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GSA14 IS A NOVEL MEMBRANE PROTEIN REQUIRED FOR PEXOPHAGY AND AUTOPHAGY

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GSA14 IS A NOVEL MEMBRANE PROTEIN REQUIRED FOR PEXOPHAGY AND AUTOPHAGY
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2008

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Ethanol ( jstor )
Fluorescence ( jstor )
Jury sequestration ( jstor )
Microscopy ( jstor )
Nitrogen ( jstor )
Peroxisomes ( jstor )
Pichia ( jstor )
Starvation ( jstor )
Vacuoles ( jstor )
Yeasts ( jstor )

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University of Florida
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University of Florida
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Copyright the author. 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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8/8/2003
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GSA14 IS A NOVEL MEMBRANE PROTEIN REQUIRED FOR PEXOPHAGY AND AUTOPHAGY By TINA CHANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by Tina Chang

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In Loving Memory of In Ho Park You are greatly missed by everyone. Although you have passed on, you will be in my heart, always and forever. To weep is to long for my beloved, To speak often of is to remember, To smile is to share in the priceless memories. In my heart, I shall always carry those precious memories, For your life meant the world to me and you taught me so much, You taught me to live, enjoy, cherish, laugh, dream above all To remember OUR time together Author Unknown

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iv ACKNOWLEDGMENTS I would like to thank the following people who helped to make this research possible: my mentor, W. A. Dunn Jr., Ph.D., for his teaching, guidance, time, energy, and continued support; my committee members, J. Aris, Ph.D., and C. West, Ph.D., for their time and expertise; M. J. Thomson for his invaluable source of knowledge and assistance in sequencing and molecular cloning; and last, but not least, my colleagues, family, and friends for their love and support.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS..................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES..........................................................................................................viii ABSTRACT....................................................................................................................... .x CHAPTER 1 INTRODUCTION...........................................................................................................1 The Vacuole and Its Characteristics................................................................................2 Proteinases A and B.................................................................................................3 Vacuolar Protein Sorting ( VPS ) Genes....................................................................3 The Peroxisome and Its Characteristics..........................................................................4 Alcohol Oxidase.......................................................................................................4 PEX Genes................................................................................................................6 Autophagy...................................................................................................................... .6 Mammalian Autophagy............................................................................................6 Yeast Autophagy......................................................................................................9 Pexophagy..............................................................................................................10 Signaling event................................................................................................11 Early sequestration event................................................................................11 Intermediate sequestration event.....................................................................14 Late sequestration event..................................................................................15 Degradation event...........................................................................................15 Pichia pastoris as a Study Model For Autophagy........................................................15 Human Diseases............................................................................................................16 2 MATERIALS AND METHODS..................................................................................18 Yeast and Bacterial Strains and Media.........................................................................18 Isolation of Glucose-Induced Selective Autophagy-Deficient ( gsa ) Mutants by Restriction Enzyme-Mediated Integration (REMI) Mutagenesis.................................20 Measurements of Peroxisome and Protein Degradation...............................................21 Electron Microscopy.....................................................................................................22 Molecular Biology.........................................................................................................23 Construction of Gsa14 Expression Vectors............................................................23

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vi Plasmid Isolation and DNA Sequencing................................................................26 Yeast Transformation....................................................................................................27 Western Blot Analysis...................................................................................................27 Fluorescence Microscopy..............................................................................................28 3 RESULTS..................................................................................................................... .29 Gsa14 Is Required For Both MicroAnd Macroautophagy of Peroxisomes in Pichia pastoris ..........................................................................................................................29 Degradation of Alcohol Oxidase Induced by Glucose and Ethanol Adaptation....30 Protein Degradation Induced by Nitrogen Starvation............................................30 Morphological Studies of gsa14 Mutant................................................................33 GSA14 Encodes a Unique 102-kDa Protein.................................................................36 Cellular Localization of Gsa14.....................................................................................39 Recombinant GFP-GSA14 Expression in gsa14 and DMM1 Cells.......................39 Gsa14 Complementation of gsa14 mutants with GFP-Gsa14 Constructs.............44 GSA14 Localization......................................................................................................46 Gsa14 Localization in DMM1 Cells.......................................................................46 Localization of GFP-Gsa14 in gsa Mutants...........................................................50 Co-localization of GFP-Gsa14 and BFP-Gsa11...........................................................58 4 CONCLUSIONS...........................................................................................................61 REFERENCES..................................................................................................................65 BIOGRAPHICAL SKETCH............................................................................................70

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vii LIST OF TABLES Table page 1-1. Autophagy genes.......................................................................................................12 2-1. Pichia pastoris strains...............................................................................................19 2-2. Primer sequences......................................................................................................26 3-1. Vacuole morphology during micropexophagy.........................................................34 3-2. Predicted number of transmembrane domains by protein databases........................38 3-3. Quantitation of the localization of GFP-Gsa14........................................................49

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viii LIST OF FIGURES Figure page 1-1. Methanol utilization pathway in methylotrophic yeast...............................................5 1-2. Autophagy pathway....................................................................................................8 2-1. Restriction Enzyme-Mediated Integration................................................................20 2-2. Structures of GSA14 expression vectors..................................................................24 2-3. Structures of GSA14 and GSA11 expression vectors...............................................25 3-1. Glucose and ethanol adaptation in wildtype GS115 and mutant strains gsa1 , gsa7 , gsa9 , gsa10 , gsa12 , gsa14 , gsa18 , gsa19 , gsa20 , and pep4 / prb1 .........................31 3-2. Protein degradation during nitrogen starvation in wildtype GS115 and mutant strains gsa1 , gsa7 , gsa9 , gsa11 , gsa12 , gsa14 , and pep4 / prb1 ........................................32 3-3. Morphology of gsa14 and GS115 during glucose and ethanol adaptation...............35 3-4. Amino acid alignment of Gsa14 and Apg9...............................................................37 3-5. GFP-Gsa14 expression in g14 mutant and DMM1..................................................40 3-6. Western blot analysis of TC10 ( gsa14 his4 ::pTC1 (PGAPDH GFP-GSA14, HIS4)), TC14 ( gsa14 his4 ::pTC2 (PGSA14 GSA14-GFP, HIS4)), and TC16 ( gsa14 his4 ::pTC3 (PGAPDH GSA14-GFP, HIS4))............................................................42 3-7. Western blot analysis of TC10 ( gsa14 his4 ::pTC1 (PGAPDH GFP-GSA14, HIS4)) and TC14 ( gsa14 his4 ::pTC2 (PGSA14 GSA14-GFP, HIS4))........................................43 3-8. Complementation of gsa14 mutants with GFP-Gsa14 constructs............................45 3-9. GFP-Gsa14 expression in DMM1 cells grown in methanol medium.......................47 3-10. Glucose adaptation in TC3 cells..............................................................................48 3-11. Nitrogen Starvation in TC3 cells.............................................................................51 3-12. Western blot analysis of pTC1 expression in gsa7 , gsa7 , gsa9 , gsa10 , gsa11 , gsa12 , gsa14 mutants and DMM1 cells...............................................................52

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ix 3-13. GFP-Gsa14 localizes to an area near the vacuole membrane in gsa7 , gsa9 , gsa11 mutants during growth in methanol medium........................................................54 3-14. GFP-Gsa14 localizes to an area near the vacuole membrane in gsa12 , gsa14 , and SMD1163 ( pep4 / prb1 ) mutants during growth in methanol medium...................55 3-15. GFP-Gsa14 localization in gsa7 , gsa9 , and gsa11 cells during glucose adaptation Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…Â…...56 3-16. GFP-Gsa14 localization in gsa11 , gsa12 , gsa14 , pep4/prb1 mutants during glucose adaptation..............................................................................................................57 3-17. Co-localization of BFP-Gsa11 and GFP-Gsa14 under methanol conditions...........59 3-18. Co-localization of BFP-Gsa11 and GFP-Gsa14 during glucose adaptation............60 4-1. Proposed conserved domains present in Gsa14........................................................61 4-2. Model for the insertion of Gsa14 into the sequestering membrane of the vacuole..63

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x Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science GSA14 IS A NOVEL MEMBRANE PROTEIN REQUIRED FOR AUTOPHAGY AND PEXOPHAGY By Tina Chang August 2002 Chair: William A. Dunn Jr., Ph.D. Major Department: Anatomy and Cell Biology Cells adapt to environmental and nutritional changes by synthesizing and degrading various cellular proteins and organelles. The degradation of proteins via autophagy pathway has attracted much attention because the mechanism of protein turnover still remains unclear. Our lab has utilized the methylotrophic yeast Pichia pastoris as a study model for the autophagy of peroxisomes during glucose adaptation, ethanol adaptation, and nitrogen starvation. Many g lucose-induced s elective a utophagy-deficient ( gsa ) mutants have been isolated and characterized. This study characterizes Gsa14, which is a novel membrane protein required for pexophagy and autophagy. My results indicate that Gsa14 is required for an early event in microand macropexophagy as well as nonselective autophagy induced by nitrogen starvation. Under methanol conditions, this protein localizes to vesicles that are adjacent to the vacuolar membrane. However, during glucose-induced autophagy of the peroxisomes, these vesicles form patches at the

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xi vacuole surface. The forming of these patches requires the interaction of another protein, Gsa9. The study of micropexophagy in P. pastoris has enabled us to better understand protein degradation by autophagy pathway. The study that I have done will help provide further understanding and knowledge of the molecular mechanism of microautophagy in eukaryotic cells.

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1 CHAPTER 1 INTRODUCTION Cellular activities are regulated and maintained as a well-controlled balance between protein synthesis and degradation of various proteins. Protein lifetimes usually last from a few minutes to more than ten days and most long-lived proteins are believed to be degraded in a lytic compartment, the lysosome or vacuole (Ohsumi, 2001). The degradation pathway to this compartment has attracted much attention. Autophagy has been described to be the major pathway used to transport endogenous proteins and organelles for degradation (Klionsky and Emr, 2000). Methylotrophic yeasts such as Hansenula polymorpha and Pichia pastoris are able to utilize methanol as the sole carbon and energy source for their growth. They metabolize methanol by enzymes in the peroxisomes and cytoplasm whose synthesis is induced under methanol-growth conditions (Hill et al., 1985). These enzymes, as well as the whole peroxisome, are rapidly degraded when the carbon source is switched to glucose or ethanol. The study of the degradation processes of peroxisomes has been important because the inability to break down proteins in the peroxisomes poses serious health consequences in humans (Moser and Moser, 1996). Genetic screens for peroxisome degradation mutants in yeast have been successfully utilized to identify many genes required for degradative events (Sakai et al., 1998; Tuttle and Dunn, 1995). Furthermore several genes cloned from yeast have been found to have structural and functional homologues in various invertebrates and vertebrates including humans (Tuttle and Dunn, 1995). These findings

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2 show that the yeast model is beneficial and significant for our studies on peroxisome degradation. In the following sections, I will discuss the existing knowledge related to autophagy and peroxisome degradation. I will also discuss the methods available to study the mechanisms of autophagy in yeast peroxisomes and the previous work that has been done in our lab. The Vacuole and Its Characteristics The vacuole plays an important role in pHand ion-balance and functions as a storage compartment for ions (Jones et al., 1997). In yeast, the vacuole is a major degradative organelle and the counterpart of the eukaryotic lysosome. It contains various degradative enzymes such as endoand exoproteinases, lipases, nucleases, and glycosidases that are able to degrade any subcellular constituents (Klionsky and Emr, 2000; Thumm, 2000). About 85% of the total intracellular protein breakdown occurs in the vacuole for removal of cellular components or supply of amino acids upon nutrient deprivation (Teichert et al., 1989). To better understand the role of the vacuole and its function, it is important to also understand how it is synthesized. Proteins involved in vacuolar biogenesis in yeast have been identified by genetic screens (Bryant and Stevens, 1998). These proteins get to the vacuole as proenzymes by various pathways including sorting of vacuolar proteins from the Golgi apparatus and are synthesized on the endoplasmic reticulum and cytoplasm-tovacuole (Cvt) pathways which are synthesized on polyribosomes in the cytosol. Each route is unique from one another, yet there are significant overlaps of the genetic requirements within one another (Bryant and Stevens, 1998). For example in S. cerevisiae, Cvt7 has been shown to overlap with Apg9 and Aut9.

