK 83 109 0.04 98 VEEIHPTGALTIAGSVSAAQSLISSLR 119 128 0.06 52 RGEDMSMQAL 119 131 0.06 57 RGEDMSMQALSTL D eamidation (Q); Phospho (S) 129 137 0.06 40 STLVGNFLR 138 159 0.09 55 SGGFYVVQPILGGVDETGPHIY 214 235 0.07 99 DLASGNGINIAVVTEDGVDIQR 236 243 0.03 45 HQNFEGLE a T, trypsin digest; TC, trypsin and chymotrypsin digested proteasomal proteins with the percent coverage of the deduced protein sequence. bResidue number of deduced protein sequence, difference between the calculated and observed re, and peptide sequence are reported.

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117 Table 3 4. Summary of MS/MS spectral counting and integration with a calculated ratio of Ac1 Hfx. volcanii wild type cells (DS70 pJAM204). Peptide Sequence 1 Mass Score E va lue Integral MQGQAQQQAYDR + N Acetyl Ac1 733.34 (2+) 87 2.4e 007 7.218 QGQAQQQAYDR N2 646.81 (2+) 69 1.3e 005 7.093 e 2 Ac1:N2 103:1 1 1 protein was purified from Hfx. volcanii cells expressing psmA his6 (DS70 pJAM204) by Ni2+affinity chromatogra phy unassembled and assembled into CPs.

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118 Table 3 5. N terminal modifications Nacetylation and MAP cleavage pattern in 20S proteasomes and whole cells and influence 20S proteasome mediated peptidase activity terminal forms a Specific activity b N1 N2 Ac1 Ac2 37 65 wild type (1 % ) 100 % (99 % ) 74 1 363 15 12 (G13) 100 % 252 7 1770 70 Q2A 100 % 78 3 621 41 Q2D (20 % ) 100 % (80 % ) n.d. n.d. Q2P (18 % ) (13 % ) 100 % (69 % ) n.d. n.d. Q2S 100 % 50 4 421 50 Q2T (10 % ) 100 % (90 % ) 44 13 370 60 Q2V 100 % 16 2 274 25 aNacetylat CPs (and whole cells in parenthesis if different from CPs purified by the twostep affinity method) as determined by MS integration and MS/MS spectral counting of the N ral counting was validated by multiple reaction monitoring (MRM) initiated detection of peptides. bSpecific activity was determined for proteasomal CPs purified by the two step affinity method using the chymotrypsinlike peptide substrate Suc LLVY AMC. A ctivity was monitored by the release of 7 amino 4methyl coumarin. Assay conditions were in 20 mM Tris HCl buffer at pH 7.2 with 2 M NaCl at the temperatures indicated. Activity was monitored in triplicate and is reported as nmols of product released min1 mg1 protein. n.d., not determined.

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119 Table 3 6. Three additional proteins identified by ESIQTOF MS scans of proteasomeenriched samples. ORF no. p I / Mr a (Da) Amino acid position, Pfam domain and description, E value Putative amino acid sequence of ORF ( % His of total protein)b HVO_2645 4.49 / 2075 2.38 41 184 aa, PF00994 molybdopterin binding, Expect 1.3e 35 MSD H DQ H YGADD H GRDQ HH GEGDGDDD H G HHHH DVDDLEAAVL TISSTRDVDDDPAGDAIAELLH EAGH SVSVRRVVDDDYDEIQI AVARMADRGDVDVTVTTGGTGVTPDDRTVEAATQLFEKTLPGF GELFRRLSYDDIGTKVVGTRATAGVVDGMPTFCLPGSENAARL GTAEIIVPEAP H LTGLAR RDAE (6.7 %) HVO_2108 5.96 / 32702.80 54 101 aa, PF01022 HTH_5 bacterial regulatory protein arsR family with helix turn helix motif, Expect 1.1e 03 167 292 aa, PF01614 IclR bacterial transcription regulator family, Expect 1.9e 39 MGSPFYN H RSDGVGGSL H C LTESNSCSARWNKLFTLPRR H AVM TENAPNRIETSRKTIRVLDALREH GSSSVTFLARELGMNKSTV H N H LSTLEAEKFVVRDGTDYELSLRLLGFGGYVQH R H PLYQVA KREVH RLASQTGELANLMVEEYGQGVYLASEQGERAVDLDIYP GLRRPLH AIGLGKAILAH LPSERVDEIIEEH GLPAETDQTITD RAELDAQLATVRDRGYAVDNEELIRGLRCVGAPIIAKDGTVLG AISVSQPVSRMNNERFT DEVPDIVQSAANVIEL H TN H (4.7 %) HVO_1577 4.60 / 19377.70 5 173 aa, PF03444 DUF293 Domain of unknown function may be distantly related to HrcA heat inducible transcriptional repressor, Expect 2.9e 67 13 68 aa, PF08279 HTH_11 HTH helix turn helix domain in a wide variety of proteins, Expect 3.5e 06 13 126 aa, PF02082 Rrf2 Transcriptional regulator family related to Y_phosphatase2 and other transcription regulation families, Expect 5.1e 04 MSSIELTSSQKTILTALINLYRDSEDAVKGEDIAAEVNRNPGT IRNQMQSLKALQLVEGVPGPKGGYKPTANAYEALDVDKMDEPA FVPLFH NDEEVEGVNVDEIDLSSVHH PELCRAEIH VQGSVREF H EGDKIRVGPTPLSKLVIDGTLDGKDDTSNILILRIDDMQAPV GEPQH (3.4 %) aFor comparison, calculated pI and Mr of proteasomal proteins are: 1 (4.47 and 27594.16 Da) 2 (4.29 and 26727.17 Da) pre (4.56 and 25993.77 Da) and mature (4.52 and 20584.99 Da). bDeduced a mino acid sequence communicated by J. Eisen, TIGR with histidine residues underlined (April 2007 version, ).

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120 Table 3 7. MS/MS analysis of 20S proteasomes purified by twostep affinity purification. Protein Residue no. MASCOT E value Sequence 1 12 73 3.4e 06 MQGQAQQQAYDR + Acetyl (N term) 44 55 65 1.5e 05 TPEGVVLAADK 58 68 42 3.5e 03 SPLMEPTSVEK 58 68 46 1.4e 03 SPLMEPTSVEK + Oxidation (M) 72 88 83 3.5e 07 ADDHIGIASAGHVADAR 172 183 25 2.4e 01 GDHQEHLEENFR 12 22 59 5.8e 05 GTSLFSPDGR 23 29 35 1.5e 02 IYQVEYAR 54 67 64 2.6e 05 STPSELMEAESIEK 54 67 77 1.0e 06 STPSELMEAESIEK + Oxidation (M) 71 87 67 1.4e 05 LDDALGAATAGHVAD AR 104 115 68 4.5e 06 YGEPIGVETLTK 149 162 68 7.5e 06 LFATDPSGTPQEWK 57 68 48 7.9e 04 TEEGVVLATDMR 57 68 45 1.7e 03 TEEGVVLATDMR + Oxidation (M)

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121 Table 3 8. MS/MS identification of the 30kDa i soform chain predominant in Hfx. volcanii c ells a. Protein Residue no. MASCOT E value Sequence 13 24 44 1.3e 05 GITIFSPDGR 58 68 52 2.7e 03 SPLMEPTSVEK + Oxidation (M) 72 88 49 4.4e 03 ADDHIGIASAGHVADAR b. Protein Residue no. MASCOT E value Sequence 1 12 33 2.0e 03 M T GQAQQQAYDR + Acetyl (N term) 25 32 30 1.4e 03 LYQVEYAR 58 68 42 5.5e 03 SPLMEPTSVEK 72 88 49 1.7e 05 ADDHIGIASAGHVADAR c. Protein Residue no. MASCOT E value Sequence 13 24 34 5.5e 03 GITIFSPDGR 25 32 61 3.7e 05 LYQVEYAR 58 68 57 2.2e 05 SPLMEPTSVEK 72 88 45 1.9e 06 ADDHIGIASAGHVADAR d. Protein Residue no. MASCOT E value Sequence 25 32 61 4.1e 03 LYQVEYAR 58 68 37 1.7e 03 SPLMEPTSVEK + Oxidation (M) 72 88 60 2.2e 03 ADDH IGIASAGHVADAR

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122 Figure 31. I His were purified from recombinant H fx volcanii and separated by 2DE (45 and 25 Isoforms of 1 and/or 1 His (a to h), (i to m) and 2 (n and o) proteins were identified by MS analysis of protein spots. A) Molecular mass standards on left and p I range of 3.9 to 5.1 on top are indicated. B) Magnified region of 2DE gel.

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123 Figure 32. MS/MS Fragmenta tion of Acet MoxQGQAQQQAYDR, an N terminal fragment of 741.34 Da which corresponds to the mass of the defined fragment plus oxidation (16.00 Da for methionine sulfoxide; Mox) and acetylation (42.01 Da; Acet ) of the N terminus with a delta value < 0.1 Da. The individual Mascot ion score was 76 with an E value of 5.5e 7. Labeled peaks include b and yions as well as dominate internal ion fragments. Loss of ammonia (*, 17.03 Da) is indicated.

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124 Figure 33. MS/MS Fragmentation of Acet MNRN(D)DKQAYDRGTSLFSPDGRIYQVE, an N terminal fragment of the 2 protein of 20S proteasome s The peptide was generated from a Glu C endoprotease digest of 2 protein purified via association with 1 His. The peptide ha d an acetyl group at the N terminus (Acet ) and a deamidation at the asparginine residue (N(D)) in the fo urth position. The m/z of the parent ion was 776.62 Da The i ndividual M ascot ion score was 24 with an E value of 0.1. Labeled peaks include band yions.

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125 Figure 34. 20S proteasome CPs are rapidly isolated by two step affinity purification f rom Hfx. volcanii A ) SDS PAGE gel of cell lysate (lane 1) and chromatography fractions after purification by Ni2+Sepharose (lane 2) and CPs (lane 3) isolated after the two step affinity purification using Ni2+Sepharose and StrepTactin chromatography. Molecular mass standards indicated on left. B ) Size exclusion chromatogram of two step purified CPs Proteasomal CPs were purified from Hfx. volcanii H26 pJAM2545 cells expressing the synthetic psmB strepII psmA his6 subunit s of proteasomal CPs with C terminal StrepII and His6 affinity tags, respectively.

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126 Figure 35. an acetylated N terminal initiator methionine (Ac1) were dominant in wild type cells. MS spectral integration was performed to quantify the various N after purification by Ni2+Sepharose from Hfx. volcanii wild type cells (DS70pJAM204). A ) Isotopic envelope of the MS scan for a 733.79, 2+ (m/z) peptide corresponding wi th the MS/MS of the Ac1 peptide. B ) MS scan for a 647.17, 2+ (m/z) peptide corresponding with the MS/MS of the N2 peptide. C ) Total MS scan for the MRM selecting for two peptides 733.79, 2+ for Ac1 and 647.17, 2+ for N2.

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127 Figure 36. Q2P, and Q2T proteins accumulate in cells as heptameric rings. A ) SDS PAGE of cell lysate and Ni2+Sepharose fractions of Hfx. volcanii GZ130 ( psmA Q2T (lanes 5 and 6), respecti vely. B ) Size exclusion (Superose 6) chromatography of Ni2+P type (.). proteins (data not shown). C ) qRT PC R analysis of the psmA specific transcripts of Hfx. volcanii GZ130 cells expressing the psmA his6 and psmA Q2T his6 genes in trans (see methods for details).

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128 Figure 37. f a chromosomal psmA deletion or exacerbate it. Hfx. volcanii GZ130 ( psmA ) with ), 212) ( ). For inoculums c ells were grown in ATCC 974 medium with novobiocin. Cells were streaked from 80 C glycerol stocks onto fresh plates, grown twice to logphase in 2 ml medium and used as an inoculum for final analysis of growth in ATCC 974 medium with novobiocin and NaCl concentrations from 1 to 2.25 M as indicated. Each subculture was inoculated to a final OD600 of 0.01 to 0.02. Growth is represented as a percentage relative to growth for that strain at 2.25 M NaCl (100 % growth was ~1 109 CFUml1). Experiments were pe rformed in triplicate and the mean S.D. was calculated.

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129 Figure 38. Hfx. volcanii phenotype and A) grew to higher cell density and B) at higher rates and that those expressing wildty Hfx. volcanii GZ130 ( psmA ( ).