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3 Proteinases A and B The vacuole contains proteinases A and B which are required for activating the proenzymes by proteolytic cleavage or the propeptide once they have entered into the vacuole (Betz and Weiser, 1976). Synthesis of proteinases A and B is increased 2-3 fold during carbon or nitrogen starvation or when cell growth enters into stationary phase (Betz and Weiser, 1976). Proteinase A, also known as PEP4 in yeast, is required to initiate a vacuolar protease activation cascade by triggering the maturation event of proteinase B to yield the active enzyme (Teichert et al., 1989). The activation of proteinase A and B in turn activates other hydrolytic proteinases. Mutation of proteinase A results in the accumulation of inactive proenzymes, deficient activity of several hydrolases including PrB, and the inability to degrade long-lived proteins (Rendueles and Wolf, 1988; Teichert et al., 1989; Zubenko and Jones, 1981). The proteinase B precursor in S. cerevisiae is encoded by the PRB1 gene (Teichert et al., 1989). PrB mutants have also shown to be defective in protein degradation; however there is very little difference in protein degradation rates between pep4 and prb1 mutants (Teichert et al., 1989). Vacuolar Protein Sorting ( VPS ) Genes Vacuolar protein sorting ( VPS ) genes have also been isolated in yeast. These genes are important in regulating the transport of proenzymes from the rough endoplasmic reticulum to the vacuole. They are also required for cell growth and sporulation, and some are also required for endocytosis (Raymond et al., 1992; Stack et al., 1995). Some Vps proteins such as Vps18, Vps11, and Vps16 have been shown to physically interact with one another and to be required for starvation-induced autophagy for protein transport to the vacuole (Rieder and Emr, 1997). Isolating and analyzing the vacuole mediated Vps proteins enable us to further understand its role in autophagy in yeast.

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4 The Peroxisome and Its Characteristics The peroxisomes are essential organelles present in all eukaryotic cells. They vary from crucial roles in the -oxidation of very long chain fatty acids to biosynthesis of cholesterol in man. They also play major roles in the mobilization of storage oil and photorespiration in plants, penicillin production in filamentous fungi and function in the primary metabolism of various carbon and/or nitrogen sources in yeast (Veenhuis et al., 1983). Alcohol Oxidase When P. pastoris is grown in methanol-containing media, the number and size of the peroxisomes increase dramatically, sometimes accounting for 80% of the total cytoplasmic volume (Veenhuis et al., 1978). These organelles contain alcohol oxidase (AOX) and other enzymes involved in methanol metabolism. Methanol is first metabolized by AOX in the yeast methanol utilization pathway (Waterham et al., 1997). AOX is a homooctomeric protein made up of eight identical subunits of ~74kD each, which contain a flavin adenine dinucleotide molecule (FAD) as a prosthetic group (vander-Klei et al., 1991). Methanol is oxidized by using oxygen as its electron acceptor and produces hydrogen peroxide (H2O2) and formaldehyde (CH2O) as a result of this chemical reaction (Gleeson and Sudbery, 1988). The peroxisomal catalase reduces H2O2 to H2O and O2. Formaldehyde finally leaves the peroxisome and is catalyzed in the cytosol by formaldehyde dehydrogenase and formate dehydrogenase (FDH) to CO2 and

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5 CYTOSOL PEROXISOMECH3OH CH3OH HCHOO2 H2O2 ½ O2+H2O HCHO GAP DHA DHA DHAP ADP HCOOH NADH NAD CO2 NAD NADH ATP Xu5P F6P F3P P1 3 5 2 4 8 6 7 CYTOSOL PEROXISOMECH3OH CH3OH HCHOO2 H2O2 ½ O2+H2O HCHO GAP DHA DHA DHAP ADP HCOOH NADH NAD CO2 NAD NADH ATP Xu5P F6P F3P P1 3 5 2 4 8 6 7 Figure 1-1. Methanol utilization pathway in methylotrophic yeast. When methanol enters the cell, it is first metabolized in the peroxisome by using oxygen as its electron acceptor to produce H 2 O 2 and CH 2 O. Peroxisomal catalase reduces H 2 O 2 to H 2 O and O 2 . Formaldehyde is further degraded in the cytosol by formaldehyde dehydrogenase to form CO 2 and energy in the form of NADH. Formaldehyde enters the xylulose 5-phophase cycle for the biosynthesis of amino acids. 1 = Alcohol oxidase; 2 = catalase; 3 = formaldehyde dehydrogenase; 4 = formate dehydrogenase; 5 = dihydroxyacetone synthase; 6 = dihydroxyacetone kinase; 7 = fructose 1, 6bisphosphate aldolase; 8 = fructose 1, 6-bisphosphate phosphatase.

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6 H2O, providing an energy source in the form of 2-NADH, and formaldehyde enters the xylulose 5-phosphate cycle for the biosynthesis of amino acids. PEX Genes Within the past decade, much research has been made on the biogenesis and degradation of peroxisomes resulting from studies of yeast mutants that are defective in these functions. Twenty-three genes (known as PEX genes) have been identified to be required for peroxisome biogenesis (Veenhuis et al., 2000). The PEX gene products – called peroxins – are believed to play a role in the import of matrix and membrane proteins or in the proliferation of peroxisomes (Veenhuis et al., 2000). PTS1 and PTS2 are two peroxisomal targeting signals that function independently and are required for proper sorting of protein to the peroxisome (Veenhuis et al., 2000). PEX5 and PEX7 encode the PTS1 and PTS2 receptors, respectively (Veenhuis et al., 2000). Pex5p is mostly cytosolic with a smaller portion associated with peroxisomes (de Walque et al., 1999; Dodt et al., 1995; Elgersma et al., 1996; Gould et al., 1996; van der Klei et al., 1995). The protein binds to PTS1 signals and is required for the import of peroxisomal proteins in the PTS1 class of proteins (Purdue and Lazarow, 2001). Pex7p is a cytosolic protein (Elgersma et al., 1998; Rehling et al., 1996; Zhang and Lazarow, 1995) that acts directly with the import of PTS2 class of proteins (Purdue and Lazarow, 2001). Cells that are deficient in Pex5p and Pex7p have shown to be completely impaired or defective in protein import to the peroxisomes. Autophagy Mammalian Autophagy In mammalian cells, selective autophagy is chaperone-mediated by a receptor at the lysosomal membrane identified as LAMP-2 protein (Cuervo and Dice, 1996). Specific

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7 cytosolic proteins are transported directly through the lysosomal membrane into the lysosomal matrix where they are eventually degraded (Cuervo and Dice, 1998; Dice, 2000). Mutations of the LAMP-2 protein have been shown to cause a LAMP-2 deficiency in patients with Danon disease (discussed later) (Saftig et al., 2001). Nonselective autophagy has not been shown to exist in mammalian cells. At a morphological level, autophagy can be differentiated into two modes: macroautophagy and microautophagy. Both pathways are multi-step processes that control protein degradation events (Fig. 1-2). The autophagic degradation can be specific by degrading organelles (Veenhuis et al., 2000) or a nonspecific breakdown of bulk cytoplasm, including proteins, nucleic acids, and lipids (Klionsky and Emr, 2000). The role of microautophagy in mammalian cells has not been clearly established, and will accordingly not be discussed in this paper. Macroautophagy is the main pathway for sequestration of cellular components to the autophagosomes (Dunn, 1990a). It involves portions of cytoplasm to be sequestered within double-membraned vesicles known as autophagosomes (Dunn, 1990a; Dunn, 1990b). The outer membrane then fuses with the vacuolar membrane and the innermembrane and its contents are degraded by the acid hydrolases (Dunn, 1990b). The origin of the autophagosome membrane is still unknown in yeast but is likely formed from parts of the endoplasmic reticulum in mammalian cells (Dunn, 1990a; Dunn, 1990b).

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8 Figure 1-2: Pathways of Autophagy. Our data suggest the selective degradation of peroxisomes by microautophagy proceeds through five morphologically and genetically defined events. We now propose a similar sequence of events for starvation-induced macroautophagy. These events include: 1) Signaling events; 2) Early sequestration events that include peroxisome recognition and the association of the “preautophagosome” (pAP) with the vacuole or association of the “pre-autophagosome” with the sequestering membranes; 3) Late sequestration events and transport of proteins (e.g., Gsa14) from the “pre-autophagosome” to the vacuole membrane or the sequestering membranes; 4) Homotypic fusion events which results in the formation of a microautophagic body or a autophagosome that then fuses with a lysosome forming a macroautophagic body; and 5) Degradation events within the vacuole or autolysosome. Gsa11 Gsa14 Gsa10 Gsa12 Gsa18 Gsa19 Vacuole Gsa1 Gsa9 Gsa21 Gsa14 Gsa11 Peroxisomes Gsa7 Gsa15 Prb1 2. Early Sequestration 3. IntermediateSequestration 4. Late Sequestraton/ Homotypic Fusion 5. Degradation 1. Signaling Microautophagic Body pAP pAP pAP pAP AP Macroautophagic Body Autolysosome Lysosome Gsa17 Docking and Fusion Rough Endoplasmic Reticulum or Sequestering Membranes AutophagosomeMicroautophagyMacroautophagy Gsa11 Gsa14 Gsa10 Gsa12 Gsa18 Gsa19 Vacuole Gsa1 Gsa9 Gsa21 Gsa14 Gsa11 Peroxisomes Gsa7 Gsa15 Prb1 2. Early Sequestration 3. IntermediateSequestration 4. Late Sequestraton/ Homotypic Fusion 5. Degradation 1. Signaling Microautophagic Body pAP pAP pAP pAP AP Macroautophagic Body Autolysosome Lysosome Gsa17 Docking and Fusion Rough Endoplasmic Reticulum or Sequestering Membranes AutophagosomeMicroautophagyMacroautophagy