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130 Figure 39. Two dimensional gel electrophoresis of cell lysate of Hfx. volcanii GZ130 expressing kDa isoform chain, type) and identified as

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131 CHAPTER 4 N TERMINAL PROTEOME AN D ACETYLTRANSFERASES IN H ALOFERAX VOLCANII Introduction In the yeast Sacchar omyces cerevisiae Nacetylation is catalyzed by at least three N terminal acetyltransferases (NATs), NatA, NatB and NatC which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively (Polevoda and Sherman, 2003a) These enzymes act co translationally on separate groups of substrates, each with degenerate motifs (Polevoda et al. 1999; Polevoda and Sherman, 2000; Polevoda and Sherman, 2003b; Polevoda et al. 2003) NatA typically acts on N terminal residues such as Ser, Al a, Gly and Thr after the i nitiator Met is removed cotranslationally by methionine aminopeptidase (Moerschell et al. 1990) In contrast, NatB and NatC often modify the N terminal initiator Met when it precedes either Glu, Asp, Asn or Met residues (NatB) or the bulky hydrophobic residues Ile, Leu, Trp or Phe (NatC). In E. coli three NATs have been identified (RimI, RimJ and RimL); however, these enzymes are only known to modify the ribosomal proteins S18, S5 and L12 with N terminal Ala Arg Ala His and Ser Ile residues, respectively (Yoshikawa et al. 1987; Tanaka et al. 1989) There is no evidence that the Rim proteins act co translationally. Instead, at least the RimL enzyme appears to act post translational based on the finding that the ribosomal proteins L7 and L12 have identical N termini and yet only L12 is acetylated (Tanaka et al. 1989) Thus, RimL is thought to recognize a certain protein structure and not just the extreme N terminus of the substrate protein. Originally, only a few archaeal proteins were known to be modified acetylation including: glutamate dehydrogenase 2 (Maras et al. 1992) and Alba1 chromatin proteins of Sulfolobus solfataricus (Bell et al. 2002) ; ribosoma l proteins S7P, L31e, and S19P of Haloarcula marismortui (Kimura et al. 1989; Hatakeyama and Hatakeyama, 1990; Klussmann

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132 et al. 1993) ; and halocyanin of Natronobacterium pharaonis (Mattar et al. 1994) In addition based on our studies subunits of 20S proteasome s from H f x volcanii as well as the HrcAlike HVO_1577 were also acetylated (p resented in Chapter 3) (Humbard et al. 2006) Even with identification of three additional N terminally acetylated protein s from Hfx. volcanii i t was unclear whether this modification is common or rare in archaea or Hfx. volcanii Nothing is known regarding the mechanism or function of N terminal acetylation in archaea. The majority of these modifications are on N terminal serine residues, exposed by a presumed methionine aminopeptidase and followed by either Ser or Ala residues (SerSer/Ala ). Halocyanin represents an example for a relatively large group of predicted archaeal lipoproteins in which an internal Cys appears to be modified by a diphytanyl (glycerol)diether, converted to the N terminal residue via cle avage by a signal peptidase II like protease, and then Nacetylated (Mattar et al. 1994) Glutamate dehydrogenase 2 (GDH2) is the single example (prior to this study) of an archaeal protein acetylated directly on its initiating N terminal methionine residue (Maras et al. 1992) Although the presence of six internal N epsilon methyl lysine residues is speculated to be responsible for the extreme thermostability of GDH 2 (Maras et al. 1992) the role of the N acetyl g roup remains to be determined. Regarding the archaeal enzymes which catalyze Nacetylation, recent phylogenetic analysis suggests a family of uncharacterized bacteria and archaea acetyltransferases (BAAs) may be responsible for this type of modification (Polevoda and Sherman, 2003a; Polevoda and Sherman, 2003b) Hfx. volcanii are both acetylated on the N terminal methionine and follow ed by bulky polar amino acids (glutamine and asparagine) (Humbard et al. 2006) This motif is closer to the NatB motif of enzymatic activity found in yeast cells (Polevoda and Sherman, 2003b) In contrast to Nacetylation in which the modifying

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133 enzymes and function(s) remain to be elucidated, acetylation of Alba1 on an internal lysine residue (Lys16 of the mature protein) is known to be mediated by Sir2 (Bell et al. 2002) and alleviate repression of minichromosome maintenance activity in S. solfataricus (Marsh et al. 2006) Recently, a systematic study of the N terminal peptides from the haloarchaea species Halobacterium salinarum and Natronomonas pharaonis has demonstrated that there is likely a generalized Nacetylation pathway at work in the haloarchaea (Falb et al. 2006; Aival iotis et al., 2007) To further investigate this, we examine d the N terminal proteome of H fx. volcanii and measured the frequency of methionine aminopeptidase cleavage and Nacetylation The frequency of modifications were compared to the occurance in distantly related Hb. salinarum and N. pharaonis In addition, genomic and phylogenetic studies were performed to examine the function different acetyltransferases may serve in Hfx. volcanii physiology. Results and Discussion Phylogenetic D istribution of GNAT F amily in Haloferax volcanii Phylogenetic analysis of acetyltransferase homologs from a variety of organisms reveals seven different groups : Ard1p, Nat3p, Mak3p, Nat5p, C amello, Bacterial and Archaeal acetyltransferases (BAA), and lysine acetyltransferases (Figure 41) Three of these families, Ard1p, Nat3p, and Mak3p, are related to the dominant yeast N terminal acetyltransferase activities (NatA, NatB, and NatC). The C amello family is most likely related, distantly, to the Mak3p family, but examples of the Camello1 and Camello2 family are currently restricted to mouse, rats, Xenopus and humans (Popsueva et al. 2001; Polevoda and Sherman, 2003b) Both the Nat5p and Bacterial and Archaeal acetyltransf erase families have no known substrates or function and the lysine acetyltransferases represent a distant family of unrelated function.

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134 Interestingly, Nat5p is part of the NatA complex, comprised of Nat1p and the catalytic subunit Ard1p (Gautschi et al. 2003) Out of the three major families of N terminal acetyltransferases that are responsible for the majority of the activity in yeast, Ard1p Nat3p and Mak3p, Hfx. volcanii only encodes one clear member of the Nat3p family, HVO_2320 (Figure 41). Interestingly, HVO_2320 is one of the only acetyltransferase genes that could not be knocked out in Hfx. volcanii using standard methods (Zhou et. al., unpublished). Cells with a conditional knockdown constructs of HVO_2320 using a PtnaA promoter fusion had severely retarded growth and eventually lost the fusion (Zhou et. al., unpublished). The Nat5p family of acetyltransferases is a relatively new and mysterious family. Hfx. volca nii encodes several putative acetyltransferases that belong to this family, HVO_1972, HVO_0201, HVO_2930, HVO_2423, HVO_2804, HVO_2326, HVO_ 2795, and HVO_2734 (Figure 4 1)(Table 4 2) There is no current data on the substrate specificity of this family of acetyltransferases. However, Nat5p appears in complex with Ard1p and Nat1p of NatA. Nat5p subunits are archored to the ribosome through Nat1p, similar to Ard1p (Gautschi et al. 2003; Arnesen et al. 2006) Homologues of Nat5p have been identified in yeast, humans, and fruit flies. Based on sequence similiarity, Nat5p is believed to have acetyltransferase activity (Arnesen et al. 2006) Efforts to identify and isolate substrates in yeast have been unsuccessful. In addition to Hfx. volcanii proteins and Nat5p from yeast, the Salmonella typhimurium YgjM was also present in this clad. It is interesting that Hfx. volcanii acetyltransferases are so heavily conce ntrated in this family. Perhaps the haloarchaea will be a good system to study the mechanism and effects these different genes have on acetylation in the cell. All eight of the

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135 acetyltransferases in this family encoded by Hfx. volcanii have been deleted individually from the chromosome (Zhou et. al., unpublished). Members of the uncharacterized BAA N terminal acetyltransferase family (e.g. HVO_2709, HVO_1954) make up a well isolated branch of the phylogenetic tree (Figure 4 1). There is currently no data that shows that these proteins have activity acetyltransferase and otherwise. Therefore, there are no known substrates or any in vivo role purposed for these enzymes. These proteins are distantly related to the Rim proteins, specifically RimI, of Escherichia coli RimI acetylates a ribosomal protein in E. coli Out of all the Rim proteins, RimI shares the most similarity to eukaryotic N terminal acety ltransferases (Polevoda and Sherman, 2003b) Likewise, the Hfx. volcanii proteins HVO_2709 and HVO_1954 share the most homology with RimI and other Rim proteins. Reversible post translational modification of primary amines (lysine residues) by acetylation is common in eukaryotes, bacteria, and archaea. Hfx. volcanii encodes for three separate protein a cetyltransferases that act on lysine residues, HVO_1756, HVO_1821, and HVO_2888. There is an additional putative Gcn5 family protein acetyltransferase, HVO_2886, that is proximal to HVO_2888 (Figure 41). All four of the different lysine acetyltransferases belong to the Gcn5 family but HVO_2888 also shares high homology to the yeast Elp3 (Altman Price and Mevarech, 2009) Unlike N terminal acetylation, acetylation amino groups of lysine residues is reversible. The reverse reaction, or deacetylation is carried out by Sir2 family HVO_2194 and HdaI family HVO _0522 deacetylases. The proposed target of these different acetyltransferases and deacetylases are DNA binding proteins such as Alba (Bell et al. 2002; Wardleworth et al. 2002; AltmanPrice and Mevarech, 2009) Acetylation and deacetylation of

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136 Alba affects it s affinity for DNA. Parallels are drawn between histone acetylation, of the H3 / H4 histone proteins, and Alba (Zhang et al. 2003b; AltmanPrice and Mevarech, 2009) There are two annotated ORFs in the Hfx. volcanii genome that did not fit into the different acetyltransferase families, HVO_2677 and HVO_2510 The N terminal domain of HVO_ 2510 aligns well with the GNAT Pfam but the C terminus does not show any significant homology to any other proteins. HVO_2677 is a short protein, only 82 amino acids in length, and shows only weak homology to the Gcn5family of acetyltransferases (Table 4 2) Other Nacetylated P roteins in Haloferax volcanii Our i nitial observations indicated that the frequency of N terminal acetylation in Hfx. volcanii may be more common than is seen in bacterial species such as E. coli As an outgrowth of the study of 20S proteasome s in Hfx. volcanii we discovered a contaminating protein HVO_1577, a putative transcriptional regulator, was also N terminally acetylated. Interestingly, this protein was found to have an N terminal serine residue modified by an acetyl group based on t he detection of a modified tryptic peptide spanning residues 2 to 11 (Acet SSIELTSSQK) ( Figure 4 3). The N terminal serine residue suggests the substrate is first cleaved by a methionine aminopeptidase and then acetylated at an N terminal serine residue o f a Ser Ser/Ala motif (similar to eukaryal NatA substrates). In contrast to HVO_1577, acetylation at initiating N common among archaea yet consistent with t he S. solfataricus GDH2. The consensus of these three archaeal proteins is Acetyl Met ( Glu/Gln/Asn ) and, thus, somewhat similar to eukaryal NatB substrates. Regarding the Hfx. volcanii terminal acetylated and nonacetylated proteins (the latter of which appears to be cleaved by a methionine aminopeptidase) was detected, suggesting these modifications are regulated or vary between complexes of different quaternary structure.

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137 To generate a more comprehensive understanding of acetylation in Hfx. volcanii a variety of methods were used to separate cellular proteins and these fractions were analyzed by MS (Kirkland et al. 2008b) Based on this shotgun proteomics approach, nearly 30 % of soluble protein was found to be N terminally acetylated. The Hfx. volcanii proteomic dataset was sys tematically searched for N terminal peptides and their derivatives including those modified co and/or post translational. Overall, 297 unique peptides were identified that mapped to the N termini of 236 proteins representing 18 % of the MS/MS detected pr oteome ( Table 4 3). None of the MS/MS detected peptides were formylated, consistent with previous findings that archaea like eukaryotes initiate translation with methionine, in contrast to bacteria which use formyl methioinine (Ramesh and RajBhandary, 2001) Instead, the majority of N terminal peptides detected in the Hfx. volcanii proteome had patterns consistent with modification by methionine aminopeptidase and/or N terminal aminotransferase. In fact, over 70 % of the proteins with MS/MS detectable N termini included peptides in which the initiator methionine was removed (N2) and/or the initiator methionine and/or penultimate (second) residue of the deduced polypeptide was Nacetylated (Ac1 and Ac2, respectively ) ( Table 4 3 ). The remaining ~30 % of the proteins detected by MS/MS had retained their initiator methionine and were not modified (N1). Multiple N terminal peptide variants were detected for 22 % of the 236 total proteins. Most of these variants were apparent processing intermediates ( i.e ., mixtures of N1 and N2, N1 and Ac1, N1 and Ac2, N2 and Ac2 or N1, N2 and Ac2). Only a small percentage (5.5 % ) of the 236 total proteins with MS/MS detected N termini appeared to be protein isoforms including m ixtures of N2 and Ac1 as well as Ac1 and Ac2 ( Table 4 3). Although Nacetylation is relatively rare in Bacteria with only a few protein

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138 e xamples (Isono and Isono, 1980; Cumberlidge and Isono, 1979; Arai et al. 1980) it is becoming apparent that this type of modification is common among Archaea (at least haloarchaea ) (Aivaliotis et al. 2007; Falb et al. 2006) as well as Eukarya (Polevoda and Sherman, 2003b) Over one fourth (29 % ) of the proteins with MS/MS detectable N termini were Nacetylated in the Hfx. volcanii proteome ( Table 4 3). M ost of these Nacetylated proteins were singly modified with a relatively equal ratio (30:33) of Ac1 to Ac2 forms. Interestingly, of the five proteins with both Ac1 and Ac2 isoforms, three were previously found to accumulate at high levels when Hfx. volcanii is grown in the presence of the irreversible proteasome inhibitor clasto lactacystin lactone (Kirkland et al. 2007) These included H VO_ 0860, H VO_ 1545 and H VO_ 2784 annotated as a SufB like FeS assembly protein, dihydroxyacetone kinase L subunit, and R psM ribosomal protein S13p/S18e respectively (Table 43). Although the N end rule (in which the half life of a protein is determined by its N terminal residue) is conserved in Bacteria and Eukarya (Mogk et al. 2007) ; its function including the relationship of protein stability to Nacetylation is not known in Archaea. Thus, the relationship of these Ac1 and Ac2 isoforms to protein stability and susceptibility to proteasome mediated degradation is highly speculative. For the Nacetylated proteins, the majority (nearly 80 % ) of th ose in an Ac2 form appeared to products of a NatA like acetyltransferase, which in yeast preferentially transfers terminal residues after the initiator methionine has been removed (Polevoda et al. 2003) In addition, a number of the Ac1 modified proteins detected in the Hfx. volcanii proteome appeared to be a result of a yeast like NatB acetyltransferase activity in which the initiator methionine residue preceding a penultimate Asp, Glu, Asn or Met was Nacetylated (Polevoda et al. 2003; Huang et al. 1987) However, none

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139 of Nacetylated peptides detected in the Hfx. volcanii proteome were related in N terminal sequence to products of the yeast NatC acetyltransferase ( i.e., Ac1 forms with penultimate Ile, Leu, Trp or Phe residues ) (Polevoda et al. 2003) Instead, the majority of Ac1 modified proteins (over 80 % ) had small penultimate residues suggesting an additional acetyltransferase activity distinct from the yeast like NatA, NatB and NatC is present. Occurrence of Dif ferent Amino Acids in the Penultimate and Antepenultimate Positions The H fx volcanii genome does have coding capacity for a methionine aminopeptidase ( HVO_2600 ). The frequency of the second, penultimate, amino acid of the entire, deduced, proteome favors small amino acids such as serine (21 % ), threonine (14 % ) alanine (10 % ) valine (5 % ) but there is also a relatively frequent occurrence of larger, charged, amino acids such as aspartic acid (6 % ) and arginine (7 % ) These proteins with small amino acids in the second position are all potentially MAP substrates. In the baseline proteome data, t he relative abundance of Hfx. volcanii proteins with small residues ( i.e ., Gly, Ala, Pro, Val, Ser, Thr) in the penultimate position of their deduced primary se quence was significantly greater for N terminal MS/MS detected peptides modified by methionine aminopeptidase and/or N terminal aminotransferase (87 % ) compared to those that were unmodified (N1 alone) (32 % ) (Table 4 3). In particular, the penultimate residue that was dominant for the modified proteins was serine (at 40 % of the total proteins modified) compared to its limited presence in this same position of unmodified proteins (16 % of total proteins unmodified). This bias, of small residues at the pe nultimate position, contrasts with the theoretical Hfx. volcanii proteome in which about 60 % of the deduced proteins have small residues and 21 % have serine residues at this position. Based on analysis of the MS/MS detected N terminal peptides, initiator m ethionine removal occur red nearly exclusively when the penultimate residue of the protein was small consistent with the substrate preferences of methionine aminopeptidases characterized from