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9 Yeast Autophagy A variety of autophagy pathways have been also been discovered in yeast. As seen in mammalian cells, proteins and organelles are also transported to the vacuole by selective and nonselective pathways. In S. cerevisiae , the cytoplasm-to-vacuole targeting (Cvt) pathway is a selective transport pathway which overlaps autophagy (Klionsky and Emr, 2000). It transports cytosolic aminopeptidase I (API) to the vacuole (Klionsky and Emr, 2000). API is synthesized as a precursor in the cytosol and then oligomerized into a homododecamers which then associates into a higher-order protein assembly called a Cvt complex (Kim and Klionsky, 2000; Teter and Klionsky, 2000). After formation of this complex, the vesicle fuses with the vacuole and then an intravacuolar autophagic or Cvt body is released into the lumen (Kim and Klionsky, 2000). Subsequent breakdown allows recycling of cellular components and maturation of the API precursor (Kim and Klionsky, 2000). The APG pathway -induced by nitrogen starvation -is an example of nonselective autophagy in S. cerevisiae . Autophagic bodies are nonselectively sequestered from the cytoplasmic components of the double-membraned autophagosomes in response to nitrogen starvation (Baba et al., 1994; Egner et al., 1993). These autophagic bodies are accumulated in the vacuole and degraded by proteinases (Baba et al., 1994; Takeshige et al., 1992). Cells that are defective in genes required for this pathway do not accumulate autophagic bodies during nitrogen starvation and eventually die (Tsukada and Ohsumi, 1993). In P. pastoris , selective autophagy is induced by either glucose or ethanol adaptation. Transport to the vacuole by autophagy is the primary mode for degradation of cytoplasmic constituents. The selective degradation of peroxisomes referred to as

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10 pexophagy has been shown to occur only in yeast. As with general autophagy, there are two types of pexophagy known as macropexophagy and micropexophagy. Macropexophagy is a selective process induced by ethanol adaptation in which peroxisomes are first sequestered into autophagosomes, which then fused with the vacuoles. Micropexophagy is a selective process induced by glucose adaptation. It has been thought to occur by a mechanism in which the vacuolar membrane invaginates, resulting in protrusions that surround and engulf a cluster of peroxisomes (Sakai et al., 1998; Tuttle et al., 1993). Both pathways of peroxisome degradation seem to be specific because other organelles like the mitochondria are not degraded (Veenhuis et al., 2000). Non-selective autophagy is a microor macroautophagy process induced by nitrogen starvation. Pexophagy Pexophagy can be viewed as a multistage process that includes the following events: sequestration signaling, early sequestration, intermediate sequestration, late sequestration, and degradation. A number of mutants defective in micropexophagy have been isolated. Currently, 15 g lucose-induced s elective a utophagy (GSA) genes have been identified (Table 1-1). The functional roles of the proteins required for autophagy in P. pastoris have been determined by examining the movements of the vacuole by fluorescence and electron microscopy as it sequesters the peroxisomes during glucose adaptation and by the cellular location of GFP-GSA constructs. In the following sections, I will describe the putative roles of the Gsa proteins that have been identified and characterized to date for each event.

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11 Signaling event Previous work from our lab has shown that GSA1 encodes the alpha subunit of the phosphofructokinase enzyme complex. This protein is required for the delivery of peroxisomes to the vacuole during glucose adaptation but not ethanol adaptation nor nitrogen starvation (Yuan et al., 1997). The finger-like extensions from the vacuole were absent in gsa1 mutants, suggesting a defect at the onset of micropexophagy (Yuan et al., 1997). The data suggest GSA1 is required for an early signaling event of vacuole sequestration of the peroxisomes. Early sequestration event Recent data show that PpVps is another protein required at an early stage in selective peroxisome degradation as well as activating Vps34 (phosphatidylinositaol 3kinase) for vacuolar protein sorting (Stasyk et al., 1999). PpVps15 is structurally and functionally homologous to Vps15 in S. cerevisiae (Stasyk et al., 1999). The protein localizes peripherally to the cytoplasmic face of a Golgi or vesicle compartment (Herman et al., 1991a; Herman et al., 1991b; Kohrer and Emr, 1993). P. pastoris vps15 mutants grown in methanol-containing media sustained high levels of peroxisomal alcohol oxidase after addition of glucose or ethanol (Stasyk et al., 1999). Vacuole morphology by electron microscopic studies revealed the vacuole arms starting to surround theperoxisomes but they are not completely taken up by the vacuoles, suggesting that PpVps15 is required at an early stage in pexophagy (Stasyk et al., 1999).

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12 Table 1-1. Autophagy Genes Pichia pastoris Saccaromyces cerevisiae GSA1 PAZ2 CVT3 APG4 Comments Mammalian Homologues 1 Phosphofructokinase (PFK1) PFKP, PFKL, PFKM 7 12 2 7 E1-like enzyme responsible for the conjugation of Apg12 to Apg5 and Apg8 to phosphatidylethanolamine HsGSA7 9 6 9 Coiled-coil protein found at the vacuolar surface 10 1 10 1 Serine/threonine protein kinase that complexes with Apg13, Apg17, Cvt9, and Vac8 ULK1 11 7 2 Soluble protein associated with a “preautophagosomal” compartment AB007864 12 18 WD40 protein associated with the vacuolar surface AL080155 AF151808 13 Putative transmembrane protein FLJ10847 14 9 7 9 Transmembrane protein associated with a “pre-autophagosomal” compartment AK025822 15 14 Endopeptidase (Proteinase A, Pep4) 16 Transmembrane protein: P-type ATPase II BAA77246 17 Zinc-finger protein AK001901 18 WD40 protein AAH00464 19 PpVps15 13 Serine/threonine protein kinase anchored to a membrane via myristoylation and complexes with Vps34, Apg6 and Apg14 p150

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13 Table 1-1 Continued 20 3 E2-like enzyme responsible for the conjugation of Apg8 to phosphatidylethanolamine T46276 6 Vps30 Soluble protein associated with Apg14, Vps15 and Vps34. Beclin 2 5 8 Soluble protein conjugated to phosphatidylethanolamine at the surface of the autophagosome MAP-LC3 GATE-16 21 PpVac8 Armadillo-repeat protein (ScVac8) anchored to the vacuole surface via myristoylation and palmitoylation BAB71463.1 NP_115526 Gsa12 protein is required for an early sequestration event of pexophagy in P. pastoris (Guan et al., 2001). The ultrastructural morphology of gsa12 mutants revealed the vacuole remaining round with no apparent finger-like extensions surrounding the peroxisomes hence stalling micropexophagy at an early stage (Guan et al., 2001). GFPconstructs of GSA12 show localization of the protein at the vacuole surface. The gsa12 mutants show no initiation of vacuole sequestration or peroxisome labeling by Gsa9 further verifying that Gsa12 functions upstream of Gsa9 and downstream of Gsa11 and Gsa7 in the pexophagy pathway, as described later. Ethanol-induced degradation of AOX and starvation-induced protein degradation in gsa12 mutants was suppressed providing evidence that Gsa12 is also required for macropexophagy and nonselective autophagy (Guan et al., 2001). GSA9 has also been identified and shown to be required for glucose-mediated pexophagy but not nonselective starvation-induced macroautophagy in P. pastoris (Kim et al., 2001). Previous electron microscopy data suggested that Gsa9 may be required 1 Genes of P. pastoris required for glucose-induced selective autophagy of peroxisomes (micropexophagy). 2 Genes of P. pastoris required for peroxisome autophagy (micropexophagy). 3 Genes of S. cerevisiae required for cytosolic to vacuole sorting of aminopeptidase I (selective autophagy). 4 Genes of S. cerevisiae required for starvation-induced autophagy (nonselective autophagy).

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14 during a middle/late stage of micropexophagy because the vacuolar finger-like projections only partially surrounded the peroxisome and unable to complete the engulfment process (Kim et al., 2001). Sequence and biochemical analysis revealed that GSA9 is a structural and functional homologue of CVT9 in S. cerevisiae (Kim et al., 2001). Recent data also revealed that Gsa9 localization does not require Gsa11. This protein appears to be concentrated in a unique compartment at the vacuole membrane suggesting that this protein may mediate the vacuole and peroxisome interactions during microautophagy (Kim et al., 2001). Intermediate sequestration event GSA11 and GSA10 are likely required for the intermediate sequestration events of autophagy of the peroxisomes during glucose adaptation. Biochemical data revealed GSA11 is required for glucoseand ethanol-induced pexophagy as well as nonselective autophagy in P. pastoris because the degradation of AOX was severely impaired in these pathways (Stromhaug et al., 2001). In gsa11 mutants, the vacuole formed a “cup-like” structure adjacent to the peroxisomes, partially surrounding them suggesting that Gsa11 is likely required for an intermediate event in macropexophagy (Stromhaug et al., 2001). This protein has no affect on the localization of Gsa9. GFP/HA-Gsa11 was distributed predominantly throughout the cytosol during normal methanol growth conditions. However during glucose adaptation, GFP/HA-Gsa11 localized to one or more cytoplasmic structures that were close to the vacuole (Stromhaug et al., 2001). The localization of Gsa11 to this compartment required Gsa10, Gsa12, PpVps15, and Gsa14, but not Gsa7. GSA10 encodes a serine/threonine protein kinase homologous to APG1 in S. cerevisiae (Stromhaug et al., 2001). It is required for micropexophagy and nonselective

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15 autophagy in P. pastoris (Stromhaug et al., 2001). GSA10 is associated with the intermediate event in peroxisome sequestration and has been shown to be necessary for peroxisome sequestration events downstream of peroxisome recognition by Gsa9 and upstream of the assembly of Gsa11 compartment (Stromhaug et al., 2001). Late sequestration event Gsa7 is a 71-kDa protein required for degradation of peroxisomal AOX during glucose adaptation or nitrogen starvation (Yuan et al., 1999). Morphological data suggests that Gsa7 is required for a late sequestration event because “finger-like” extensions of the vacuole were almost completely surrounding the peroxisomes during glucose adaptation (Yuan et al., 1999). Gsa7 is not required for Gsa9 and Gsa11 localization (Yuan et al., 1999). It has been proposed by Yuan et al. that Gsa7 is required for bringing together the opposing vacuolar membrane arms for fusion to complete the peroxisome sequestration. Degradation event Proteinases A and B are two vacuole endopeptidases that are required for the degradation of AOX during glucose or ethanol adaptation (Tuttle and Dunn, 1995). During pexophagy, the peroxisomes accumulate within the vacuoles of cells lacking these proteinases. Pichia pastoris as a Study Model For Autophagy Yeast is one of the best-characterized eukaryotic models of autophagy because it possesses many characteristics that make it an especially useful system for molecular and genetic studies. The Saccharomyces cerevisiae genome was sequenced by a worldwide collaboration of scientists in April 1996. The genome project identified about 6,300 putative open reading frames in yeast. Since then, researchers have characterized many

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16 novel genes. Pichia pastoris in particular has been the desirable model for the study of pexophagy because of their ability to produce large peroxisomes under methanol conditions which enables us to visually characterize the cellular morphology of gsa mutants during pexophagy. This rapid degradation is not seen in S. cerevisiae . Human Diseases In recent years, autophagy has been linked to a growing number of human diseases including neurodegenerative disease, cardiovascular disease, and breast cancer (Klionsky and Emr, 2000). Evidence shows lower levels of autophagy genes such as beclin 1 in mammalian cells has been linked to breast cancer (Saftig et al., 2001). The deficiency of Lamp-2 protein in humans have been associated with a heart disease known as Danon disease (Saftig et al., 2001). This disease is characterized by a cardiomyopathy, myopathy and variable mental retardation (Saftig et al., 2001). Elevated expression of autophagy is associated with neurodegenerative diseases such as ParkinsonÂ’s (Klionsky and Emr, 2000). Several of the molecular components underlying the autophagy process have been characterized including the specific roles of genes within the autophagy pathway. About 30 genes have been identified as part of this pathway of which 17 are listed in Table 1-1. Beclin 1 is the first identified mammalian gene required for autophagy. It has structural similarity to the yeast autophagy gene APG6/VPS30 and is deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Studies have shown that beclin 1 induces autophagy in apg6 mutants of S. cerevisiae . Its autophagic activity in MCF7 cells is associated with the inhibition of MCF7 cellular proliferation and tumorigenesis in nude mice (Liang et al., 1999).