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140 Bacteria Archaea and Eukarya (Ben Bassat et al. 1987; Tsunasawa et al. 1985; Huang et al. 1987; Miller et al. 1987) and similar to other archaeal proteomes (Falb et al. 2006) Of the 16 proteins detected in the Hfx. vo lcanii proteome that were exceptional to this rule ( i.e., in which the initiator methionine was removed from a bulkier residue), most resulted in exposure of an acidic N terminal residue (i.e ., Asp or Glu) (Table 4 3) and, thus, may be a reflection of the increased number of acidic residues used for hydration of halophilic proteins in high salt (Mevarech et al. 2000) Conclusions N terminal processing involves multiple steps activities, most notably the activities of methionine aminopeptidases and N terminal acetyltransferases. Following up on the obs ervation type subunits from 20S proteasomes in Hfx. volcanii and HVO_1577 were N terminally acetylated, a large proteomic survey of N terminal peptides from Hfx. volcanii reveals evidence of a generalized protein acetylation pathway in the haloarchaeon. N termini were identified from 236 different proteins. Over 70 % of those proteins identified were modified by a methionine aminopeptidase and 21 % were modified by an N terminal acetyltransferase. Several proteins had multiple N terminal peptides identified, 297 peptides for 236 proteins. These data are in good agreement with the recently published N terminal proteomes of two other haloarchaea (Falb et al. 2006; Aivaliotis et al. 2007) Hfx. volcanii encodes for acetyltransferases that align with Nat3p, Nat5p, BAA, and lysine acetyltransferase families. Altho ugh no activity has been demonstrated for any of these enzymes, HVO_2320, a member of the Nat3p family, may be essential based on the finding that a conditional knockdown displays severely inhibited growth under standard laboratory conditions (Zhou, unpublished) These data indicate that N terminal acetylation of proteins could be an important cellular process in the haloarchaea.

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141 Table 4 1. Strains and plasmids used in this study Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli F recA1 endA1 hsdR17 (r k m k + ) supE44 thi 1 gyrA relA1 Life Technologies GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL 136 dam13::Tn9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 Ne w England Biolabs BL21 (DE3) F ompT [ lon ] hsd S B (r B m B ) (an E. coli B strain) polymerase gene Novagen Haloferax volcanii H26 pyrE2 (Allers et al. 2004) GZ130 psmA (Zhou et al. 2008) GZ110 2677 (Zhou, unpublished) GZ111 (Zhou, unpublished) GZ115 (Zhou, unpublished) GZ116 (Zhou, unpublished) GZ121 (Zhou, unpublished) GZ122 (Zhou, unpublished) GZ123 (Zhou, unpublished) GZ124 (Zhou, unpublished) GZ125 (Zhou, unpublished) GZ127 (Zhou, unpublished) GZ128 (Zhou, unpublished) GZ129 ( Zhou, unpublished) GZ135 (Zhou, unpublished) H26 P tnaA HVO_2320 (Zhou, unpublished) MH2085 This study. MH2086 This study. Plasmid pJAM2018 Ap r ; pTA131 containing HVO_2677 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_2677 coding region (Zhou, unpublished) pJAM2019 Ap r ; pTA131 derived HVO_2677 suicide plasmid (Zhou, unpublished) pJAM2020 Ap r ; pTA131 containing HVO_2510 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_2510 coding region (Zhou, unpublished) pJAM2021 Ap r ; pTA131 derived HVO_2510 suicide plasmid (Zhou, unpublished) pJAM2014 Ap r ; pTA131 containing HVO_ 1756 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 1756 codi ng region (Zhou, unpublished)

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142 Strain or Plasmid Genotype; oligonucleotide used for amplification Source pJAM2015 Ap r ; pTA131 derived HVO_1756 suicide plasmid (Zhou, unpublished) pJAM2016 Ap r ; pTA131 containing HVO_ 2930 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 2930 coding region (Zhou, unpublished) pJAM2017 Ap r ; pTA131 derived HVO_2930 suicide plasmid (Zhou, unpublished) pJAM2034 Ap r ; pTA131 containing HVO_ 2623 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_2 623 coding (Zhou, unpublished) pJAM2035 Ap r ; pTA131 derived HV O_2623 suicide plasmid (Zhou, unpublished) pJAM2036 Ap r ; pTA131 derived HVO_2795 suicide plasmid (Zhou, unpublished) pJAM2037 Ap r ; pTA131 containing HVO_2 795 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_2 795 coding region (Zhou, unpublished) pJAM2039 Ap r ; pTA131 containing HVO_2 886 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_2 886 coding region (Zhou, unpublished) pJAM2040 Ap r ; pTA131 derived HVO_2886 suicide plasmid (Zhou, unpublished) pJAM2048 Ap r ; pTA131 containing HVO_ 0201 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 0201 coding region (Zhou, unpublished) pJAM2049 Ap r ; pTA131 derived HVO_0201 suicide plasmid (Zhou, unpublished) pJAM2041 Ap r ; pTA131 containing HVO_ 1821 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 1821 coding region (Zhou, unpublished) pJAM2042 Ap r ; pTA131 derived HVO_1821 suicide plasmid (Zhou, unpublished) pJAM2043 Ap r ; pTA131 containing HVO_ 2804 with ~500 bp of genomic DNA fla nking 5 and 3 of the HVO_ 2804 coding region (Zhou, unpublished) pJAM2044 Ap r ; pTA131 derived HVO_2804 suicide plasmid (Zhou, unpublished) pJAM2045 Ap r ; pTA131 containing HVO_ 2326 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 2326 coding region (Zhou, unpublished) pJAM2046 Ap r ; pTA131 derived HVO_2326 suicide plasmid (Zhou, unpublished) pJAM2050 Ap r ; pTA131 derived HVO_2423 suicide plasmid (Zhou, unpublished) pJAM2516 Ap r ; pTA131 containing HVO_ 2709 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 2709 coding region (Zhou, unpublished) pJAM2518 Ap r ; pTA131 derived HVO_2709 suicide plasmid (Zhou, unpublished) pJAM2519 Ap r ; pTA131 containing HVO_ 1954 with ~500 bp of genomic DNA flanking 5 and 3 of the HVO_ 1954 coding region (Zhou, unpublished) pJAM2520 Ap r ; pTA131 derived HVO_1954 suicide plasmid This study. pJAM2056 Ap r Nv r ; Insert gene HVO_ 2320 into Nde I and Kpn I of pJAM809, Hvo2320 StrepII This study. pJAM2058 Ap r ; pTA1 31 insert 323 bp P tnaA into Xba I and Nde I This study.

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143 Strain or Plasmid Genotype; oligonucleotide used for amplification Source site of pJAM2056, in front of hvo 2320 pJAM2060 Ap r ; pTA131 insert about 500 bp upstream P tnaA HVO_2320 This study.

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144 Figure 41. GNAT protein phylogenetic tree. Haloferax volcanii proteins compa red to bacterial and eukaryotic homologues. Ap Aeropyrum pernix, Aa Aquifex aeolicus, At A. thaliana, Af Archaeoglobus fulgidus, Ce C. elegans, Cco Campylobacter coli, Cpn Chlamydia pneumoniae, Dr Deinococcus radiodurans, Dm D. melanogaster, Hae Haemophilus influenzae, Hn Halobacterium sp. NRC 1, Hv Haloferax volcanii, Hs H. sapiens, Mth Methanobacterium thermoautotrophicum, Mj Methanococcus jannaschii, Mm M. musculus, Pae Pseudomonas aeruginosa, Pa Pyrococcus abyssi, Ph Pyrococcus horikoshii, Rn Rattus nor vegicus, Rr R. rattus, Sc S. cerevisiae, Sty Salmonella typhimurium, Sp S. pombe, Sty Streptomyces coelicolor, Ss S. solfataricus, Syn Synechocystis sp., Ta Thermoplasma acidophilum, Up Ureaplasma urealyticum.

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149 ORF no. Pfam domain, description, E value; NCBI Conserved Domain Database or Clusters of Orthologous Groups, description, E va lue Putative amino acid sequence of ORF a HVO_2886TG_gha_455 COG2153, ElaA, Predicted acyltransferase Expect 9e 10 MTNSDVRVATGDAERDDAFGVRKAVFVDEQGVDEELEWDEHDDPD AEAVHFVASRDGDAVGAARLREYEPGVGKVERVAVLESARGEGWG RRLMEALEAEARERGFDSLLLHGQTTAEGFYRGLGYETKSDEFDE AGIPHVEMRKSL HVO_2930TG_gha_455 PF00583, acetyltransferase (GNAT) family, Expect 7e 12 COG1247, Sortase and related acyltransferases Expect 7e 36 MTVRLRAATPSDLPAIREIYAPFVENTAISF AYDPPSVADLETKL EQKTDYPWLVCELDGEVAGYAYAGAIRERIAYRWAVETSIYVRPE FQRRGVARGLYTALLDLLERQGYVSAVAVITTPNPASIAFHESFG FERVGRFERVGYKGDAWHDVEWWSLDLDDRPDDPDAPLSVADARD RDWWDDALTRGAALVENSA aDeduced a mino acid sequence communicated by J. Eisen, TIGR April 2007 version ( )

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150 Frequency of Amino Acids in Penultimate Position A 10% C 1% D 6% E 4% F 2% G 4% H 2% K 4% L 4% M 1% N 5% P 5% Q 2% R 7% S 21% T 14% V 5% W 0% Y 2% I 2% Figure 42. Frequency of amino acids in the p enultimate position of the N termin i of proteins from the deduced proteome of H fx. volcanii

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151 Figure 43. MS/MS Fragmentation of Acet SSI ELTSSQK, an Nterminal fragment of HVO_1577. This peptide had a Mascot individual ion score of 39, an E value of 3.1e 3, and an MS/MS spectra that had a complete y ion series and 4 of 9 bions. N terminal a cetyl group (Acet ).

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152 Table 4 3. Summary of N termina l peptides and associated proteins of Hfx. volcanii detected by MS/MS. Protein Accession Number and Name N terminal Form Peptide Sequence Group I. N1 alone Penultimate residue small ( e.g. Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0028 hypo thetical protein N1 MTRAELER HVO_0041 argF ornithine carbamoyltransferase N1 MLETTHFTDIDDISASELDR HVO_0062 dipeptide ABC transporter dipeptide binding N1 MPDTNK HVO_0196 conserved hypothetical protein N1 MADLIVK HVO_0203 rfcS replication factor C small subunit N1 MSEAAESGDAPAGR HVO_0274 conserved hypothetical protein N1 MSGRKQGDSAVYAAAQGR HVO_0455 cctB Thermosome subunit 2 N1 MSQRMQQGQPMIIMGEDAQR HVO_0874 epf mRNA 3'' end processing factor homolog N1 MSSVDKQLENLK HVO_0887 porB 2 oxoglutar ate Fd oxidoreductase beta N1 MSSNVR HVO_0888 porA 2 oxoglutarate Fd oxidoreductase N1 MPADFNWAIGGEAGDGIDSTGK HVO_1009 aad oxidoreductase N1 MSLDYRR HVO_1054 gatA Glutamyl tRNA(Gln) amidotransferase N1 MSLNAFITK HVO_1145 RPS1A 30S ribosomal protein S3Ae N1 MSERSVSKQKR HVO_1684 thrS threonyl tRNA synthetase N1 MSDIAVILPDGTELSVEEGATVR HVO_1799 FAD dependent oxidoreductase putative N1 MTDNYDVIIAGAGPAGGQAAR HVO_1914 3 ketoacyl CoA thiolase N1 MPVPVIAAAYR HVO_2110 arcR transcription regulator N1 MATNKPR HVO_2158 mdh nadp dependent malic enzyme N1 MTLGDDAR HVO_2222 ppiA peptidyl prolyl cis trans isomerase N1 MSDNPTATLHTNKGDITVELFEDK HVO_2564 rplC ribosomal protein L3 N1 MPQPSRPRKGSMGFSPR HVO_2740 ndk Nucleoside diphosphate kinase N1 MSDAERT FVMVKPDGVQR HVO_A0266 transcriptional regulator putative N1 MVNSVLRLAMNER Penultimate residue other HVO_0138 tyrS tyrosyl tRNA synthetase N1 MDAYDLITR HVO_0199 pmm phosphoglucomutase/phosphomannomutase N1 MDEISFGTDGWR HVO_0435 phosphoribosyl ATP pyrophosphohydrolase N1 MDADTPTDEVLDELFATIESR HVO_0452 leuS leucyl tRNA synthetase N1 MDYDPQELEAR HVO_0826 TET aminopeptidase homolog N1 MEFDFDR HVO_1381 mdmC caffeoyl CoA O methyltransferase N1 MDILTDETR

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153 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_1402 pmu phosphomannomutase N1 MELF GTAGIR HVO_1568 HydD putative N1 MDLDYGMLGGR HVO_1585 acs Acetyl coenzyme A synthetase N1 MDVPDTDTVVHEPDPAFVEDANVTR HVO_1678 eIF 2 Translation initiation factor N1 MDYDDQLDR HVO_1896 Ribosomal protein S24e N1 MDIEIISEEENPMLHR HVO_2091 gabT 4 amin obutyrate aminotransferase N1 MDRDSVEPTVTSLPGPK HVO_2554 Chain J Trigger Factor Ribosome Binding N1 MEALKADITKGVAR HVO_2601 hit histidine triad protein N1 MDQLFAPWR HVO_2712 RtcB RNA terminal phosphate cyclase oper N1 METREFGDIELR HVO_2716 acd Acy l CoA dehydrogenase N1 METVGSATGLTDEQR HVO_2790 mrp Mrp protein homolog N1 MDEADVR HVO_2796 conserved hypothetical protein N1 MDWPHDPDGEEGSEGGR HVO_2899 conserved domain protein N1 MELAAIEDLIESHIEDADATVSRPR HVO_A0286 unknown N1 MEFAIWAYPWDVLDAGPR H VO_B0268 alkanal monooxygenase like N1 MDFSIVDLSPVPK HVO_0387 conserved hypothetical protein N1 MNDEELDELR HVO_0581 cell division protein FtsZ N1 MQDIVREAMER HVO_0790 conserved hypothetical protein N1 MNTDVGLSAR HVO_0806 pyk pyruvate kinase N1 MRNA KIVCTLGPASFDR HVO_0819 sirR transcription repressor N1 MLSDVMEDYLK HVO_0973 aspB aspartate aminotransferase N1 MNFDFSDR HVO_0999 cobyrinic acid ac diamide synthase N1 MNTVLVTSTGESTGK HVO_1082 orotate phosphoribosyltransferase N1 MKNVDDLIASAAELADR HVO_1099 dapD 23 4 5 tetrahydropyridine 2 carboxy N1 MNLESDVR HVO_1198 syrB universal stress protein N1 MYSHILFPTDGSDCADAALDHAIEHAR HVO_1344 conserved hypothetical protein TIGR00291 N1 MISLDEAVTAR HVO_1377 Hypothetical UPF0145 protein N1 MIVTTTETVIG R HVO_1380 mcmA methylmalonyl CoA mutase subunit alpha N1 MFDPDELEEIR HVO_1382 alpha NAC homolog N1 MFGGGGMNPR HVO_1527 glucose 1 phosphate thymidylyltransferase N1 MQAVVLAAGK HVO_1830 gndA 6 phosphogluconate dehydrogenase N1 MQLGVIGLGR HVO_2057 glucose 1 phosphate thymidylyltransferase N1 MKGVLLSGGTGSR HVO_2068 ftsZ cell division protein N1 MNVFCFGAGK HVO_2313 nirH heme biosynthesis protein N1 MIQADADLSR HVO_2373 ribosomal protein S8.e N1 MKDQGRSK HVO_2543 rpmD ribosomal protein L30P N1 M QAIVQLR

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154 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_2699 bcp Bacterioferritin comigratory protein N1 MLEPGDDAPDFELPDQHGDLVSLSDFR HVO_2706 eif2bd translation initiation factor eIF N1 MIDETIEEIR HVO_2869 DNA binding domain similar to hmvng1864 N1 MYDLTGFQR HVO_A0586 rffH1 glucose 1 P thymid ylyltransferase N1 MQTVILAAGR HVO_B0064 unknown function N1 MFGEYLTR Group II. N2 and intermediates Penultimate residue small ( e.g. Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0024 tssB thiosulfate sulfurtransferase N2 VDVVSPTWLADR HVO_0027 tr anscription anti termination factor N2 SELLDTLRDDHETPLSR HVO_0069 arylsulfatase N2 TAESPENVLFVVMDTVR HVO_0104 radA DNA repair and recombination protein N2 AEDDLESLPGVGPATADK HVO_0107 SUF system FeS assembly protein NifU family N2 GIGSDMYR HVO_011 8 Ribosomal LX protein N2 SQFIITGSFTSR HVO_0123 srp Signal recognition 54 kDa protein N2 VLDNLGSSLRGSLDK HVO_0167 ahcY adenosylhomocysteinase N2 SEHYAPVSEHLDDVEAAR HVO_0177 arsC arsC protein N2 TDAADVDVDADTDATTTR HVO_0304 etfA Electron transfer fl avoprotein alpha N2 SDVLAVVEHR HVO_0329 ilvE branched chain amino acid aminotran N2 GFDEMDVSTIWQSGEYK HVO_0353 ribosomal protein S23 (S12) N2 ANGKYAAR HVO_0491 Pyridoxamine 5' phosphate oxidase family N2 SLDQQTELSPDEIR HVO_0541 acnA aconitate hydra tase 1 N2 SDTETLDAIR HVO_0704 dgs dolichol P glucose transferase N2 SQSVGVVVPAYRPDPER HVO_0736 Domain of unknown function DUF302 superf N2 ALPIDPSAIKPEDIGEER HVO_0738 conserved hypothetical protein N2 ADEEADEAPAVELGTGASVEGAPLAR HVO_0870 proS prolyl tRNA synthetase N2 SDEQELGITESK HVO_0880 coiled coil protein of COG 134 N2 VTKQEVLSEFDVQELDEAR HVO_1000 acetyl CoA synthetase N2 GELSELFAPNR HVO_1020 PBS lyase HEAT like repeat domain protein N2 SDGDDDTTELSPESFDER HVO_1022 nadh dependent fla vin oxidoreductase N2 TDSLFTPLSLR HVO_1053 gatC glutamyl tRNA(Gln) amidotransferase N2 SDTPVDADEVR HVO_1077 conserved protein N2 TSEWVSLFSGGK HVO_1132 purA adenylosuccinate synthetase N2 TVTIVGS QLGDEGK HVO_1174 TFIIE alpha subunit N2 AFEELLNDPVIQK HVO_1257 moxR methanol dehydrogenase regulatory N2 SDNPDRFGDDGQR

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155 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_1311 dehydrogenase putative (TBD) N2 AFDDVDFEGR HVO_1451 Glutamate dehydrogen ase N2 ASAAPSTATDDEPTEETALETAR HVO_1453 gdhA Glutamate dehydrogenase N2 AQEANPFESLQEQIDDAATYLDVR HVO_1492 conserved hypothetical protein N2 AHWTDSIVGDRMTVDR HVO_1493 conserved hypothetical p rotein N2 PQIHLDDETVAR HVO_1506 ilvC ketol acid reductoisomerase N2 TELTTEVYYDDDADR HVO_1541 glpK glycerol kinase N2 SGETYVGAIDQGTTGTR HVO_1595 conserved hypothetical protein N2 SDDTAPDENWGQQFDR HVO_1626 metallo beta lactamase N2 TVSDWSDWLPR HVO_1745 hypothetical protein N2 VAPISHADELDPFIALELPPGWVR HVO_1857 conserved protein N2 SGSPDDERLEEL R HVO_1871 Chlorite dismutase family N2 VEAPQTDEGWFALHDFR HVO_1946 translation initation factor SUI1 putative N2 SEVCSTCGLPEELCVCEDVAK HVO_1973 conserved hypothetical protein N2 TESESDTEAEAESER HVO_2029 t rh transcription regulator AsnC family N2 TYENLDSDLINALLEDGR HVO_2256 Protein of unknown function DUF262 family N2 AIYVDDPLNSNDEDWDISK HVO_2265 hypothetical protein N2 TDDPEDIPDPVKPTTLQK HVO_2300 eif5A transla tion initiation factor eIF 5A N2 AKEQKQVR HVO_2371 conserved hypothetical protein N2 VVDSLSDGTR HVO_2400 conserved hypothetical protein N2 SDLEIER HVO_2413 tuf translation elongation factor EF 1 s... N2 ADKP HQNLAIIGHVDHGK HVO_2436 tme nadp dependent malic enzyme N2 GLDDDSRKYHR HVO_2452 ribonucleoside diphosphate reductase ade N2 SDANLSTDELVLPVK HVO_2557 rpmC ribosomal protein L29 N2 AILYTEEIR HVO_2558 30S ribosomal protein S3P N2 ADEHQFIENGLQR HVO_2559 rplV ribosomal protein L22 N2 GINYSVEADPDTTAK HVO_2624 pyrG CTP synthase N2 PTDEYDPEMGRK HVO_2677 acetyltransferase (gnat) family N2 AELQTQTVSSGK HVO_2723 snp snRNP homolog N2 SGRPLDVLEASLDEPVTVLLK HVO_2737 RPL8A 50S ribosomal protein L7Ae N2 AVYVDFDVPADLADSAVEALEVAR HVO_2743 methionine synthase vitamin B12 independent N2 TELVSTTPGLYPLPDWAK HVO_2753 Domain of unknown function N2 SESEQR HVO_2758 ribosomal protein L11 N2 AGTIEVLVPGGK HVO_2774 eno phosphopyruvate hydratase N2 TRITSVALR HVO_2783 rpsD ribosomal protein S4 N2 TTGNNTKFYETPNHPFQGER HVO_2945 valS valyl tRNA synthetase N2 PSGEYDPETVEAK

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156 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_2961 lpdA dihydrolipoamide dehydrogenase N2 VVGDIATGTELLVIGAGPGGYVAAIR HVO_2981 upp uracil phosphoribosyltransferase N2 PIEDRDDAYLITHALAK HVO_A0406 hypothetical protein N2 GLFDSGDVNPEAQR HVO_A0472 trxB2 thioredoxin reductase N2 ADVIVIGGGPAGLTTALFAAK HVO_A0503 amidohydrolase N2 TVLDWVDEPR HVO_ B0113 Luciferase like monooxygenase superfam. N2 SDISFEYNVPVFAGAPDEGTEPTHR HVO_B0295 ugpC sn glycerol 3 phosphate transport N2 SDITIQNLR HVO_B0376 oxidoreductase N2 TTLEDIDLDFVPFGQTGLQTSELQFGTWR HVO_C0038 hypothetical prote in N2 TEYDEDSIPSHTLESNGR HVO_0213 Ferritin like domain subfamily N1/N2 MSEDVTALLK / SEDVTALLK HVO_0313 ATP synthase (E/31 kDa) subunit N1/N2 MSLDNVVEDIR / SLDNVVEDIRDEAR HVO_0354 rpsG ribosomal prot ein S7 N1/N2 MSESDAPEPESPASSEEAK / SESDAPEPESPASSEEAK HVO_0359 tuf translation elongation factor EF 1 s... N1/N2 MSDKPHQNLAIIGHVDHGK / SDKPHQNLAIIGHVDHGK HVO_0711 conserved hypothetical protein N1/N2 MGLLDTISS LWK / GLLDTISSLWK HVO_0812 ppsA phosphoenolpyruvate synthase N1/N2 MAVVWLDDVR / AVVWLDDVR HVO_0964 trxC thioredoxin N1/N2 MSDDELTDIR / SDDELTDIRK HVO_1577 imd inosine 5'' monophosphate dehydrogenase N1/N2 MSSIELTSSQKTILTALINLYRDSEDAVK / SSIELTSSQK HVO_1699 aminotransferase classes I and II superf N1/N2 MTATFPGIPYLEWIVDR / TATFPGIPYLEWIVDR HVO_1858 Ribosomal protein S19e N1/N2 MVTIYDVPADALIEEVAGR / VTIYDVPADALIEEVAGR HVO_1869 hypothetical protein N1/N2 MSLIDFIR / SLIDFIR HVO_2361 carB carbamoyl phosphate synthase large N1/N2 MTDEDTSTGPQEGTEDR / TDEDTSTGPQEGTEDR HVO_2464 sucD succinyl CoA synthase alpha subunit N1/N2 MSIFVDDDTRVVVQGITGGEGK / SIFVDDDTR HVO_2547 rpl32e ribosomal protein N1/N2 MSEE ITELEDISGVGPSK / SEEITELEDISGVGPSK HVO_2577 pyrF orotidine 5' phosphate decarboxylase N1/N2 MGFFDDLR / GFFDDLR HVO_2625 guaA GMP synthase C terminal domain N1/N2 MVNVDEFIEDATASIR / VNVDEFIEDATASIR HVO_A0377 hydantoin racemase putative N1/N2 MTEILWVDPV GHDDFSGDIEALLQNAAR / TEILWVDPVGHDDFSGDIEALLQNAAR HVO_A0488 cobO cob(I)alamin adenosyltransferase N1/N2 MTDAPDGTPETGDETTPDSVR / TDAPDGTPETGDETTPDSVR Penultimate residue other HVO_1238 oxidoreductase aldo/keto reductase family N2 EYTTLGDTGMEVSR HVO_1824 RecJ like exonuclease N2 EDELIDSSQLSLPR HVO_B0167 narJ chaperonin like protein N2 DTNTNAETRTDDTNRDTDR HVO_1654 cysK Cysteine synthase N1/N2 MDDSILDAIGSPLVR / DDSILDAIGSPLVR HVO_1443 ABC transporter ATP binding protein N2 ITVENLR HVO_26 21 ppc phosphoenolpyruvate carboxylase N2 WYSYSSRMR

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157 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_A0529 ilvB3 acetolactate synthase large N2 NTSERLVESLERLGVER Group III. Ac1 and intermediates Penultimate residue small ( e.g. Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0133 cctA Ther mosome subunit 1 Ac1 Ac MSQRMQQGQPMIILGEDSQR HVO_0424 rli RNase L inhibitor homolog Ac1 Ac MADDSIAVVDLDR HVO_0481 gap glyceraldehyde 3 phosphate dehydrogenase Ac1 Ac MSEKSYLSAGENVDESDVVR HVO_0827 conserved protein Ac1 Ac MSDTESQLR HVO_1164 HD doma in protein Ac1 Ac MSDTEDTLNGGR HVO_1716 exsB protein Ac1 Ac MTTEPTSDDK HVO_2598 ppk polyphosphate kinase Ac1 Ac MSDDEVRGGDSQGR HVO_2779 rpl18e Chain O Ac1 Ac MSKTNPRLNSLIAELK HVO_2902 gatE glutamyl tRNA(Gln) amidotransferase Ac1 Ac MTEYDYDYEDLGLVAG LEIHQQLDTETK HVO_A0021 unknown Ac1 Ac MANYEVIERVVVDRWGDEEGR HVO_B0053 hypothetical protein Ac1 Ac MACAELEALR HVO_C0043 hypothetical protein Ac1 Ac MTDDGLVMER HVO_0536 Nutrient stress induced DNA binding prot Ac1/N1 Ac MSTQKSVLKEAGSVGDNPVRLDTEK / MSTQK SVLKEAGSVGDNPVR HVO_2336 pyridoxine biosynthesis protein Ac1/N1 Ac MPEETDLEELR / MPEETDLEELR HVO_B0117 unknown Ac1/N1 Ac MGTDQSTNRR / MGTDQSTNR HVO_B0217 livK 2 branched chain amino acid ABC Ac1/N1 Ac MTDQPALTDESRR / MTDQPALTDESR Penultimate resi due NatB like ( e.g ., Asp, Glu, Asn, Met) HVO_1120 conserved hypothetical protein Ac1 Ac MDDDDPRRRPR HVO_2923 psmC Proteasome subunit alpha2 Ac1 Ac MNRNDKQAYDR HVO_B0045 L 2,4 diaminobutyrate decarboxylase Ac1 Ac MNGVGDVDGERR HVO_0572 lpl lipoate p rotein ligase Ac1/N1 Ac MNDSQGSIADREWRVIR / MNDSQGSIADR Penultimate residue other HVO_1967 pgi glucose 6 phosphate isomerase Ac1 Ac MQVDLGNVLDTAPAHGVSR Group IV. Ac1 and N2 isoforms and intermediates Penultimate residue small ( e.g. Gly Ala, Pro, Val, Ser, Thr, Cys) HVO_0291 conserved protein Ac1/N2 MSSNEIPTREVAR / SSNEIPTREVAR HVO_1250 thiol disulfide isomerase/thioredoxin Ac1/N2 Ac MVLLESDSELER / VLLESDSELER