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17 The Cancer Genome Anatomy Project database shows very few chromosome abnormalities mapped around Gsa14, which is located at 2q36. However many human cancers have been linked to a region on the chromosome near Gsa11. Further studies in this region may help provide clues for drug development that would stimulate autophagy in cancer cells.

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18 CHAPTER 2 MATERIALS AND METHODS Yeast and Bacterial Strains and Media The parental yeast strain GS115 ( his4 ) was a generous gift from Dr. J. M. Cregg (Oregon Graduate Institute, Beaverton, OR) and was routinely cultured at 30 C in YPD (1% Bacto yeast extract, 2% Bacto peptone, and 2% dextrose). P. pastoris was grown in YNM (0.67% yeast nitrogen base, 0.4 mg/liter biotin, and 0.5% methanol) to induce peroxisome biogenesis and then transferred YND (0.67% yeast nitrogen base, 0.4 mg/liter biotin, and 2% glucose) or YNE (0.67% yeast nitrogen base, 0.4 mg/liter biotin, and 2% ethanol) to induce the degradation of the peroxisomes. If histidine (40mg/L) is added, the media is called YNMH or YNDH. The electroporation medium (EP) is composed of 1M sorbitol, 2% glucose, 0.1% yeast nitrogen base (YNB) and 0.4% mg/L biotin. Luria broth (LB) media is composed of 0.5% yeast extract, 1% tryptone, 0.5% NaCl. All media contained 2% agar when made as plates. Zeocin was added at 25 g/ml when culturing Escherichia coli (DH5 ) and 100 g/ml when culturing P. pastoris . Ampicillin is added to LB media at a final concentration of 100 g/ml when selection is needed for E. coli growth. A list of yeast strains used in the study is included in Table 21.

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19 Table 2-1: Pichia pastoris strains Name Genotype GS115 his4 SMD1163 his4 p e p 4 p rb1 DMM1 GS115:: p DM1 ( PAOX1GFP-SKL, HIS4 ) WDY7 his4 g sa 7 WDKO7 PPF1 g sa 7 ::ARG4 R13 GS115 g sa9-1 ::Zeocin R11 GS115 g sa10-1 ::Zeocin R22 GS115 g sa11-1 ::Zeocin R2GS115 g sa12-1 ::Zeocin R19 GS115 g sa14-1:: Zeocin P p v p s15 PPF1 v p s15 ::ARG4 TC1 GS115 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC2 SMD1163 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC3 DMM1 his4 :: p TC1 ( PGAPDH GFP-GSA14, HIS4 ) TC4 WDY7 his4 :: p TC1 ( PGAPDH GFP-GSA14, HIS4 ) TC5 WDKO7 his4 :: p TC1 ( PGAPDH GFP-GSA14, HIS4 ) TC6 R13 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC7 R11 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC8 R22 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC9 R2 his4 :: p TC1 ( PGAPDH GFP-GSA14, HIS4 ) TC10 R19 his4 :: p TC1 ( PGAPDHGFP-GSA14, HIS4 ) TC11 P p vs p 15 his4 :: p TC1 ( P GAPDH GFP-GSA14 , HIS4 ) TC12 P p vac8 his4 :: p TC1 ( P GAPDH GFP-GSA14 , HIS4 ) TC13 DMM1 his4 :: p TC2 ( PGSA14 GSA14 ( NT ) -GFP, HIS4 ) TC14 R19 his4 :: p TC2 ( PGSA14GSA14 ( NT ) -GFP, HIS4 ) TC15 DMM1 his4 :: p TC3 ( PGAPDHGSA14-GFP, HIS4 ) TC16 R19 his4 :: p TC3 ( PGAPDHGSA14-GFP, HIS4 )

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20 Restriction Enzyme-Mediated IntegrationGrow the replica-plated colonies on methanol plates and then switch to glucose plates Identify gsa mutants by Direct Colony Assay of AOX Isolate g enomic DNA and p erfo r m restriction di g estion and li g ation Amplify the resulting vector in E. coli and sequence the genomic DNA Growth selection on Zeocin plates Transform GS115 or STW1 cells (pREMI plus BamHI or DpnII) Isolation of Glucose-Induced Selective Autophagy-Deficient ( gsa ) Mutants by Restriction Enzyme-Mediated Integration (REMI) Mutagenesis Figure 2-1 displays the basic steps required for isolating REMI mutants. G lucoseinduced s elective a utophagy ( gsa ) mutants were generated by random insertion of pREMI into the parental strain GS115. The plasmid DNA pREMI contains a Zeocin resistance gene behind the transcription elongation factor I (TEF) and a synthetic prokaryotic (EM7) promoters which allow for selection in both yeast and E. coli , respectively. The pREMI was linearized by BamHI and randomly incorporated into GS115 cells by electroporation, aided by 1.0 unit of BamHI or 0.5 units of DpnII which randomly cleaves the genomic DNA. Zeocin-resistant transformants were selected for growth on YPD plates containing 1 M sorbitol and 100 g/ml Zeocin and then screened and identified by their inability to degrade peroxisomal AOX by direct colony assay as Figure 2-1. Restriction Enzyme-Mediated Integration. Steps in isolating REMI mutants in P. pastoris .

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21 previously described (Tuttle and Dunn, 1995). Mutants were isolated and verified by liquid medium AOX assay. Measurements of Peroxisome and Protein Degradation The degradation of peroxisomes in P. pastoris was quantified by measuring the loss of AOX activity during glucose or ethanol adaptation. Cells were first cultured in YNM(H) and switched to YND(H) or YNE(H) for 6 hours. Samples were collected at 0 and 6 hours after shifting to glucose or ethanol. The cells were collected by centrifugation and resuspended in 1mL ice cold breaking buffer (20 mM Tris/Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA) containing 1 uL PIC and broken by vortexing four times at 1 minute intervals in the presence of glass beads. After centrifugation at 4 C, 2500 rpm for 5 minutes, the supernatant was assayed for AOX activity. Alcohol oxidase was measured in a reaction coupled with horseradish peroxidase and the oxidation of 2,2Â’-azino-bio (3-ethylbenz-thiazoline-6-sulfonic acid), ABTS. The reaction mixture contained 3.4 units/ml horseradish peroxidase and 0.53 mg/ml ABTS in 33 mM potassium phosphate buffer, pH 7.5. The above supernatant (50 uL) was added to the reaction buffer, vortexed, and incubated at room temperature for 30-60 minutes until green color developed. The assay was stopped by adding 0.2 mL 4N HCl to the reaction and the absorbance was measured at 410 nm. The degradation of cellular proteins during nitrogen starvation was performed as described previously (Tuttle and Dunn, 1995). Cellular proteins were radiolabeled with 1 Ci/ml [14C] valine for 16 h in 0.67% YNB, 2% glucose, 0.4 mg/L biotin, and 4 mg/L histidine (if needed). The cells were then washed and switched to nitrogen starvation medium containing 0.17% YNB (without amino acids and NH4SO4) and 2% glucose and

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22 supplemented with 10mM valine. Aliquots were removed at 2-24 h of chase, and production of trichloroacetic acid (TCA)-soluble radioactivity was measured by the addition of 20% TCA. Acid-soluble and acid-insoluble radioactivity was separated by centrifugation and the radioactivity present measured by scintillation counting. The rates of protein degradation were calculated from the slopes of the linear plots of TCA-soluble radioactivity versus time of chase and expressed as the percentage of total 14C-labeled protein. Electron Microscopy The ultrastructural analyses were done as previously described (Veenhuis et al., 1983). P. pastoris strains were grown for 40 h in YNM(H) and then transferred to YND(H) or YNE(H) for 3 h. Cells were harvested briefly by centrifugation, washed in water, and fixed in 1.5% KMnO4 in veronal-acetate buffer (0.3 mM sodium acetate, 0.3 mM sodium barbital, pH 7.6) for 20 minutes at room temperature. The specimens were dehydrated by washing with increasing concentrations of ethanol followed by two washes with 100% propylene oxide. The cells were then infiltrated with 50:50 mix of propylene oxide and the POLY/BED 812 resin (Polysciences, Inc., Warington, PA) for two days. The preparations were transferred to 100% POLY/BED with accelerator 2, 4, 6-Tri (dimethylaminomethyl) phenol (DMP-30, Polysciences, INC.) for another two days and then incubated in the oven overnight at 60°C. The resulting blocks were sectioned by D. Player, Department of Anatomy and Cell Biology, University of Florida, College of Medicine, and examined using JEOL 100CX II transmission electron microscope.

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23 Molecular Biology Construction of Gsa14 Expression Vectors Previously in the lab, the gene for the green fluorescent protein (GFP) was inserted behind of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter into the EcoRI site of pIB2. The resulting expression vector was then used to construct the GFP fusion protein of Gsa14 being expressed by the constitutive and glucose-inducible GAPDH promoter (Fig. 2-2). GSA14 was amplified from genomic DNA by PCR with ID-PROOF polymerase (ID Labs Biotechnology) using a forward primer of WID298 that contained a KpnI site. The sequences for each primer are listed in Table 2-2. The reverse primer WID266 contained a XhoI site. GSA14 was inserted behind the GFP gene. Sequencing was done to verify that GFP and Gsa14 were in frame. The resulting pTC1 vector was linearized by cutting within the HIS4 gene (e.g. SalI) and used to transform by electroporation in GS115, DMM1, SMD1163, Pp vps 15 , WDK07, WDY7, R5, R12, R15, R2, and R19 cells. A second GFP-vector was created previously in the lab inserting GFP at the SphI site in the multi-cloning site (MCS) of pIB1 (Fig. 2-2). This vector was used to create a second Gsa14-GFP fusion protein vector inserting Gsa14 with its endogenous promoter in front of GFP resulting in the expression vector, pTC2. GSA14 was amplified from genomic DNA by PCR using a forward primer WID267 that contained KpnI site and a reverse primer WID305 with a XhoI site. This expression vector was also sequenced to verify that the fusion protein was in frame.

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24 Figure 2-2. Structures of GSA14 expression vectors.

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25 Figure 2-3. Structure of GSA14 and GSA11 expression vectors.

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26 Table 2-2. Primer sequences WID202 5Â’CCCAACGAAAAGAGAGACCA 3Â’ WID203a 5Â’GCTGACAGAAAATTTGTGCCCAT 3Â’ WID266 5Â’GTTTTGGACTCGAG GGTACTAATGCTTCATT 3Â’ XhoI WID267 5Â’GCAGGCTAGGGTACC GGTACTGGCACATT 3Â’ KpnI WID298 5Â’CTCTCACATTGTCGGTACC ATGCATAAGAATAACACGAC 3Â’ KpnI WID305 5Â’CAAGATCTATGCTCGAG AACAAATAATGCCTTATGCTGTTGACTAA 3Â’ XhoI A third vector was made using GSA14 with a GAPDH promoter in place of GSA14 endogenous promoter by amplifying it from genomic DNA using a forward primer WID298 containing KpnI site and reverse primer WID305 with XhoI site (Fig. 2-3). It was inserted in front of the GFP gene in pIB1-GFP, constructed previously in the lab. The final expression vector, pTC4 (Fig. 2-3) was assembled by excising Gsa11 from pPS69 by cutting with KpnI and inserted at the KpnI site in front of the blue fluorescent protein (BFP) in pBFP-GAPZ. Sequencing was done to ensure that fusion protein was in frame. Plasmid Isolation and DNA Sequencing All plasmids were isolated from the E. coli cultures by Promega Wizard Plus Miniprep (Promega, Madison, WI). Direct sequencing of the plasmid was done to ensure that the gene was in frame with the vector by using self generated primer WID202 (reverse) and WID203a (forward) which sequences across the Cand Nterminus of GFP respectively. See Table 2-2 for list of primer sequences. The primers for sequencing were synthesized by GEMINI Biotech Company and sequencing was performed by ABI PRISM Genetic

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27 Analyzer at the University of Florida. The DNA sequences were assembled using DNAman software. Yeast Transformation Cells were grown overnight in YPD to a density of A600 = 1.0 and then harvested and treated with 10mM dithiothreitol (DTT) in YPD containing 25 mM HEPES, pH 8, for 15 minutes at 30 C with gentle shaking. The cells (500 mL) were washed twice in ice-cold water and once in 1 M sorbitol and then resuspended into 0.5 mL of 1 M sorbitol. Cells (40 L) were mixed with 0.2-1.0 g of linearized vector DNA and transferred to a 0.2-cm gap cuvette (Bio-Rad, Hercules, CA). DNA was introduced into the cells by electroporation at 1.5 kV, 25 F, 400 (Gene Pulser, Bio-Rad Corp.). The cells were then plated onto selection EP plates and incubated at 30 C for 3-5 days before colonies appeared. Western Blot Analysis Cells were grown in 2 mL YPD or YNM(H) and then adapted to YND(H) or YNE(H). The cells were collected by centrifugation and prepared for SDS-PAGE. The cells were pelleted and lysed in 150 uL of 67 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, 1.5% DTT solution and 2 uL of PIC (200 mM phenylmethylsulfonyl fluoride (PMSF), 14.5 mM pepstatin A, 105 mM leupeptin in DMSO) by vortexing (four times at one-minute intervals) with glass beads. The samples were heated at 100°C for 5 minutes and cell debris and glass beads were removed by centrifugation. Fifteen microliters of each sample were loaded onto 6% or 8% SDS-PAGE. The proteins were then transferred to nitrocellulose by Trans-Blot SD-Dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA) for 1 h. The blots were blocked in 5% nonfat dried milk in

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28 PBS containing 0.1% Tween 20 (PBS-T) overnight at 4°C, incubated with mouse antiGFP primary antibody (Covance Inc., Princeton, NJ) and then in 2% nonfat dried milk with secondary goat anti-mouse antibody conjugated with HRP (Covance Inc., Princeton, NJ) for 2 h at room temperature. After each step the blots were washed four times in PBS-T. HRP was detected by ECL method (Amersham, Piscataway, NJ). Fluorescence Microscopy Cells expressing GFP fusion proteins were grown in YND(H) for 2-24 h or YNM(H) for 8 h. FM4-64 (Molecular Probes, Eugene, OR) was added to a final concentration of 20 g/ml, and the cells were incubated for an additional 16 h. Cells grown in YNM(H) medium were then transferred to YND(H), YNE(H), or nitrogen starvation medium containing 0.17% YNB (without amino acids and NH4SO4) and 2% glucose for 1-4h. The cells were examined immediately using a Zeiss Axiophot fluorescence microscope. Image capture was done using SPOT camera (Diagnostics Instruments, Inc., Sterling Heights, MI) with Adobe Photoshop software.

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29 CHAPTER 3 RESULTS Gsa14 Is Required For Both MicroAnd Macroautophagy of Peroxisomes in Pichia pastoris When P. pastoris is grown on methanol as its sole carbon source, peroxisome biogenesis is induced and peroxisomal enzymes such as AOX are synthesized for the assimilation of this compound. The metabolic shift from methanol to a different carbon source such as glucose or ethanol induces microor macropexophagy, respectively, causing the rapid degradation of AOX and peroxisomes which are not needed for glucose or ethanol metabolism (Dunn, 1994; Tuttle and Dunn, 1995). A novel approach to identify molecular events required for these degradative processes was developed in our lab. This method allowed us to disrupt gene expression and rapidly identify GSA genes required for glucose-induced selective autophagy. GS115 cells were transformed by electroporation with BamHI or DpnII and pREMI linearized by BamHI. Those putative mutants unable to degrade AOX during glucose adaptation were identified by direct colony assay and verified by liquid medium assay. Several mutants defective in peroxisome degradation upon glucose adaptation were identified. R16 and R19 are two such mutants whereby pREMI has been inserted into the open reading frame of GSA14. The pREMI vector was inserted into a single gene locus in each of these mutants and verified on southern blots (D. Maatouk and W. Dunn, unpublished data).

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30 Degradation of Alcohol Oxidase Induced by Glucose and Ethanol Adaptation Glucose-induced degradation of AOX was dramatically reduced in the gsa mutants (Fig. 3-1, panel A). Over 80% of the AOX activity was lost in parental GS115 cells. This degradation was blocked in SMD1163 ( pep4 / prb1 ) cells suggesting that the peroxisomes were degraded by the vacuole. The amount of AOX remaining after 6 h of glucose adaptation in gsa14 was comparable to those in gsa9 and gsa18 with about 40% AOX remaining due to partial blockage of microautophagy. The amount of AOX remaining in gsa10 , gsa11 , gsa12 , and gsa20 were about 10-20% higher. However gsa1 , gsa7 , gsa19 , and SMD1163 ( pep4 / prb1 ) mutants show more than 60% AOX activity remaining suggesting a nearly complete blockage of microautophagy. Degradation of AOX in gsa mutants and GS115 cells was also examined during ethanol adaptation (Fig 3-1, panel B). The degradation of AOX in SMD1163 ( pep4 / prb1 ) was severely impaired verifying that AOX was degraded by the vacuole. At 6 h of ethanol adaptation, less than 10% AOX remained in GS115 and again about 40% AOX activity remained in gsa14 . The amount of AOX remaining in gsa14 cells was comparable to that seen in gsa11 and gsa12 mutants. The data suggest that Gsa14 is required for glucose-induced and ethanol-induced pexophagy. Protein Degradation Induced by Nitrogen Starvation The rates of protein degradation during nitrogen starvation were evaluated for each of the mutants (Fig. 3-2). The nonselective delivery of cellular components to the

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31 Figure 3-1. Glucose and ethanol adaptation in wildtype GS115 and mutant strains gsa1 , gsa7 , gsa9 , gsa10 , gsa12 , gsa14 , gsa18 , gsa19 , g sa20 , and pep4 / prb1 . P. pastoris cells were cultured in YNM(H) for 36 h and then switched to 2% glucose (A) or 0.5% ethanol (B). Aliquots were removed at 0 and 6 h of adaptation, the cells lysed, and AOX activities assayed as described in METHODS AND MATERIALS. The data is expressed as a mean +/S.D. of a percentage of AOX activity remaining at 6 h relative to 0 hour. N=3-5 determinations. A B

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32 Figure 3-2. Protein degradation during nitrogen starvation in wildtype GS115 and mutant strains gsa1 , gsa7 , gsa9 , gsa11 , gsa12 , gsa14 , and p ep4 / prb1 . Cells radiolabled with [14C] valine were grown in YNM(H) and then switched to nitrogen starvation medium. Aliquots were removed at 2, 6, 12, and 24 h of chase. Radioactivity was measured by scintillation counting.

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33 vacuole by microautophagy and macroautophagy is induced by nitrogen starvation (Kim and Klionsky, 2000; Klionsky and Emr, 2000). The degradation of AOX in SMD1163 ( pep4 / prb1 ) was severely impaired during nitrogen starvation verifying that AOX was degraded by the vacuole. Starvation-induced protein degradation was significantly inhibited in gsa14 mutants compared to GS115 cells. This inhibition is comparable to other mutants such as gsa7 , gsa11 and gsa12 . However, protein degradation in gsa1 and gsa9 mutants was not impaired as was previously published by our lab [Kim, 2001 #6021;Yuan, 1997 #5814]. The data suggest that Gsa14 is required for selective and nonselective microand macroautophagy in P. pastoris . Morphological Studies of gsa14 Mutant I examined the vacuolar morphology of GS115 cells by electron microscopy and DMM1 cells expressing GFP-Gsa14 (TC3) by fluorescence microscopy. GS115 and TC3 cells were grown in YNMH and then adapted to YNDH for 3 h. The stages of micropexophagy were categorized into four stages defined by the vacuole morphology (Table 3-1). In methanol-grown GS115 cells, the majority of the cells examined by electron microscopy were in Stage 0 or Stage 1 as expected since ongoing pexophagy is minimal. A majority of the TC3 cells visualized by fluorescence microscopy also revealed the vacuoles in Stage 0 and 1. The addition of glucose to the medium enhanced micropexophagy. This correlated with an increase GS115 and TC3 cells containing vacuoles in Stages 2 and Stage 3. In order to better assess the site of blockage in microand macropexophagy, I examined the cellular morphology of the gsa14 mutants during glucose and ethanol

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34 Peroxisomes VacuoleTable 3-1. Vacuole morphology during micropexophagy.1 Cell Strain Growth Condition Stage 0Stage 1Stage 2Stage 3Total GS115 MeOH632011 Glucose00549 DMM1 MeOH11279350 Glucose311162050 1 Cells were grown in YNM(H) and then switched to YND(H) to induce micropexophagy. The stages of vacuole morphology in GS115 cells were visualized and determined by electron microscopy. The stages of vacuole morphology in DMM1 cells were visualized and determined by fluorescence microscopy.

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35 gsa14 GS115 Methanol 3h Glucose 3h EthanolAB C D F EV V V V V V P P P P P P N Figure 3-3. Morphology of gsa14 and GS115 during glucose and ethanol adaptation. gsa14 mutant and GS115 cells were grown in YNMH (A and B) and then adapted to glucose (C and D) or ethanol (E and F) for 3 h. Cells were harvested, fixed in potassium permanganate, and prepared for electron microscopy as described in MATERIALS AND METHODS. N, nucleus; V, vacuole; P, peroxisome; arrowhead, autophagosome.