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158 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_1560 Uncharacterized protein conserved Ac1/N2 MSNYLVAMEAAWLVR / SNYLVAMEAAWLVR HVO_2056 spsK spore coat polysaccharide synthesis Ac1/N2 Ac MPNIHDVEVR / PNIHDVEVRDLQVNADQR HVO_2809 sdhB succinate dehydrogenase chain B hom Ac1/N2 Ac MSTQVPETVEEESATEAASAGK / STQVPETVEEESATEAASAGK HVO_0628 dppD dipeptide ABC transpo rter ATP binding Ac1/N1/N2 Ac MSQDGNDVSRR / MSQDGNDVSRR / SQDGNDVSR HVO_0783 ATP dependent protease Lon protease Ac1/N1/N2 Ac MSNDTNTDDSLHER / MSNDTNTDDSLHER / SNDTNTDDSLHER HVO_2959 2 oxoacid decarboxylase E1 beta chain Ac1/N1/N2 Ac MSSQNLTIVQAVR / MSSQNLTIVQAVR / SSQNLTIVQAVR Penultimate residue other HVO_1091 psmA proteasome subunit alpha1 Ac1/N2 Ac MQGQAQQQAYDR / QGQAQQQAYDRGITIFSPDGR Group V. Ac2 and intermediates Penultimate residue small/NatA like ( e.g. Gly, Ala, Pro, Val, Ser, Thr, Cys) HVO_0070 NifU like domain protein Ac2 Ac STETQDGEDDLKER HVO_0116 Ribosomal protein L31e Ac2 Ac SANDFEER HVO_0778 cctA Thermosome subunit 3 Ac2 Ac ASRMQQPLYILAEGTNRTHGR HVO_0862 conserved hypothetical protein Ac2 Ac SVGYQVSSDHQL AR HVO_1804 nadC nicotinate phosphoribosyltransferas Ac2 Ac SDFDIVGPEAIR HVO_2226 trpD anthranilate phosphoribosyltransferase Ac2 Ac AVVESEFGEWPLKR HVO_2348 conserved hypothetical protein TIGR00294 Ac2 Ac SHQLPDVQASQPDVTVGLSQVGVTGVEK HVO_2506 Iso pentenyl diphosphate delta isomerase Ac2 Ac SNAAADEATELHK HVO_2550 rpsN ribosomal protein S14p/S29e Ac2 Ac SDSETEQTGEHASR HVO_2551 rpl5p ribosomal protein L5. Ac2 Ac SEADFHEMR HVO_2562 rplW ribosomal protein L23 Ac2 Ac SIIEHPLVTEKAMNQMDFDNK HVO_A0 520 TG_gha_452 uncharacterized domain 1 putative Ac2 Ac SEQDDYAEIRER HVO_C0074 TG_gha_454 dppF dipeptide ABC transp. ATP binding Ac2 Ac SVVSETHTSQDGR HVO_0316 ATP synthase archaeal A subuni Ac2/N1 Ac SQATQDSVREDGVIASVSGPVVTAR / MSQATQDSVR HVO_0551 mut L 2 DNA mismatch repair protein mutL Ac2/N1 Ac SDIKQLDEK / MSDIKQLDEKTVQR HVO_0979 ndhG nadh dehydrogenase/oxidoreductase Ac2/N1 Ac SSEQKPFVTDDTQVQTETR / MSSEQKPFVTDDTQVQTETR HVO_0729 ppa inorganic pyrophosphatase Ac2/N2 Ac VNLWEDMETGPNAPDEIYAVVECLK / VNLWEDMETGPNAPDEIYAVVECLK HVO_0861 sufB/sufD domain protein Ac2/N2 Ac SAQLPANLSAETVR / SAQLPANLSAETVR HVO_0884 aldehyde reductase Ac2/N2 Ac TENLASADDAPR / TENLASADDAPR HVO_1637 slyD peptidyl prolyl cis trans isomerase Ac2/N1/N2 Ac SDEQQAEAEQVDEEVESG IQDGDFVR / MSDEQQAEAEQVDEEVESGIQDGDFVR / SDEQQAEAEQVDEEVESGIQDGDFVR HVO_2126 oligopeptide ABC transporter oligopeptid Ac2/N1 Ac SDDNITEVNMDR / MSDDNITEVNMDR HVO_2600 map methionine aminopeptidase type II Ac2/N2 Ac SIGPLDDETVEK / SIGPLDDETVEK

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159 Protein Accession Number and Name N terminal Form Peptide Sequence HVO_2620 rieske 2Fe 2S domain protein Ac2/N1 Ac SDSDKYPADSGR / MSDSDKYPADSGR HVO_2757 L1P family of ribosomal proteins Ac2/N1/N2 Ac ADTIVDAVSR / MADTIVDAVSR / ADTIVDAVSR HVO_A0378 hydantoin utilization protein B Ac2/N2 Ac STHDVDPATVEVIR / STHDVDPATVEVIR Pe nultimate residue other HVO_0662 DNA binding protein Ac2 Ac KFIEEIVVDAFLPTFRALLAEDLRDR HVO_1308 aroA 3 phosphoshikimate 1 carboxyvinyltr Ac2 Ac DAHVTPSR HVO_1497 FruB phosphocarrier protein Hpr Ac2 Ac ERTVTVVPEDGLHARPASK HVO_2486 bccA biotin carb oxylase Ac2 Ac FSKVLVANR HVO_C0075 TG_gha_454 oligopeptide/dipeptide ABC transporter Ac2 Ac QYELAMDSDTALDRR HVO_2242 eif probable translation initiation fact Ac2/N1 Ac EYQTALDR / MEYQTALDR HVO_A0571 hydrolase isochorismatase family Ac2/N1 Ac LDATASQAK / MLDATASQAK HVO_B0112 mandelate racemase/muconate lactonizi Ac2/N1 Ac EITDISATK / MEITDISATK Group V. Ac1 and Ac2 isoforms and intermediates Penultimate residue small ( e.g. Gly, Ala, Pro, Val, Ser, Thr, Cys) includes NatA like Ac2 forms HVO_0 136 translation initiation factor eIF 1A Ac1/Ac2 Ac MSDDENESR / Ac SDDENESRR HVO_0860 sufB FeS assembly protein SufB Ac1/Ac2/N1 Ac MSSDQDHLKETDTEAR / Ac SSDQDHLKETDTEAR/MSSDQDHLK HVO_1396 P hydroxybenzoate hydroxylase Ac1/Ac2/N1 Ac MSTDENDTAEDSSASPR / Ac STDENDTAEDSSASPR / MSTDENDTAEDSSASPR HVO_1545 dihydroxyacetone kinase L subunit Ac1/Ac2/N1 Ac MADAETQREAVLDALDNVAER / Ac ADAETQR / ADAETQREAVLDALDNVAER HVO_2784 rpsM ribosomal protein S13p/S18e Ac1/Ac2/N1/N2 Ac MSAEEPQDSSPEEEEDLRYFIR / Ac SAEEPQDSS PEEEEDLR / MSAEEPQDSSPEEEEDLR /SAEEPQDSSPEEEEDLR

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160 CHAPTER 5 PHOSPHORYLATION AND METHYLATION OF SUBUNITS FROM 20S PROTEAS OMES AND P ROTEASOME ACTIVATING NUCLEOTIDASE A IN H ALOFERAX VOLCANII Introduction Phosphorylation is one of the most common post transl ational modifications found in all three domains of life. Several studies have demonstrated that archaeal proteins are phosphorylated; however, relatively little is known regarding the identities of the proteins that are modified or the sites of modification (Kennelly, 2003; E ichler and Adams, 2005; Kirkland et al., 2008a) Furthermore, the impact phosphorylation has on the functional properties of the archaeal proteins that are modified or the kinases/phosphatases that regulate these events are poorly understood. Proteasomes are multicatalytic proteases found in all three domains of life. In many organisms, including eukaryotes and haloarchaea, the catalytic 20S core particle of proteasomes is essential for growth (Lee et al. 1992; Lee et al. 1991; Georgatsou et al. 1992; Fujiwara et al. 1990; Zhou et al. 2008) Although it is known that eukaryotic proteas omes are regulated through interactions with accessory proteins, such as the 19S cap, and the ubiquitintargeting pathway, several subunits of the 20S and 19S complex are also subject to various forms of coand post translational modification including N terminal acetylation, phosphorylation, S glutathionation, N myristoylation, and O linked glycosylation (for details see Table 1 1) Several different kinases have been used to study the in vitro phosphorylation of different 26S proteasome subunits includi ng Polokinases and casein kinase II (Bose et al. 2004; Bose et al. 1999; Castano et al. 1996; Feng et al. 2001; Fernandez et al. 2002; Horiguchi et al. 2005; Iwafune et al. 2004; Mason et al. 1996; Zhang et al. 2007) Additionally, there is evidence that some of the post translational modifications, such as phosphorylation, are regulated by growth

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161 interferon (Bose et al. 2004; Bose et al. 2001; Horiguchi et al. 2005; Rivett et al. 2001; Tokumoto et al. 2000; Wakata et al. 2004) Subunit composition of Hfx. volcanii proteasomes also changes as a function of growth phase. 20S core particles from Hfx. volcanii type subunits and Hfx. volcanii synthesizes different 20S core particles made up of three dif ferent subunits: (Kaczowka and Maupin Furlow, 2003) In addition to the three different subunits of 20S core particles, Hfx. volcanii also synthesizes two different proteasome activating nucleotidases, PanA and PanB. While the transcripts of all five genes in crease as cells transition into stationary phase, the levels of b (Reuter et al. 2004) become more prevalent or proteasomes This study elaborates on the phosphorylation state of different 20S core particle subunits and the proteasome activ ating nucleotidase PanA and connects the phosphorylation states of the Hfx. volcanii The ability of complement hypoosmotic stress was examined as well as wholecell chymotrypsin like peptidase activity of cells expressing variant subunits. A c andidate Hfx. volcanii Rio1 type kinase was purified and used to study the in vitro -32P ATP to different, recombinantly purified proteasome subunits. In addition, a new post translational modification, methyl in vivo isolated samples. Results and Discussion M odulate D uring G rowth phase T ransitions Post transcriptional modification of the predominant proteasomal proteins of Hfx. volcanii to late stationary

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162 phase. 2specific isoforms of pI 3.0 and 4.4, both of whi ch migrated at 37.5 kDa consistent with the mobility of PAGE gels (Wilson et al. 1999) isoform of pI 4.4 was is oform of pI 3.0 disappeared in late stationary phase (Figure 51A ) Affinity purification of 20S proteasomes from early stationaryphase cells expressing a hexahistidine tagged ( His6 isoform pI 3.0 and 4.4) associate d active (Suc LLVY AMC hydrolyzing) 20S proteasomes in Hfx. volcanii ( Figure 51B ). pI 4.4 was also detected in non20S proteasomal fractions. Treatment of these purified 20S proteas omes with phosphatase resulted in a basic shift in pI type subunits were phosphorylated ( Figure 51C ). type proteins of Hfx. volcanii are decorated by phosphorylati on. Based on the IEF migration and phosphatase sensitivity of both pI 4.4) was detected at all stages of growth and occurred in both free and CP assoc pI 3.0) was only detected in association with CPs and was not present in late stationary phase. In addition to Thus, Hfx. volcanii not only modulates the types of subunits present in the CP and P AN subtypes during the transition from logto stationary phase growth (Reuter et al. 2004) but appears also to post translationally modify the proteasomal proteins during this transition (Humbard and Zuobi Hasona, unpublished)

PAGE 163

163 S ubunit is Phosphorylated MS analysis 2+ affinity chromatography of His6 proteins revealed a with an individual MOWSE score of 57 and an Expect value of 2.5e4. It was 13 amino acids in length and spanned residues 119 to 131 (RGEDMSMQALS TL) (numbering includes the 49 amino acid fragment at the N terminus that is cleaved during maturation) and had a monoisotopic mass of 1518.66 Da. It eluted by RP HPLC between 69.69 minutes and 70.27 minutes as a doubly charged ion (760.340, 2+). The peptide contained a deamidation on the eighth residue (glutamine 126) and a phosphorylation on the eleventh residue (serine 129). The MS/MS fragmentation contained primarily band a ions instead of y ( Figure 52 ). The loss of phosphate on a b11 indicates that the phosphorylation site was on serine 129 and not threonine 130. The mass defect between b10 and b11 eliminated form of phosphoserine. There is also a b12 98 ion. The predominance of a b ion series (and corresponding lack of a yion series) can be attributed to the presence of an arginine at the N terminus of a peptide. It has been previously observed that the presenc e of basic amino acids (arginine, lysine, and histidine) can alter the normal fragmentation patterns seen in mass spectrometry (van Dongen et al. 1996) Specifically the presence of an N terminal arginine supports the formation of a ions (and dions) over y ions. To bolster the validity of the spectrum missing y ion series, several aions as well as several ions resulting from internal fragmentation were identified (inset Figure 52). An additional phosphopeptide was detected. That spectrum was unable to differentiate between a phosphate group on S129 and T130. The actual site of phosphorylation was determ ined to be S129 based on the spectrum shown in Figure 52. To validate the spectrum and phosphorylation site, a synthetic peptide (RGEDMSMQALpSTL) was synthesized and analyzed by direct infusion on