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36 adaptation. GS115 and gsa14 cells were grown in YNMH and then adapted to YNDH or YNEH for 3 h. Cells were harvested, fixed in potassium permanganate, and prepared for electron microscopy (Fig. 3-3). During growth in YNMH, both GS115 and gsa14 show a single large, round vacuole with about 5-10 peroxisomes next to it (Fig 3-3, panel A and B). Upon glucose adaptation, the vacuolar arms in GS115 cells extend and enwrap the gray peroxisomes, completely engulfing them (Fig 3-3, panel D). However in gsa14 mutants, the vacuole appears next to the peroxisomes and slightly indents. Arm-like extensions were also not observed (Fig 33, panel C). A similar morphology was observed in gsa12 and PpVps15 mutants (Guan et al., 2001; Stasyk et al., 1999). During ethanol adaptation, a peroxisome can be seen sequestered within an autophagosome in GS115 cells (indicated by an arrow in Fig. 3-3, panel E). These autophagosomes were not observed in gsa14 mutants during ethanol adaptation. These results suggest that Gsa14 is required for an early event in peroxisome degradation during glucose and ethanol adaptation. GSA14 Encodes a Unique 102-kDa Protein GSA14 was cloned and sequenced by J. M. Thomson and P. Stromhaug (Gene Bank Nucleotide accession number AY075105). It encodes 885 amino acids of a 102 kDa protein with structural homology to the integral membrane protein Apg9 of Saccharomyces cerevisiae . The protein sequence alignment of Gsa14 and Apg9 is shown in Figure 3-4. The alignment reveals a large central region (190-679 residues) of homology with 45% identity. This region contains 4-6 transmembrane domains, which are highlighted regions in Fig. 3-4. The C-termini of these proteins are asparagine rich.

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37 Gsa14.........MHKNNTTFLSRVFGINSRNIDVHNPLFA.......................28 Apg9merdeyqlpnshgkn-----i--lq-devnpslnsqemsnfplpdiergssllhstndsr60 Gsa14............................................................28 Apg9edvdendlrvpesdqgtsteeedevdeeqvqayapqisdgldgdhqlnsvtskenvlete120 Gsa14............DDSVIPLYDQNQSAYYDNAYSDYSSDEEDSKPRTSRQTDSNLHMTDHS76 Apg9ksnlerlvegst----pkvgqlsseeee--efinndgfdd-tplfqkskihefsskksnt180 Gsa14GDGNDPFNQMDNSSRFSNSSSFHYNNTDQEDDNPELLLLQDEDSSLNRKNLDNSDFQSFA136 Apg9iedgkrplffrhilqnnrpqrdtqklftssnaihhdkdksanngpr-ingnqkhgtky-g240 Gsa14KKTFNQIPKIKFQLPKKEDYPTSHRQPPDIENQNGTLKSSVKKQIHFLDPMDKALWMWSN196 Apg9sa-qprftgsplnntnrftklfpl-k-nlls-isvlnntpedrintlsv.ker---k-a-299 TM#1 Gsa14VSNLDTFLHQVYDYYTGNGFNCIMMNKFTELFTVVFIVWLFSFMGNCIDYDKLMNDRNVY256 Apg9-e---i--qd--n--l----y--ile-ilnic-ll-v-fvsty--h-v--s--ptshr-s359 TM#2 Gsa14QFSQVKIDKCYSKIGFFPQKLIYWLFFIGLCLKLYQIFLDYLVLKDMKLFFNLLLGLSDD316 Apg9dii...------nsitgft-ffl-m-yffvi--iv-lyf-vqk-selqn-yky--ni---416 Gsa14ELQTISWGLVVKRIMILRDKNINAIVSQNTDLTSRKRMNAHDIANRILRKENYMIAMYNK376 Apg9----lp-qn-iqql-y-k-q-amtanvvevkakn-id..---v----m-r---l--l--s474 Gsa14SILDLDIELPLIGKVQLLTNTLQWNLNIAILDYFFDSETGQINLPALKERNRHTISTELK436 Apg9d--n-slpi--frtnv.--k--e--i-lcvmgfv-nesgfikqsi.--psq-eftre--q532 TM#3 Gsa14KRLIFCGIINIVLAPILSIYFIMYYFLKFFYDFKTNPADISSREYSPYARWKLREFNELP496 Apg9--fmla-fl--i---f-vt--vll--fry-ney--s-gs-ga-q-t-i-e--f--y---y592 TM#4 Gsa14HIFNRRLNISTESSNKYINQFPKETTTALLKFIMFISGSIVGVLVIVTILDPEFFLNFEL556 Apg9---kk-isl--tla---vd-----k-nlf---vs--c--f-ai-afl-vf---n-----i652 TM#5 Gsa14TPGRTVLFYVSTLGAIFTICKNSIPDDTLVFDPEVSLRYLSQFTHYLPQEWEGKYHTEEV616 Apg9-sd-s-i--iti----wsvsr-t-tqeyh-----et-ke-yey-----k----r--k--i712 TM#6 Gsa14KNDFCKLYTLKLYLVGKEILSWLFLPYILCYKLPECADTISDFFREFSVHVDGLGYVCTF676 Apg9-le-----n-rivillr-lt-lmit-fv-wfs--ss-gr-v ----n-ey--------ky772 Gsa14AMFQFNNQHNENGNANVHQNGNGNGGVPSAKSKSKKVPNPNRFTTKPSMRDMENDDKMIK736 Apg9---nmk-idg-dths..mdedsltkkiavng-htlnskrrsk--aedhsdkdlann--lq830 Gsa14SYMYFLESYGNDE.............IVQHQQALNRSLIYSTEISPTSGDDLNDSNILGL783 Apg9--v--mdd-s-s-nltgkyqlpakkgypnnegdsflnnk--wrkqfqp-qkpelfr-gkh890 Gsa14RQRNVATTGKRQNSIGNGLIYNGQNKRLSIGEAKTNVYSNPIASTVLDKDLQYKLANSYI843 Apg9algpghnispaiy-tr-pgknwdn-nngddikng--nataknddnngnn-he-v-te-fl950 Gsa14LNGMPGL...NEANQ.PADRKNERKYSNDSPGSY.........EIGR.............877 Apg9ds-afpnhdvidh-kmlnsny-gngil-kggvlglvkeyykksdv--.............997 Asn-rich Region Figure 3-4. Amino acid alignment of Gsa14 and Apg9. The amino acid sequence of Gsa14 from P. pastoris was aligned with its structural homologue Apg9 from S. cerevisiae . Amino acid identities (-), gaps (. . .), and pREMI insertion ( ) are indicated. The alignment reveals a large central region of homology with 45% identity. Highlighted regions designate transmembrane domains predicted by TopPred.

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38 pREMI was inserted immediately after the first transmembrane domain (D245) as indicated by the arrow. The number of transmembrane domains varies from 4-6 depending upon the algorithm used (Table 3-2). A search of the National Center for Biotechnology Information database revealed structural homologues of Gsa14 in Schizosaccharomyces pombe (NP_596247), Arabidopsis thaliana (NP_180684), Homo sapiens (XP_087158), Drosophila melanogaster (AAF58018), and Caenorhabditis elegans (NP_503178). Protein Database Website Number of Predicted TM Domains Gsa14 Apg9 “DAS” http://www.sbc.su.se/~miklos/DAS/ 6 6 PSIPred http://bioinf.cs.ucl.ac.uk/psiform.html 5 6 SMART http://smart.embl-heidelberg.de/ 4 6 SSPro http://promoter.ics.uci.edu/BRNN-PRED/ 5 5 TMHMM http://www.cbs.dtu.dk/services/TMHMM-2.0 4 6 TopPred http://www.sbc.su.se/~erikw/toppred2/ 6 6 Table 3-2. Predicted number of transmembrane domains by protein databases. The amino acid sequence of Gsa14 from P. pastoris and Apg9 from S. cerevisiae was entered into various protein databases. The number of predicted transmembrane domain was returned as shown in the table above.

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39 Cellular Localization of Gsa14 Three different expression vectors were created to localize Gsa14 within the cell. These expression vectors were created by fusing the GFP reporter gene to GSA14 either at the Nor C-terminus of Gsa14 behind different promoters. The first vector, pTC1, contains GFP at the N-terminus of GSA14 under the glucose GAPDH promoter. A second vector called pTC2 was created by inserting GFP at the Cterminus expressed under the endogenous promoter of GSA14 . GFP was also fused at the C-terminus of GSA14 under GAPDH promoter, resulting in the expression vector pTC3. This was done to see if there was a difference in expression when fusing GFP to the front or back of GSA14 . The expression vectors were transformed into gsa14 mutants and DMM1 cells and assayed for AOX degradation to verify if each of the recombinant GSA14 did indeed rescue gsa14 mutants. Recombinant GFP-GSA14 Expression in gsa14 and DMM1 Cells I first examined the expression of these constructs in gsa14 mutants and DMM1 cells by transforming the cells with pTC1, pTC2, and pTC3 (Fig. 3-5). I observed positive GFP-Gsa14 expression by all three vectors. All three constructs of Gsa14 were present in vesicles throughout the cytosol of the rapidly growing and dividing cells (See arrowheads in Fig. 3-5) and the number of dots per cell varies ranging from one to five. Data suggest that the GFP at the Nor C-termini did not affect Gsa14 localization. Next, I examined GFP-Gsa14 expression in TC10 ( gsa14 his4 ::pTC1 (PGAPDH GFP-GSA14, HIS4)), TC14 ( gsa14 his4 ::pTC2 (PGSA14 GFP-GSA14, HIS4)), and

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40 gsa14 DMM1pTC1(PGAPDHGFP-GSA14, HIS4)pTC2(PGSA14GSA14-GFP, HIS4)pTC3(PGAPDHGSA14-GFP, HIS4) gsa14 DMM1pTC1(PGAPDHGFP-GSA14, HIS4)pTC2(PGSA14GSA14-GFP, HIS4)pTC3(PGAPDHGSA14-GFP, HIS4) Figure 3-5. GFP-Gsa14 expression in g14 mutant and DMM1. gsa14 and DMM1 cells expressing pTC1, pTC2, and pTC3 were grown in YPD for 24 h and the cellular distribution of GFP-GSA14 was visualized as dots by (arrowheads) in situ fluorescence microscopy during logarithmic growth.

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41 TC16 ( gsa14 his4 ::pTC3 (PGSA14 GFP-GSA14, HIS4)) cells growing in YPD medium. These yeast strains were analyzed at various time points by Western blotting as described in Materials and Methods (Fig. 3-6). TC10, TC14, and TC16 cells were grown on YPD and 1 mL aliquots were removed at 6 h (OD600 0.6010.748), 24 h (OD600 2.600-2.681), and 48 h (OD600 2.803-2.946). Protein extracts were prepared and analyzed on SDS-PAGE. The Gsa14-GFP and GFP-Gsa14 fusion proteins were identified using monoclonal antibodies that recognized GFP. Gsa14GFP and GFP-Gsa14 migrated as a 128 kDa protein as expected (indicated by the arrow in Fig. 3-6). In all three strains, a small amount of Gsa14-GFP expression is detected at 6 h with a dramatic increase at 24 h. However when the cultures reached stationary growth phase at 48 h, the intensity of the bands seem to completely disappear and expression was significantly reduced to very little or none. The significant increase in expression at 24 h may be due to increased cell numbers during exponential growth that is reflected by a 4-fold increase in cell density (OD600). To the contrary, the lack of Gsa14-GFP expression at 48 h may be due to lack of glucose or because the expression may be short term. Finally, I examined Gsa14 expression during glucose adaptation. TC10 and TC14 cells were grown in YNM(H) and then adapted to YND(H). Equal volumes of cells were removed at 0, 2, 4, 6, and 8 h and protein extracts were prepared and separated by SDS-PAGE. Monoclonal antibodies that recognized GFP were used to identify the 128 kDa GFP-Gsa14 protein as indicated by the arrow in Figure 3-7. At 0 h, small amount of GFP-Gsa14 or Gsa14-GFP protein is detected in both strains. The intensity of the bands appeared to increase at 2 h suggesting that there is an increase

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42 Figure 3-6. Western blot analysis of TC10 ( gsa14 his4 ::pTC1 (PGAPDH GFP-GSA14, HIS4)), TC14 ( gsa14 his4 ::pTC2 (PGSA14 GSA14-GFP, HIS4)), and TC16 ( gsa14 his4 ::pTC3 (PGAPDH GSA14-GFP, HIS4)). Cells were grown in YPD medium and samples were taken at 6, 12, and 24 h. Protein extracts were prepared as described in MATERIALS AND METHODS and separated by SDS-PAGE. The GFP-Gsa14 and Gsa14-GFP proteins were identified using monoclonal antibodies that recognized GFP. GFP-Gsa14 and Gsa14-GFP migrated as a 128-kDa protein (arrow). GFP-Gsa14 TC10TC14TC166 h 6 h 6 h 24 h 24 h 24 h 48 h 48 h 48 h 120 kDa 205 kDa

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43 Figure 3-7. Western blot analysis of TC10 ( gsa14 his4 ::pTC1 (P GAPDH GFP-GSA14, HIS4)) and TC14 ( gsa14 his4 ::pTC2 (P GSA14 GSA14-GFP, HIS4)). Cells were grown in YNM(H) and then adapted to YND(H) or YNE(H). Aliquots were removed at 0, 2, 4, 6, and 8 h. Protein extracts were prepared as described in METHODS AND MATERIALS and the proteins were separated by SDS-PAGE. After transfer to nitrocellulose, the GFP-Gsa14 and Gsa14-GFP proteins were identified using monoclonal antibody that reco g nized GFP. GFP-Gsa14 and Gsa14-GFP mi g rated as a 128-GFP-Gsa14 GFP-Gsa14Glucose Ethanol TC10 TC14 0 h 2 h 4 h 6 h 8 h 0 h 2 h 4 h 6 h 8 h 205 kDa 120 kDa0 h 2 h 4 h 6 h 8 h TC100 h 2 h 4 h 6 h 8 hTC14 205 kDa 120 kDa GFP-Gsa14 GFP-Gsa14Glucose Ethanol TC10 TC14 0 h 2 h 4 h 6 h 8 h 0 h 2 h 4 h 6 h 8 h 205 kDa 120 kDa0 h 2 h 4 h 6 h 8 h TC100 h 2 h 4 h 6 h 8 hTC14 205 kDa 120 kDa

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44 in protein expression. The GFP-Gsa14 expression gradually decreased at 4 and 6 h, and eventually disappearing at 8 h. There also appears to be second band at about 100 kDa in TC14 cells during glucose and ethanol adaptation. This second protein may be a fragment of Gsa14-GFP processed in vivo during or in vitro during the processing of cell extracts. Since this cell strain contains GFP fused at the Cterminus of Gsa14, the clip likely occurred at the N-terminus. This is consistent with the absence of this band in TC10 cells that express GFP-Gsa14 because GFP was fused at the N-terminus of Gsa14 in TC10 cells. Further analysis would need to be done to verify this observation and its significance. Gsa14 Complementation of gsa14 mutants with GFP-Gsa14 Constructs The functionality of these GFP-Gsa14 constructs was determined by testing their ability to complement the Gsa14 phenotype. gsa14 /pTC1 (TC10), gsa14 /pTC2 (TC14), gsa14 /pTC3 (TC16), and DMM1/pTC1 (TC3) cells were cultured in YNM(H) medium and then switched to YND(H) medium for 3 h to induce micropexophagy. Alcohol oxidase assays were preformed as described in Methods and Materials to see if the gsa14 mutants could be rescued by the Gsa14 expression vectors (Fig. 3-8). As shown earlier, gsa14 mutants reveal a partial blockage of micropexophagy because 30-40% AOX still remained after glucose induction. However, the level of AOX activity still remaining in gsa14 mutants expressing pTC1, pTC2, and pTC3 after 3 h glucose adaptation was considerably lower (2-fold lower) than the gsa14 mutant but comparable to GS115 wildtype and DMM1 cells expressing pTC1. This data suggest that the Gsa14 constructs were able to rescue the Gsa14 phenotype. Therefore, my data show that GFP fusion at the Nor Cterminus were functional and localized in the cell.

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45 Figure 3-8. Complementation of gsa14 mutants with GFP-Gsa14 constructs. gsa14 mutant and DMM1 cells expressing GFP-Gsa14 by the various GSA14 expression vectors (pTC1, pTC2, pTC3) were cultured in YNM(H) for 36 h and then switched to YND(H). Aliquots were removed at 0 and 6 h of adaptation, the cells lysed, and AOX activities were assayed as described in METHODS AND METHODS. The data is ex p ressed as a mean + / -S.D. p ercenta g e of AOX activit y

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46 GSA14 Localization The cellular distribution of Gsa14 was examined to better understand how it functions in pexophagy and autophagy. Since all three Gsa14 constructs were shown to be functional and proved its ability to rescue gsa14 mutants by AOX assay, pTC1 expression vector was used to carry out the rest of the experiments. Gsa14 Localization in DMM1 Cells GFP-Gsa14 was expressed in DMM1 ( his4 ::pDM1 (PAOX1BFP-SKL, Zeocin)) cells that express the blue fluorescent protein (BFP) with the SKL peroxisomal targeting signal at its C-terminus (Fig. 3-9). The DMM1 cells expressing GFP-Gsa14 appear as dots (arrowheads) and patches (arrows) in YNMH medium (Fig. 3-9). The dots are near or adjacent to the vacuole membrane. The patches are visible at the vacuole surface, which are labeled in red by FM4-64. The peroxisomes are labeled in blue in DMM1 cells. The dots and patches were quantified by fluorescence microscopy (Table 3-3). The majority of GFP-Gsa14 localization in DMM1 cells appeared as dots during methanol condition. When the cells are adapted to glucose induction medium, GFP-Gsa14 was predominantly found in dots at the vacuole or in patches. During glucose-induced pexophagy, the profiles of the involuted vacuole with arm-like protrusions that extend out and around the peroxisomes can be seen (Fig. 310, panel A and B). Peroxisomes completely engulfed by the vacuole can also be observed (Fig. 3-10, Panel C). DMM1 cells expressing GFP-Gsa14 (TC3) were grown in YNMH in the presence of FM4-64 and then adapted to YNDH to induce microautophagy. When the cells were visualized by fluorescence microscopy, GFPGsa14 appeared as patches at the vacuole membrane, near the site of peroxisome

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47 BFP-SKL GFP-Gsa14 FM4-64 Merge Figure 3-9. GFP-Gsa14 expression in DMM1 cells grown in methanol medium. DMM1 cells expressing GFP-Gsa14 were grown in YNMH in the presence of FM4-64 and then visualized in situ by fluorescence microscopy. GFP-Gsa14 localizes as dots (arrowheads) and patches (arrows) on the vacuolar membrane.

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48 BFP-SKL GFP-Gsa14 FM4-64 Merge A B C Figure 3-10. Glucose adaptation in TC3 cells. DMM1cells expressing GFP-Gsa14 were grown in YNMH in the presence of FM4-64 and then switched to YNDH medium for 3 h and visualized by fluorescence microscopy. GFP-Gsa14 localized as patches (arrows) at the vacuolar arms and next to the peroxisomes during the pexophagy.

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49 Table 3-3. Quantitation of the localization of GFP-Gsa141 Cell Strain Growth Condition Dot2 Dot at Vacuole3 Patch4 DMM1 MeOH Glucose 23 4 22 15 10 33 TC10 MeOH ( gsa14 ) Glucose 20 9 22 3 12 38 TC6 Glucose ( gsa9 ) 51 3 0 TC8 Glucose ( gsa11 ) 3 14 34 1 Cell were grown in YNM(H) and then adapted to YND(H). The dots or patches of GFP-Gsa14 were visualized by fluorescence microscopy and quantified in 50 cells under each condition. 2 Dots vary in size with distinct boundaries and are found throughout the cytoplasm. 3 Dots juxtaposed to the vacuolar surface. 4 Patches at the vacuolar surface having diffused borders.

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50 sequestration (see arrows in Fig. 3-10, panel B). As the vacuole proceeds to sequester the peroxisome, GFP-Gsa14 sometimes co-localized with the vacuolar arms as shown by the arrows in Figure 3-10, panel A. The blue peroxisomes are present next to the vacuole as the vacuolar arms surround and engulf the peroxisomes. These micropexophagy events could be visualized by the progression of peroxisome sequestration as well as changes in vacuole morphology. I next examined the nonselective degradation of peroxisomes induced by nitrogen starvation in DMM1 cells (Fig. 3-11). During nonselective autophagy, GFP-Gsa14 is expressed as patches and slightly cytosolic. These patches appear to be found near the vacuole membrane as the vacuole slightly indents and extend its arms around the peroxisomes, which are in close proximity to the vacuole. Localization of GFP-Gsa14 in gsa Mutants My results suggest that Gsa14 is a membrane protein that localizes to vesicles near the vacuole membrane and required for an early event in pexophagy. However, the localization of Gsa14 to the perivacuolar vesicles or to the vacuole membrane may require other Gsa proteins. For example, previous data in the lab has shown that Gsa14 is required for the localization of Gsa11 suggesting Gsa14 may act upstream of Gsa11. To determine whether other proteins are required for the localization of Gsa14 to the vacuole membrane, the cellular localization of GFP-Gsa14 was examined in different gsa mutants. gsa7 , gsa7 , gsa9 , gsa10 , gsa11 , gsa12 , gsa14 mutants and DMM1 cells expressing GFP-Gsa14 were grown on YPD and protein extracts were prepared and analyzed on SDS-PAGE as shown previously (Fig. 3-12). The GFP-Gsa14 protein

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51 BFP-SKL GFP-Gsa14 FM4-64 Merge Figure 3-11. Nitrogen Starvation in TC3 cells. DMM1 cells expressing GFP-Gsa14 were grown in YNMH in the presence of FM4-64 and then switched to nitrogen starvation medium for 3 h and visualized by fluorescence microscopy. The vacuole begins to indent and form arms that start to surround the peroxisomes. GFP-Gsa14 appear as patches and dots at the arms on the vacuolar membrane and near the i

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52 Figure 3-12. Western blot analysis of pTC1 expression in gsa7 , g sa 7 , gsa9 , gsa10 , gsa11 , gsa12 , gsa14 mutants and DMM1 cells. Cells were grown in YPD medium until exponential growth. Protein extracts were prepared as described in METHODS AND MATERIALS and the proteins were separated by SDS-PAGE. The GFP-Gsa14 proteins were identified using monoclonal antibodies that recognized GFP. GFP-Gsa14 migrated as a 128-kDa protein (arrow). gsa7 gsa9 gsa11 GFP-Gsa14 205 kDa 120 kDa gsa7 gsa10 gsa12 gsa14 DMM1 gsa7 gsa9 gsa11 GFP-Gsa14 205 kDa 120 kDa gsa7 gsa10 gsa12 gsa14 DMM1