PAGE 164

164 the QSTAR. Again, the bion series was favored over the y ion series and the characteristic b11 and b12 neutral loss sequence ions ( 98 Da) were observed ( Figure 53 ). NetPhos (Blom et al. 1999) did not predict S129 or T130 to be phosphorylation sites. S129 had a NetPhos score of 0.020 and T130 had a score of 0.095. NetPhos predictions are based on eukaryotic phosphorylation motifs so perhaps its not surprising that this algorithm did not identify the site. Currently, little is known about the s pecificity or phosphorylation motifs of archaeal kinases. hexahistidine Since this phosphopeptide was isolated in a Ni2+ affinity col umn using a into an active 20S proteasome along with unphosphorylated subunits. Using the crystal structure of the 20S proteasome from Thermoplasma acidophilum S129 was modeled to be at the It is unlikely in that position that it could interact with the This phosphorylation event may have something to do with the assembly of the ring itself. S129 is a partially conserved residue in archaea. H fx volcanii was aligned against the 20 closest homologues in archaea. Out of the 20 closest homologues, 11 have a serine in that position, the remaining 9 have alanine Two of the closest re subunits ( Q18GX3 and B9LTS6 ) from Haloquadratum walsbyi and Halorubrum lacusprofundi have a serine at the corresponding position. If the modification is conserved on that residue, and the predicted location of the phosphorylation site i rings in Haloquadratum walsbyi and

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165 Halorubrum lacusprofundi Broadening the BLAST results shows that S129 is also conserved in eukaryotes. The serine was con all contain proteolytic function. Currently there are no phenotypes associated with various phosphorylation events on 20S proteasome subunits. It has been demonstrated that phosphorylation events on the 19S cap in yeast can promote the assembly of the 20S proteasome and 19S cap into 26S proteasome s (Satoh et al. 2001) PanA is Phosphorylated A phosphopeptide specific to PanA was detected in several ESI Q TOF experiments of tryptic digestions of hexahistidine tagged Pan A chromatography fractions. The PanA phosphopeptide eluted from RP HPLC at 69.05 minutes as a quadrupoly charged ion (m/z 684.8). The peptide has a Mascot score of 70 and an expect (E) value of 1.4e 7. The phosphopeptide is composed of amino acid residues 337 361 ( MNVpSDDVDFVELAEMADNASGADIK). There is only one serine or threoni ne in the peptide, S340 ( Figure 5 4) MS/MS fragmention confirmed that S340 was the phosphorylated amino acid. Despite the large, 25 amino acid, peptide. A high Mascot score and low expect value were obtained due to the assignment of 9 yions and 6 bions in the MS/MS fragmentation. Additional peaks were assigned that increased the confidence of this peptide including a neutral loss peak ( 98 for phosphorylation) and several internal fragmentation ions. The nonphosphorylated peptide was detected in the sam e preparations as well. It is important to note, that the phosphopeptide for S340 in PanA was detected in fractions from both hexahistidine tagged PanA and hexahistidine tagged PanB fractions. This indicates that the phosphorylated PanA is in complex with PanB in Hfx. volcanii cells. Interestingly, PanA and PanB differ in this residue. While PanA has a serine in position 340, the corresponding residue in this position in PanB is an alanine.

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166 S ubunit is Phosphorylated A specific phosphopeptide was de tected by precursor ion scanning of a tryptic digest of 20S proteasome s purified by nickel chromatography of His6. The phosphopeptide eluted from RP HPLC at 37.9 minutes as a doubly charged ion (m/z 558.2). The peptide has a Mascot score of 35 and an e xpect (E) value of 1.4e 3. The peptide is composed of amino acid residues 12 21 (GTSLFSPDGR). Unfortunately, the MS/MS could not distinguish between the T13 and S14 as the phosphosite ( Figure 55 ). The MS/MS fragmentation contains 6 unique y ions and 3 unique bions. The proximity of the phosphosite to the N terminus of the peptide made the assignment more difficult. The precursor ion scan was performed on the Q qQ instrument (QTRAP). It was set to detect the neutral loss of a phosphate group. Severa l of the ion bions on the MS/MS fragmentation show the characteristic neutral loss (98) for a phosphate, but the differientiating ions, b2 or y8 are both missing in the spectrum. Since this phosphopeptide was detected by a precursor ion scan, the unphos phorylated peptide was not dectected in the same run. Subsequent experiments have found the unphosphorylated form of the peptide in chromatography fractions S ubunit is Phosphorylated A specific phosphopeptide was detected by an ESI QTOF run of a tryptic digest of 20S proteasome s purified by nickel affinity chromatography of His6. It was a doubly charged ion (m/z 479.3) with a Mascot score of 30 and an expect (E) value of 0.001. It was composed of amino acid residues 137 149 (ALLIGGVENGpTPR) of T147, is predicted to be phosphorylated in that peptide and the MS/MS annotation confirmed that T147 was the site of phos phorylation ( Figure 56) The MS/MS fragmentation contained 7 yions and 6 out of the 7 showed the characteristic neutral loss ( 98) indicative of a phosphorylation. Several other fragments ions could be assigned to internal sequencing ions of

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167 the peptide. These peak assignments bolster the MS/MS assignment. The peptide, although generated by a tryptic digest of a 20S proteasome preparation, was a semi tryptic fragment, an internal chymotryptic like cut on the N terminus of the peptide. An additional scan u sing an error tolerant algorithm for this phosphorylation site hit on two additional peptides; LIGGVENGpTP + 22 Da and LIGGVENGpT + 155 Da. The Mascot score for these two peptides were 50 and 58 and the E values were 0.0006 and 0.001, respectively. The unphosphorylated form of the peptide was also detected in the same fraction. E (0.851) it was not the most likely residue. There are several other predicted phosphorylation sites that were more likely than Thr147. For example, Ser58 was predicted (0.996) to be phosphorylated as well as Thr158 (0.981). Based on the 2D than one residue. Therefore, s ite directed mutations were made in the psmA gene that resulted in amino acid substitutions Tyr28Phe, Ser58A la, Thr147Ala, and Thr158Ala. C arotenoid Pigmentation and Cell Viability Knockout strains of both psmA panA (PanA) have a dimished capacity to adapt to low salt environments (Zhou et al. 2008) This phenotype can be complemented by providing a copy of the gene in tr ans panA ) expressing either panB panA or panAt1018g (PanA Ser340Ala) were grown in varying concentrations of NaCl ( Figure 57). Interestingly, over expression of panB did not rescue the sensitivity of the panA mutant to low salt conditions but expression of PanA Ser340Ala restored growth to wildtype levels. This indicates that the phosphorylation event on S er340 is not important for hypoosmotic stress response. Substitution mutations in the psmA gene that introduced amino acid substitu tions S er 58A la T hr 147A la T hr 158A la, and Tyr 28Phe were also tested with their ability to complement the hypo osmotic psmA strain along with over expression of psmC

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168 plasmid Over expression of psmC psmA a83t ( ) had no effect on stress response to low salt ; however other substitutions had severly inhibited and highly variable growth (data not shown). While each of the constructs was successfully psmA cells, after being transferred to liquid media or streaked onto a fresh medium the cells began to die (Figure 5 8A). Some of the substitutions appear ed to affect carotenoid biosynthesis as cells expressi ng were whiter than cells expressing wild Figure 58A). Whole cell chymotrypsinlike activity was measured for the different s ubstitution mutants. There were only modest differences in activity between the strains with the fold increase in activity compared to wildtype ( data not shown). It is unclear at this time if differences in activity can account for the phenotypes seen. Both the hypoosmotic stress and pigmentation phenotypes are recessive type Hfx. volcanii cells does not result in the same growth and pigmentation phenotypes (data not shown). Mutations and phenotypes are summarized in Table 5 2. H aloferax volcanii Encodes a Number of Putative Protein Kinases Based on genome sequence, Hfx. volcanii encodes for a number of putative serine/threonine kinases includin g two different Rio type protein kinases, type 1 (Hvo_0135) and a type 2 like kinase (Hvo_0569) (Figure 5 9A ) as well as PrkA1 and PrkA2. Riotype kinases are a relatively new atypical protein kinase family with four unique types, Rio1, Rio2, Rio3, and Ri oB. Rio type kinases are widespread throughout eukaryotes and archaea. Archaea typically have two different Rio type kinases, Rio1 and Rio2 (LaRonde LeBlanc et al. 2005) The Rio2 kinases can vary somewhat in their different domains. Rio1 kinases are characterized by a consensus sequence in the N terminal domain of the protein, STGKEA, and a second region of homology in the C terminal domain ID xxQ. Rio2 kinases are characterized by a helix turn -

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169 helix motif in the N terminal domain followed by the amino acid sequence GxGKES and the C ter minal sequence IDFPQ (LaRonde LeBlanc and Wlodawer, 2005a) The Rio1p kinase from Hfx. volcanii contains the signature domain sequence but the Rio2p does not ( Figure 59A) Rio1p kinase was cloned and purified for kinase studies. Rio type I Kinase from Hfx. volcanii Catalyzes Autophosphorylat ion in vitro To investigate the act ivity of Rio1, the kinase was fused to StrepII tag and purified from recombinant Hfx. volcanii (H26 pJAM2558) using StrepTactin chromatography. Rio1p was electrophoretically pure after passage through the Strep Tactin column ( Figure 59B ) and mass spectr ometry confirmed that only Rio1p could be detected in elution fractions ( Table 53). Rio1p ran as a monomer on a gel filtration column at the molecular weight of 50 kDa ( data not shown) Rio1p can autophosphorylate using either Mg2+ or Mn2+ as the divalent cation ( Figure 59C ). The ideal concentration of cation was between 10 and 50 mM Mg2+ with 2 M NaCl Rio1p chromatography fractions were dialyzed into 20 mM Tris buffer (pH 7.2) with either 2 M KCl or 2 M NaCl. There was no significant difference in autokinase activity ( data not shown). All subsequent kinase reactions were carried out in 2 M NaCl 50 mM MgCl2 20 mM Tris buffer ( pH 7.2) M ultiple bands were visible on the autoradiograph after the phosphorylation reaction. There is a doublet at the ap parent molecular weight of Rio1p (monomer) and there is another band running at approximately twice the mass. The MS results did not identify any contaminating proteins in the fraction. These other bands may represent a mixture of tagged and untagged Rio1p or a transient dimer that forms between two Rio1p proteins. It is also possible that there is a contaminating protein that is not detect ed by SYPRO staining or mass spectrometry.

PAGE 170

170 Rio type I K inase P hosphorylate s in vitro Purified Rio1p was used to pHis6 subunits purified from E. coli 20S core particles purified from Hfx. volcanii and casein (boiled). The Rio1p preparation was able casein (Fi gure 5 10A and variants all purified from E. coli were used in the kinase assay, unequal amounts of phosphorylation took place. Wildwas the most heavily phosphorylated under these conditions B phosphorylated but to a lesser extent than wild autoradiograph) (Figure 510B ). Based on these results, coupled with our e arlier findings by 2DGE, could not be phoshporylated by Rio1p in vitro There may be drastic structural changes in this t s meaningful interaction with the kinase. S ubunit is Methylesterified Five unique methylated peptides were identified in several ESI Q TOF experiments of tryptic digests of 20S proteasome s purified by nickel affinity chromatography His6 ( Figure 511 Figure 515) Each of the methylated peptides are derived peptides are composed of amino acid residues 13 22 ( GITIFSPDmethylGR), 23 30 ( LYQVEmethylYAR) 58 68 ( SPLMEmethylPTSVEK) 105 116 ( YGEPIGIEmethylTLTK) 150 163 ( LYETDPSGTPYEmethylWK) All five peptides eluted from the RP HPLC as doubly charged ions: 538.77 (m/z), 528.23 (m/z), 616.28 (m/z), 667.82 (m/z) and 567.23 (m/z). The Mascot scores for the 5 peptides were 42, 33, 38, 66, and 38, respectively, and the e xpect values were 0.0044, 0.032, 0.017, 2.5e 5, 0.026. The high Mascot scores and corresponding low E values validate the assignment of the peptides and the methylation sites. In addition to high Mascot

PAGE 171

171 scores and low E values, several of the MS/MS fragmen tations showed neutral losses of methyl groups ( 14). Specifically, peptide 23 30 had three neutral loss peaks, 58 68 had one neutral loss peak, and 105 116 had three neutral loss peaks. The MS/MS fragmentation was complete enough in all 5 spectra to definitively assign the location of the methylation. Although each of the meth peptides had high enough Mascot scores and were repeatable enough to report as methylated. Conclusions Phosphorylation. Due to the relatively low number of confirmed phosphorylation sites on archaeal proteins, t he identification of a phosphorylation site s for the proteasomes and PanA is a significant advance in our understanding of arc haeal protein phosphorylation and the proteasome system in Hfx. volcanii The Ser129 residue of the H fx volcanii that is phosphorylated is not highly conserved among archaea. Of 20 type proteins analyzed, a slight majority (11 / 20) had a serine residue at the analogous position. Broadeni ng the BLAST search revealed that the the chymotrypsinlike peptidase activity of the housekeeping 20S proteasomes (Orlowski and Wilk, 2000) interferon induction (Kloetzel, 2004) In this study, the phosphorylated form of the H fx volcanii complex with the His His enriched samples we

PAGE 172

172 (Schwede et al. 2003) Ser129 to crystal structures of analogous 20S proteasomes suggests this residue is located at the ring int Although phosphorylation of archaeal 20S proteasomes has not been previously described, a number of phosphosites of the eukaryotic counterpa rt have been mapped including: of Candida albicans (Fernandez et al. 2002) (Bose et al. 2004) ; 7 Ser243, 7 Ser250 (Castano et al. 1996) 2 Tyr120 of rat (Benedict and Clawson, 1996) (Beausoleil et al. 2004) (Claverol et al. 2002; Beausoleil et al. 2004) (Rush et al. 2005) of human ( Figures 5 1 6 and 517). Several of these phosphorylation sites are conserved i n H fx volcanii type subunits. Based on in vitro phosphorylation data with Rio1p it seems that the residue corresponding to the Ser56 in humans, Ser58 in Hfx. volcanii may very well be phosphorylated. It is also possible that these modifications do occur in H fx volcanii and may account, in part, for the additional proteasomal isoforms detected by 2D SDS PAGE ( Fig ure 3 1 and Figure 5 1A ). In analogy to eukaryotic cells, it is quite likely that the phosphorylation site of the H fx volcanii 20S proteasome subunits detected in this study regulates biological function. A number of studies in eukaryote s suggest that phosphorylation regulates the subcellular distribution and assembly of 26S proteasomes. For example, phosphorylation of regulatory particle Rpt6 subunit 6S