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53 was identified on Western blot using monoclonal antibodies that recognized GFP. GFP-Gsa14 migrated at the expected 128 kDa with no fragmented bands observed. Next, these cells were grown in YNM(H) in the presence of FM 4-64. The cells were then switched to YND(H) and examined by fluorescence microscopy at 0 and 3 h of adaptation. At 0 h (Fig. 3-13, 3-14), the GFP-Gsa14 appeared as dots at or near the vacuolar membrane in gsa7 , gsa9, gsa11 , gsa12 , gsa14, and pep4/prb1 mutants, as indicated by the arrowheads. At 3 h glucose adaptation (Fig. 3-15, 3-16), GFPGsa14 was present in patches at the arms of the vacuolar membrane that appear to engulf the peroxisomes in all mutants except gsa9 . In gsa9 cells, GFP-Gsa14 remained localized to dots near the vacuolar membrane. The localization of GFPGsa14 in dots and patches were also quantified by electron microscopy (as done previously) in DMM1 cells and gsa14 mutants expressing GFP-Gsa14 (Table 3-1). This suggests that the forming of patches requires Gsa9. This protein has been shown to be required for an early event in pexophagy. Therefore Gsa9 may function to recruit the Gsa14 vesicles to the vacuole membrane early in the pexophagy pathway. Previous data in the lab also suggested that Gsa12 is another protein required for an early event. However, the localization of GFP-Gsa14 to the vacuole membrane does not require any Gsa protein except Gsa9 suggesting that Gsa12 is expressed further downstream, possibly during an intermediate event, than previously concluded. Gsa7 shows no affect on Gsa14 as expected because this protein has been shown to be required for a late event. Pep4/Prb1 is required for the final degradation event of the peroxisomes and therefore also shows no affect on Gsa14.

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54 GFP-Gsa14FM4-64 Merge gsa7 gsa9 gsa11 Figure 3-13. GFP-Gsa14 localizes to an area near the vacuole membrane in gsa7 , g sa9 , gsa11 mutants during growth in methanol medium. gsa7 , gsa9 , gsa11 mutant cells were grown in YNMH medium in the presence of FM4-64 and then visualized in situ by fluorescence microscopy. Gsa14 localized as dots (arrowheads) in gsa7 , g sa11 , and gsa9 mutants near the vacuolar membrane.

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55 GFP-Gsa14FM4-64Mergegsa12 gsa14 pep4/ prb1 Figure 3-14. GFP-Gsa14 localizes to an area near the vacuole membrane in gsa12 , g sa14 , and SMD1163 ( pep4 / prb1 ) mutants during growth in methanol medium. gsa12 , g sa14 , and SMD1163 ( pep4 / prb1 ) mutant cells were grown in YNMH medium in the presence of FM4-64 and then visualized by fluorescent microscopy. In gsa12 and g sa14 cells, GFP-Gsa14 appears as dots (arrowheads) that localize adjacent to the vacuole membrane and patches (arrows) that localize at the vacuolar membrane (arrows). In SMD1163 cells ( pep4 / prb1 ), GFP-Gsa14 also localizes to the vacuolar membrane but appear as dots only.

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56 GFP-Gsa14 FM4-64 Merge gsa7 gsa9 gsa11 Figure 3-15. GFP-Gsa14 localization in gsa7 , gsa9 , and gsa11 cells during glucose adaptation. gsa7 , gsa9 , and gsa11 cells expressing GFP-Gsa14 were grown in YNMH in the presence of FM4-64 and then switched to YNDH medium for 3 h and visualized by fluorescence microscopy. In gsa7 and gsa11 mutants, GFP-Gsa14 localized as patches at the vacuole membrane surface (arrows) and as dots (arrowheads) adjacent to the vacuole membrane in gsa9 mutants.

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57 GFP-Gsa14FM4-64 Mergegsa12 gsa14 pep4 / prb1 Figure 3-16. GFP-Gsa14 localization in gsa11 , gsa12 , gsa14 , pep4/prb1 mutants during glucose adaptation. gsa11 , gsa14 , and SMD1163 ( pep4 / prb1 ) cells expressing GFP-Gsa14 were grown in YNMH in the presence of FM4-64 and then switched for YNDH medium for 3 h and visualized by fluorescence microscopy. In gsa11 , gsa14, and pep4/prb1 mutants, GFP-Gsa14 remained localized as patches on the vacuole surface.

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58 Co-localization of GFP-Gsa14 and BFP-Gsa11 Previous data in the lab suggested that Gsa14 is required for localization of Gsa11 during pexophagy. However, my data suggest that Gsa11 is not required for Gsa14 localization. I compared the co-localization of BFP-Gsa11 and GFP-Gsa14 in cells undergoing pexophagy. GS115 cells expressing BFP-Gsa11 and GFP-Gsa14 were grown in YNM medium in the presence of FM4-64 and then adapted to YND medium for 3 h (Fig. 3-18). During methanol conditions, BFP-Gsa11 appears as a cytosolic protein while GFP-Gsa14 appears as dots near the vacuole membrane as observed previously (arrowheads, Fig. 3-19). However after glucose adaptation, BFP-Gsa11 was found in regions throughout the cytosol (See arrows in Fig. 3-19) while GFP-Gsa14 was expressed as patches at the vacuole surface. There appears to be some but not a complete co-localization of Gsa11 and Gsa14. This may be due to the fact that there is an overexpression of both proteins. Gsa11 has been shown to be expressed as a cytosolic protein in rapidly growing and dividing cells (Stromhaug et al., 2001). Upon glucose adaptation, Gsa11 was distributed to cytoplasmic structures as dots that were near the vacuolar surface (Stromhaug et al., 2001). Since this protein appears to localize to a specific area in the cell, the overexpression of Gsa11 at the site may cause these dots to appear as centralized regions in the cell.

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59 Figure 3-17. Co-localization of BFP-Gsa11 and GFP-Gsa14. GS115 cells expressing BFP-Gsa11 and GFP-Gsa14 were grown in YNM medium in the presence of FM4-64 and then visualized by fluorescence microscopy. Gsa11 is a cytosolic protein that is required for an intermediate event of micropexophagy and an early event in macropexophagy. Gsa14 localizes as dots to areas near the vacuole under methanol condition. As pexophagy begins, GFP-Gsa14 first appears as dots adjacent to the vacuole membrane and then appears as patches at the vacuolar arms. BFP-Gsa11GFP-Gsa14 FM4-64 Merge

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60 BFP-Gsa11GFP-Gsa14 MergeMethanol Glucose BFP-Gsa11GFP-Gsa14 MergeMethanol Glucose Figure 3-18. BFP-Gsa11 and GFP-Gsa14 Co-localization During Glucose Adaptation. GS115 cells expressing BFP-Gsa11 and GFP-Gsa14 were grown in YNM and then adapted to YND for 3 h. Under methanol conditions, Gsa11 is expressed as a cytosolic protein and appears as patches and dots during glucose adaptation. Gsa14 expression in methanol medium is seen as dots (arrowheads) and begins to appear as patches (arrows) during growth glucose medium.

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61 CHAPTER 4 CONCLUSIONS GSA14 encodes a novel 102 kDa protein that is required for selective micropexophagy and macropexophagy as well as nonselective starvation-induced autophagy. Gsa14 is structurally homologous to Saccharomyces cerevisiae Apg9 that is autophagy via APG pathway in S. cerevisiae . This protein contains 4-6 putative transmembrane domains with an asparagine-rich region at the C-terminus (Fig. 4-1). Structural homologues also exist in many other eukaryotes, including humans suggesting that a similar functioning protein exist in other species. During micropexophagy, ultrastructural analysis of gsa14 mutants shows a blockage during an early event in peroxisome degradation. The vacuole appears next to the peroxisomes and slightly indents but the arm-like protrusions and complete engulfment of the peroxisomes is never completed. Upon ethanol adaptation, autophagosomes are Gsa14 TM TM TM TM TM TM N R Figure 4-1. Proposed conserved domains present the Gsa14. Gsa14 has four to six putative transmembrane (TM) domains and an asparagine-rich (NR) region.

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62 never formed around the peroxisomes hence halting macropexophagy at an early event. Biochemical data also provide further verification that Gsa14 is required for both microand macropexophagy. In order to better understand the functional role of Gsa14, I examined its cellular localization during pexophagy and autophagy. GFP was fused to either to the Nor Cterminus with or without the endogenous GSA14 promoter. All three constructs were able to complement the Gsa14 phenotype. There also appears to be no difference in expression levels among the different constructs, which was verified by Western blots. Under methanol conditions, GFP-Gsa14 localizes to vesicles that are adjacent to the vacuolar membrane. The number of dots varies between one and five in normal cells and gsa mutants. However when micropexophagy is induced by glucose adaptation, these vesicles form into patches at the vacuole surface. This association requires Gsa9. Previously, our lab has shown that GFP/HA-Gsa9 is mediates an early event in pexophagy, i.e., the interaction of peroxisomes to the vacuolar membrane. The data suggest that Gsa9 acts upstream of Gsa14 events. Other Gsa proteins tested do not appear to be necessary for the localization of Gsa14 and thus appear to act downstream. Biochemical and morphological analysis of Gsa14 under glucose adaptation, ethanol adaptation, and nitrogen starvation conditions proves that this protein is required for microand macropexophagy as well as nonselective autophagy. I have proposed a model for the insertion of Gsa14 into the sequestering membrane of the vacuole during pexophagy (Fig. 4-2). The localization of Gsa14 to the vacuolar patches does not appear to require other Gsa proteins except Gsa9. Previous

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63 Figure 4-2. Model for the insertion of Gsa14 into the sequestering membrane of the vacuole. Gsa1 Gsa1 Vacuole Gsa1 Gsa1 Peroxisomes 2. Earl y Se q uestration 3. Intermediate Se q uestration 4. Late Sequestraton/ HomotypicFusion 5. De g radation 1. Si g nalin g A P L y sosome Microautophagy Macroautophagy

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64 data in the lab has also shown that Gsa14 is required for the recruitment and localization of the cytosolic protein Gsa11. However, my data on the co-localization of BFP-Gsa11 and GFP-Gsa14 suggest that Gsa11 is not required for Gsa14 localization and may act upstream of Gsa11 but downstream of Gsa9. The Pichia pastoris genetic model has been beneficial for this study. Our data of Gsa14 suggest that this protein is required for degradation of peroxisomes. The structural homology of Gsa14 in various species seems to suggest that it may also serve an important function in all eukaryotic cells. Characterizing this protein has enabled us to further understand the autophagy pathway as well as its interaction with other proteins required for autophagy.

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70 BIOGRAPHICAL SKETCH Tina Chang was born and raised in Kenvil, New Jersey. In 1994, she came to Gainesville to attend the University of Florida from which she received her Bachelor of Science degree in microbiology. After working at the University of Florida Department of Molecular Genetics and Microbiology from 1998 to 2000, she was accepted to the same universityÂ’s graduate school. She graduated with a Master of Science degree in medical sciences.