PAGE 173

173 proteasome (Satoh et al. 2001) 26S pr oteasomes than free 20S proteasomes (Mason et al. 1996) Phosphorylation at either one is essential for association with 19S regulatory complexes, and the ability to undergo phosphorylation at both sites gives the most efficient (Rivett et al. 2001) In ad interferon treatment decreases the level of phosphorylation of proteasomes, which coincides with a decrease in the levels of 26S proteasomes and increase in PA28containing proteasomes, suggesting phosphorylation may play role in regulating form ation of proteasomal complexes in animal cells (Bose et al. 2004) Thus, the assembly and disassembly of 26S proteasomes appear regulated by kinase(s) and/or phosphatase(s). Proteasomal phosphorylation also appears to be important in development and subcellul proteasomes is preferentially phosphorylated in immature vs. mature oocytes of goldfish (Horiguchi et al. 2005) important in nuclear localization of proteasomes (Benedict and Clawson, 1996) Perturbation of Hfx. volcanii resulted in drastic change s in cell effects on cellular processes. Future stu dies are needed to elucidate the role of the phosphorylation of the H fx. volcanii proteasomal proteins observed in this study. The identification of several of these modification sites now lays a foundation for these studies. Methylesterification. Altho ugh methylation has not been previously reported as a post translational modification of a 20S core particle subunit in any organism, there are examples of reversible O linked methylation in other bacteria and archaea. The haloarchaea have several example s of O linked modification of taxis proteins in response to external stimuli (Brooun et

PAGE 174

174 al., 1997; Hildebrand and Schimz, 1990; Hou et al. 1998) Haloarchaea may regulate the activity of other systems using this strategy. T Hfx. volcanii is heavily methylated. The lower pI of halophilic proteins is due to the prevalence of aspartic and glutamic acids and the lack of positively charged residues such as arginine and lysine. The acidic nature of proteins in the halophile is a strategy called salting in. The negative charge on the surface of the protein aids in the solubility in high salt solutions by creating a hydration shell around the proteins. Methylation of the aspartic acids co uld be a mechanism of buffering the protein with the solution. Although the presence of the different methylation sites was demonstrated repeatably, the unmodified peptide was almost always present in the same preparation, suggesting that the modified and unmodified proteins existed simultaneously in the cell. O linked methylesterification is a reversible post translational modification and therefore probably regulated There is no clear correlation to growth phase or environmental conditions that incre ase or decrease the frequency of modification. Future work is needed to better understand how and why this modification takes place.

PAGE 175

175 Table 51. Strains and plasmids used in this study Strain or Plasmid Genotype; oligonucleotide used for amplification Source Strain Escherichia coli F recA1 endA1 hsdR17 (r k m k + ) supE44 thi 1 gyrA relA1 Life Technologies GM2163 F ara 14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm 6 hisG4 rfbD1 rpsL 136 dam13::Tn9 xylA5 mtl 1 thi 1 mcrB1 hsdR2 New England Biolabs BL21 (DE3) F ompT [ lon ] hsd S B (r B m B ) (an E. coli B strain) with gene Novagen Haloferax volcanii H26 pyrE2 (Allers et al. 2004) GZ109 H2 panA (Zhou et al. 2008) GZ114 psmC (Zhou et al. 2008) GZ130 psmA (Zhou et al. 2008) GZ138 H26 P tnaA psmB (Zhou et al. 2008) Plasmids pET 24b Km r ; E. coli expression vector Novagen pJAM621 Km r ; pET24b psmB his 6 H is 6 ) expressed in E. coli (Kaczowka and MaupinFurlow, 2003) pJAM622 Km r ; pET24b psmA his 6 1 H is 6 ) expressed in E. coli (Kaczowka and MaupinFurlow, 2003) pJAM623 Km r ; pET24b psmC his 6 2 H is 6 ) expressed in E. coli (Kaczowka and MaupinFurlow, 2003) pJAM2521 Km r ; pET24b psmA t172g his 6 1 S58A H is 6 ) expressed in E. coli This study pJAM2523 Km r ; pET24b psmA a 4 71g his6 T158A His 6 ) expressed in E. coli This study pJAM2554 Km r ; pET24b psmA a 4 39g his6 T147A His 6 ) expressed in E. coli This study pJAM2533 Km r ; pET 24b psmA a83t his6 1 Y28F H is 6 ) expressed in E. coli This study pJAM2529 Km r ; pET24b psmB t385g his6 S129A H is 6 ) expressed in E. coli This study

PAGE 176

176 Strain or Plasmid Genotype; oligonucleotide used for amplification Source pJAM2531 Km r ; pET24b psmB a388g h is6 T130A H is 6 ) expressed in E. coli This study pJAM25 37 Km r ; pET24b psmC a37g his6 2 T13A H is 6 ) expressed in E. coli This study pJAM2525 Km r ; pET24b psmC t40g hi s 6 2 S14A H is 6 ) expressed in E. coli This study pJAM2527 Km r ; pET24b psmC a235g his 6 2 T79A H is 6 ) expressed in E. coli This study pJAM648 Ap r ; Nv r ; pBAP5010 P2 rrn panA (PanA) expressed in Hfx. volcanii (Zhou et al. 2008) pJAM202 Ap r ; Nv r ; pBAP5010 P2 rrn psm B h is 6 ( H is 6 ) expressed in Hfx. volcanii (Kaczowka and MaupinFurlow, 2003) pJAM204 Ap r ; Nv r ; pBAP5010 P2 rrn psmA h is 6 H is 6 ) expressed in Hfx. volcanii (Kaczowka and MaupinFurlow, 2003) pJA M205 Ap r ; Nv r ; pBAP5010 P2 rrn psm C h is 6 2 H is 6 ) expressed in Hfx. volcanii (Kaczowka and MaupinFurlow, 2003) pJAM816 Ap r ; Nv r ; pJAM809 P2 rrn ps mB strepII StrepII) expressed in Hfx. volcanii (Humbard et al. 2009) pJAM2545 Ap r ; Nv r ; pJAM816 psmB strepII psmA his 6 StrepII, His6) expressed in Hfx. volcanii (Humbard et al. 2009) pJAM2522 Ap r ; Nv r ; pBAP5010 P2 rrn psmA t172g his 6 ( 1 S58A His 6 ) expressed in Hfx. volcanii This study pJAM2524 Ap r ; Nv r ; pBAP5010 P2 rrn psmA a 4 71g his 6 ( T158A H is 6 ) expressed in Hfx. volcanii This study pJAM2555 Ap r ; Nv r ; pBAP5010 P2 rrn psmA a 4 39g his 6 ( T147A H is 6 ) expressed in Hfx. v olcanii This study pJAM2534 Ap r ; Nv r ; pBAP5010 P2 rrn psmA a83t his6 ( Y28F H is 6 ) expressed in Hfx. volcanii This study pJAM2530 Ap r ; Nv r ; pBAP5010 P2 rrn psmB t385g his6 ( S129A H is 6 ) expressed in Hfx. volcanii This study pJAM2532 Ap r ; Nv r ; pBAP5010 P2 rrn psmB a388g h is6 ( T130A His 6 ) expressed in Hfx. volcanii This study pJAM2538 Ap r ; Nv r ; pBAP5010 P2 rrn psmC a37g his6 ( 2 T13A His 6 ) expressed in Hfx. volcanii This study pJAM2526 Ap r ; Nv r ; pBAP5010 P2 rrn psmC t40g hi s 6 ( 2 S14A H is 6 ) expressed in Hfx. volcanii This study pJAM2528 Ap r ; Nv r ; pBAP5010 P2 rrn psmC a235g his 6 ( 2 T79A His 6 ) expressed in Hfx. volcanii This study pJAM2553 Ap r ; Nv r ; pBAP5010 P2 rrn panA t1018g ( PanA S340A) expressed in Hfx. volcanii This study pJAM2558 Ap r ; Nv r ; pJAM816 P2 rrn H vo 0135 strepII (Rio1 StrepII) expressed in Hfx. volcanii This study

PAGE 177

177 Strain or Plasmid Genotype; oligonucleotide used for amplification Source pJAM2559 Ap r ; Nv r ; pJAM816 P2 rrn H vo 0 569 strepII (Rio2 StrepII) expressed in Hfx. volcani i This study

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178 Table 5 2. Site directed muta nts and phenotypes. Mutation Substitution Experiment Phenotype psmA a83t Homology None psmA t172g NetPhos Reduced pigmentation psmA a 439g MS / MS Slow growth, reduced pigmentation psmA a471g NetPhos Unstable cells, low growth psmC a37g MS / MS Not examined psmC t40g MS / MS Not examined panA t1018g PanA S340A MS / MS None

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179 Table 5 3. MS/MS results of Rio1pStrep fractions. Protein Residue no. MASCOT E value Sequence Rio1p (HVO_ 0135) 185 192 23 0.033 DYLEGDPR 221 233 40 0.00072 AGVRVPKPIAVQR 193 205 23 0.034 FENIGHDKGQVVR 234 249 17 0.15 NVLVMELVGVVDDRAR

PAGE 180


PAGE 181

181 Figure 51. Ph osphatase type 20S proteasomal proteins as a function of Hfx. volcanii growth. A) pI 4.4 was present at all stages of growth with a more acidic isoform of pI 3.0 present in logand earlystationary phase, but abs ent in late stationary phase. Cell lyase was prepared from various stages of growth as indicated (1 OD600 unit ~ 1 109 CFUml1), separated by 2DE and analyzed by immunoblot using anti B) pI 3.0 and 4.4 are associated in 20S proteasomes. 20S proteasomes were purified by Ni NTA chromatography from early stationary phase cells His6. Proteins that flowed through the Ni NTA column (5 mM imidazole) as well as those that bound the column (500 mM imidazole) were separated by 2 DE and analyzed by immunoblot using anti r e also assayed for peptidase activity using the fluorogenic endopeptidase substrate succinyl Leu Leu Val Tyr 7 amido 4methylcoumarin (Suc LLVY AMC), as p reviously described (Wilson et al. 1999) u.d., undetectable. C) treatment. 20S proteasomes (purified as above) were treated with (red) and without (green) phosphatase, separated by 2DE, and probed by immunoblot with anti anti Data generated by Kheir Zuobi Hasona.

PAGE 182

182 Figure 52. MS/MS Fragmentation of RGEDMSMQALpSTL, an internal phosphopeptide of the generated by a sequential trypsin and chymotrypsin digest 1His. The mass of the parent ion (1518.66 Da) corresponds to the mass of the peptide (1438.63 Da), deamination (1 Da) of glutamine (Q(E)), and phosphorylation (79.99 Da) of serine (pS). The individual Mascot ion score was 62 with an E value of 6.8e 4. A predominant bion series was detected due to the N terminal arginine of the peptide. C haracteristic CID neutral loss of H3PO4 and subsequent formation of dehydroalanine at residue 11 verified the phosphorylation site at Ser129. The b(11) and b(12) H3PO4 are b(11) and b(12) 97.99 Da respectively. Loss of ammonia (*, 17.03 Da) and H2O (, 18.01 Da) are indicated.

PAGE 183

183 Figure 53. MS/MS fragmentati on of the de novo synthesized peptide RGEDMSMQALpSTL Region corresponding to residues 119 The synthetic peptide was analyzed by direct infusion into the QSTAR.

PAGE 184

184 Figure 54. A PanA specific phosphopeptide was reproduci bly detected by ESI QTOF analysis of tryptic digestions of PanA purified from Hfx. volcanii DS70 cells. The phosphopeptide eluted from RP HPLC at 69.05 min as a quadrupoly charged ion (m/z 684.8, 4+) and was composed of amino acid residues 337 to 361 of P anA ( MNVpSDDVDFVELAEMADNASGADIK). MS/MS fragmentation confirmed that Ser340 was the phosphorylated amino acid. Despite the large, 25amino acid peptide, a high Mascot score ( 70) and low expect value ( 1.4e 7) were obtained due to the assignment of 9 yions and 6 bions in the MS/MS fragmentation. Additional peaks were assigned that increased the confidence of this peptide including a neutral loss peak ( 98 for phosphorylation) and several internal fragmentation ions. The nonphosphorylated peptide was det ected in the same preparations.

PAGE 185

185 Figure 55. specific phosphopeptide detected in purified 20S proteasomes by MS/MS. The phosphopeptide, which eluted from RP HPLC at 37.9 min, was detected as a doubly charged ion (m/z 558.2) by ESI Q ToF. The peptide had a Mascot score of 35 and an expect valu e (E) of 1.4e 3. The peptide was composed of amino acid residues 12 between Thr13 and Ser14 as the phosphosite. Although the MS/MS fragmentation contained 6 unique yions and 3 unique bions, the close proximity of the phosphosite to the N terminus of the peptide made the assignment difficult. A precursor ion scan was performed using a QQQ instrument (ABI QTRAP 4000) set to detect the neutral loss of a phosphate group. Several of the ion b ions on the MS/MS fragmentation displayed the characteristic neutral loss (98) for a phosphate, but the differentiating ions, b2 vs. y8 were missing in the spectrum. Since this phosphopeptide was detected by a precursor ion scan, the unphosphorylated peptide was not detected in the same run. However, subsequent experiments have detected the unphosphorylated form of this peptide.

PAGE 186

186 Figure 56. specific phosphopeptide was detected in purified 20S proteasomes by ESI Q ToF with precursor ion scanning. The peptide had a Mascot score of 30 and an expect (E) of 0.001. It was a doubly charged ion (m/z 479.3) mapping to a semi (ALLIGGVENGpTPR). Only one amino acid, Thr147, can be phosphorylated in that peptide, and MS/MS annotation confirmed that Thr147 was the site of phosphorylation. The MS/MS fragmentation contained 7 y ions and 6 out of the 7 showed the characteristic neutral loss ( 98) indicative of a phosphorylation. Several other fragments ions could be assigned to internal sequencing ions of the peptide. These peak assignments bolster the MS/MS assignment. The peptide, although generated by a tryptic digest of a 20S proteasome preparation, was a semi tryptic fragment, an internal chymotryptic like cut on the N terminus of the peptide. An additional scan using an error tolerant algorithm for this phosphorylation site hit on two additional peptides; LIGGVENGpTP + 22 Da and LIGGVENGpT + 155 Da. The Mascot score for these two peptides were 50 and 58 and the E values were 0.0006 and 0.001, respectively. The unphosphorylated form of the peptide was also detected in the same fraction.

PAGE 187

187 0 20 40 60 80 100 120 140 160 1 1.5 2 2.5 Conc. NaCl (molar) % survival 202c PanA PanB S340A Figure 57. HypopanA cells expressing pJAM202c, panA panA t1018g (PanA S340A) and panB at different NaCl concentrations.

PAGE 188

188 Figure 58. Phenotype of psm A ) cells expressing type subuntis from Hfx. volca nii type subunits Hfx. volcanii cells and Hfx. volcanii psmA cells.

PAGE 189

189 Figure 59. Rio kinases in Hfx. volcanii A ) Rio type kinase sequence explanation Conserved domains in bold and underlined. Rio2p kinase does not have a complete C terminal domain but contains three of the five residue. B ) Purification / gel filtration of Rio type kinases. Lane 1 is cell lysate and lane two is a electrophoretical pure Rio1p running at approximately 50 kDa. C) Autoradiographs of Rio in vitro phosphorylation with varying levels of divalent cations Mg2+ and Mn2+. Optimal autophosphorylation happene d in the presence of Mg2+ between 10 50 mM final concentration. The band labeled with the has not been identified.

PAGE 190

190 Figure 510. Strep_II tagged Rio1p purified from Hfx. volcanii His6 subunits purified from E. coli A) Rio1p used for in vitro phosphorylation of Hfx. volcanii (boiled and unboiled) and casein (boiled). B) Rio1p used for in vitro roteins. been boiled.

PAGE 191

191 Figure 511. residues 105 116, YGEPIGIEmethylTLTK Several neutral loss peaks indicating a loss of a methyl group were identified.

PAGE 192

192 Figure 512. residues 58 68, SPLMEmethylPTSVEK

PAGE 193

193 Figure 513. residues 23 30, LY QVEmethylYAR.

PAGE 194

194 Figure 514. residues 13 22, GITIFSPDmethylGR

PAGE 195

195 Figure 515. residues 150 163, LYETDPSGTPYEmethylWK

PAGE 196

196 Figure 516. type 20S proteasome proteins. Phopshorylation sites determined by biochemical studies are boxed and indicated by 7 Ser249 (Claverol et al. 2002; Beausoleil et al. 2004) ; Hs_ 7 Tyr160, Hs_ 2 Tyr23 and Hs_ 2 Tyr97 (Rush et al. 2005) ; Rat_ 2 Tyr120 (Benedict and Clawson, 1996) ; Hs_ 5 Ser56 (Beausoleil et al. 2004) ; Rat_ 7 and monkey Ser243 and Ser250 (Castano et al. 1996; Bose et al. 2004) ; and Cal_ 3 Ser248 (Fernandez et al. 2002) Abbreviations: Hs, Homo sapiens ; Cal, Candida albicans ; Rat, Rattus norvegicus ; Hvo, Haloferax volcanii Swiss Prot or GenBank accession numbers are as follows: Hs_ 1, P60900; Hs_ 2, P25787; Rat_ 2, P17220; Hs_ 3, P25789; Cal_ 3, 46441895; Hs_ 4, O14818; Hs_ 4like, Q8TAA3; Hs_ 5, P28066; Hs_ 6, P25786; Hs_ 7, P25788; Rat_ 7, 203207; Hvo_ 1, Q9V2V6; Hvo_ 2, Q9V2V5. Conserved residues are highlighted in grey and black with amino acid position numbers indicated on the right and left.

PAGE 197

197 Figure 517. Multiple sequence alignment of the central region of select type 20S proteasome proteins. Phosphorylation sites determined by biochemical studies are boxed and indicated by 7 Tyr 102 and Hs_ 2 Tyr154 (Rush et al. 2005) ; Hvo_ Ser129 (This study). Abbreviations: Hs, Homo sapiens ; Hvo, Haloferax volcanii Swiss Prot or GenBank accession numbers are as follows: Hs_ 1, P28072; Hs_ 1i, P28065; Hs_ 2, Q99436; Hs_ 2i, P40306; Hs_ 3, P49720; Hs_ 4, P49721; Hs_ 5, P28074; Hs_ 5i, P28062; Hs_ 6, P20618; Hs_ 7,P28070; Hvo_ Q9V2V4. Conserved residues are highlighted in grey and black with amino acid position numbers indicated on the right and left.

PAGE 198

198 CHAPTER 6 SUMMARY AND CONCLUSI ONS Summary of Findings The goals of this study were t o advance our understanding of how the different proteins of the proteasome system in Haloferax volcanii are modified post translationally and to understand the effect those modifications had on the function of the proteasome and cellular biology of Hfx. v olcanii Much has been learned about proteasome system s in the past five years including advances in proteasome assembly (Hirano et al. 2008; Rosenzweig and Glickman, 2008a; Sharon et al. 2007) structure of regulatory ATPases and cor e particles (Medalia et al. 2009; Dj uranovic et al. 2009) and new targeting mechanisms for degradation by prokaryotic proteasomes (Mukherjee and Orth, 2008) Specifically, t he components of the proteasome system are essential for cell viability under standard laborat ory conditions and are important in hypoosmotic stress response and heat shock in Hfx. volcanii (Zhou et al. 2008) This study demonstrated that all three of the 20S proteasomal proteins of Hfx. volcanii or co translationally. In addition, the proteasome activating nucleotidase PanA is also post contained N terminal acetyl groups. Closer examination of th was a mixture of acetylated methionine (Ac1) and unacetylated glutamine (the second amino acid of the deduced protein sequence, N2) as the N terminal residue. Quantitative mass spectrometry revealed th e 1 (Ac1) was in 100fold excess of unacetylated and cleaved forms (N2) helical gate (residues 2 12) were synthesized i n Hfx. volcanii cells devoid of the chromosomal copy of the psmA

PAGE 199

199 Gln 2Arg Gln 2A la was cl eaved and acetylated on its penultimate residue (alanine) Gln 2S er Gln 2V al were acetylated Gln 2Asp Gln2P ro and 2T hr were a mixture of unmodified, acetylated and/or c leaved forms; however, only forms acetylated on the initiating methionine were observed in 20S core particles. Interestingly, cells Gln 2Asp Gln 2T hr variants were more tolerant of hypoosmotic stress and grew at higher temperatures tha Hfx. volcanii In addition to ace tylation, each of the proteins of 20S core particles and PanA are modified by phosphorylation. To investigate the sites and role of post translational modifications on proteasomal proteins, Haloferax volcanii cell lysate was separated by 2 DE and analyzed by were detected in lag and logphase cells. Further analysis by tandem mass spectrometry (MS/MS) of proteasomal proteins purified from Hfx. volcanii revealed hr T hr 13 or S er 14 and PanA Ser340 phosphosites. To further investigate these and other putative phosphosites, a variety of protein variants were generated by siteer 58A la T hr 158A la Tyr 28Phe hr 147A la er 13A la hr 14A la hr 79A la er 129A la hr 130A la and PanA S er 340A la ability to complement the hypoosmotic sensitive phenotype of psmA panA (PanA) strains. While the PanA S er 340A la Tyr 28Phe variants were able to complement the mutant strains to wild er 58A la hr 147A la hr 158A la variants did not. Whether the failure to complement was caused by an inability to phosphorylate those individual

PAGE 200

200 resi dues is unclear. A RIOtype kinase (Hvo_0135) purified from the native host Hfx. volcanii was able to catalyze autophosphorylation as well as the phosphorylation of wildS er 58A la hr 147A la purified from recombinant E. coli -32P ATP Stoi chiometry of er 58A la hr 147A la (about 40% wild hr 158A la This could indicate that uch that it can no longer be phosphorylated by Rio1p or that the phosphorylation events are sequential and the phosphorylation of Thr158 is the first site of modification. In addition to phosphorylation, MS/MS scans revealed multiple methylation sites on reproducible. The exact role and the enzyme responsible for this latter modification remain to be seen. In addition to specific work done on the proteasome system in Hfx. volcanii advances in the understanding of phosphorylation and acetylation as global processes in the cell have also been made (Kirkland et al. 2008b; Kirkland et al. 2008a) A large subset of the proteome was found to N terminally acetylated. This is evidence of a generalized acetylation pathway in Hfx. volcanii Several putative N terminal acetyltransferases genes were identified in the genome sequence and some of them clustered with known acetyltransferases from yeast All together, the proteins of the proteasomal system in Hfx. volcanii are heavily modified co and / or post translationally. Altering some of these modifications has drastic effects on cell viability and proteasome function. Further study of the mec hanisms and phenotypes of these modifications will give researchers greater insight into proteasome systems in the haloarchaea as well as basic biology of the cell

PAGE 201

201 This work has resulted in the publication of three peer reviewed primary author papers wi th a fourth in preparation as well as three secondary author papers one patent application, and three review articles. Future Directions Future investigation as a continuation of this work will focus on further characterizing the post translational modifi cations by examining the enzymology of the separate modifications. Hfx. volcanii synthesizes several putative N terminal acetyltransferases, but no activity has been assigned to any of them. Biochemical characterization and establishing substrate specifi city as well as identification of in vivo substrates is required to better understand this process. The mechanism of N terminal acetylation is still unknown, whether it is co translational or posttranslational. Great advances have taken place over the p ast few years with respect to the identification of sites of phosphorylation on archaeal proteins. In vivo work with different kinases, analyzing different substrates to assign substrate specificity to different kinases may help to better understand the t argeting pathway proteins take to the proteasome or how the proteasome is specifically regulated. Finally, identifying the environmental or physiological signals that trigger changes in modification, specifically with respect to phosphorylation and methyl ation of proteasomes, is important in understand the role the se modifications are playing in the cell.

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202 APPENDIX A PRIMERS USED IN THIS STUDY Table A 1. Oligonucleotide primers used in this study. Mutation (protein variant) Primer Sequencea psmA (wild ty pe) 5 GCTCTAGA CATATG CAGGGACAAGCG 3 psmA* ( 1 Q2A) 5 GCTCTAGA CATATG GCGGGACAAGCG 3 psmA* ( 1 Q2D) 5 GCTCTAGA CATATG GACGGACAAGCG 3 psmA* ( 1 Q2P) 5 GCTCTAGA CATATG CCGGGACAAGCG 3 psmA* ( 1 Q2S) 5 GCTCTAGA CATATG TCGGGACAAGCG 3 psmA* ( 1 Q2T) 5 GCTC TAGA CATATG ACGGGACAAGCG 3 psmA* 5 GCTCTAGA CATATG GTGGGACAAGCG 3 psmA 12) 5 CGGGATCC CATATG GGGATTACGATCTTCTCGCCGGAT 3 psmA his6 rev 5 CCGAT GCTCAGC TTA GTGGTGGTGGTGGTGCTCTTCGGTCTGTTCGTCGTCCTCGG 3 psmC 5 GCTCTAGA CATATG ACCCGAAA CGACAAGC 3 psmC his6 rev 5 CCGAT GCTGAGCTTA 3 psmB strepII for 5 CTTACCT CATATG CGTACCCCGACTC 3 psmB strepII rev 5 T GGTACC TTCAAGGCCTTCGAAGTTC 3 aNde I, Blp I and Kpn I restriction sites used for cloning are underlined. The start and stop codons of psmA and psmB are highlighted in grey.

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204 T able A 3. Oligonucleotide p rimer pair s for cloning of kinase genes from Hfx. volcanii Gene Number Primer Sequence (5' 3') HVO_0135 (Rio1p) 5 CGTAGCCCTAGT CATATG ACTGACGAG 3 5 GGGTTG GGTACC CTCGTCTCCGTCTCCGTCC 3 HVO_0569 (Rio2p) 5 CGGACGGACG CATATG G TACGGAACGTCGCC 3 5 CGGC GGTACC GACGGCGAACTCGTCGACGCTC 3 HVO_2849 (PrkA1) 5 GGCTCCACGAA CATATG ACCGGCG 3 5 CATGGTCA GGTACC CCCTTCGAGTTCGGTC 3

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205 Table A 4. Oligonucleotide p rimer pair s for N terminal acetyltransferase knockouts. Construct Description Primer Sequence (5' 3') HVO_1954 500 bp 5 CGCCTCTAGAACTTCACGCTC 3 5 CGAGGCCCTCGAGAATCGTG 3 HVO_2709 500 bp 5 CGAGGACGTCTAGATTGAACGGG 3 5 GTGCATCTCGATGGCGATGTCCAC 3 5 TCGACGTGGCGGAGTGG 3 5 CGACTCGCCTGACGACC 3 5 GCTCTCGGAGTTCCTCC 3 5 CTTCGGTATGGGATTCACG 3

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239 BIOGRAPHICAL SKETCH Matthew Adam Humbard was born in February of 1980 in Pittsburg, Kansas. He received a Bachelor of Science in Microbiology from Arizona State University in August of 2003 in Tempe, Arizona. He wo rked as an undergraduate research assistant for Dr. Rajeev Misra on protein protein interaction of the multidrug resistance efflux pump AcrAB TolC of Escherichia coli and toxin colicin E1 import mechanism across the outer membrane of E. coli until he joine d the graduate program at the Department of Microbiology and Cell Science at the University of Florida in August of 2004. He began working with Dr. Julie MaupinFurlow on post translational modification of 20S proteasomes from Haloferax volcanii He marr ied Kristina Marie Palmer on October 14th, 2006 in Gainesville, Florida. He attended several national and international scientific meeting s and was a member of United States delegation to the 57th Meeting of Nobel Laureates in Lindau, Germany in 2007. Matthew Humbard plans on continuing his work in proteolysis at the National Cancer Institute at the National Institutes of Health campus in Bethesda, Maryland.