Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins.

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37 degrees C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1Delta temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1Delta cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Molecular characterization of a gene required for entry into S phase of the mitotic cell cycle in the yeast {\it Saccharomyces cerevisiae\/}

A strain of Saccaromyces cerevisiae (SC3B) with a temperature sensitive defect in the synthesis of DNA has been isolated. This defect is due to a single recessive mutation in a gene named INS1 required for the initiation of S phase. Arrested cells carrying the ins1$\sp{ts}$ allele are defective in the completion of G1 to S phase transition events including SPB duplication or separation, initiation of DNA synthesis, normal control of budding, and bud neck stability. The mutation and a gene which complements the mutation were mapped to chromosome IV. The complementing gene was proved to be the wild type allele of the temperature sensitive mutation by genetic linkage of an integrated clone. A very low abundance 4.2 kb RNA message was observed in the strain SC3B which increased greatly in this strain transformed with a multiple copy plasmid carrying the complementing clone. The wild type gene was sequenced and found to encode a 1268 amino acid protein of with a molecular weight of 142,655 Daltons. Computer assisted searches for similar DNA sequences revealed no significant homology matches. However, searches for protein sequence homology revealed a protein (the DIS3 gene product of S. pombe) with a similar sequence over a 534 amino acid stretch to the predicted INS1 gene product. A later search revealed a near identical sequence for a gene (SRK1) also isolated from S. cerevisiae. ^

Regulation of phospholipid biosynthesis in {\it Saccharomyces cerevisiae\/} by CDP-diacylglycerol synthase

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS) while phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol-1-phosphate). In this study a gene (CDS1) encoding CDP-DAG synthase in S. cerevisiae was isolated and identified. The CDS1 gene encodes the majority, if not all, of the synthase activity, and is essential for cell growth. Overexpression of the CDS1 gene resulted in an elevation in the apparent initial rate of synthesis and also steady-state level of PI relative to PS in both wild type yeast and the cds1 mutant. Down-regulation of CDS1 expression resulted in an inositol excretion phenotype and an opposite effect on the above phospholipid synthesis in the cds1 mutant. This regulation of phospholipid biosynthesis is mediated by changes of the phospholipid biosynthetic enzymes via a mechanism independent of the expression of the INO2-OPI1 regulatory genes. Reduction in the level of CDP-DAG synthase activity resulted in an increase in PS synthase activity which followed a similar change in the CHO1/PSS (encodes PS synthase) mRNA level. INO1 (encodes inositol-1-phosphate synthase) mRNA also increased but only after CDP-DAG synthase activity fell below the wild type level. PI synthase activity followed the decrease of the CDP-DAG synthase activity, but there was no parallel change in the level of PIS1 mRNA. A G$\sp{305}$/A$\sp{305}$ point mutation within the CDS1 gene which causes the cdg1 phenotype was identified. A human cDNA clone encoding CDP-DAG synthase activity was characterized by complementation of the yeast cds1 null mutant. ^

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

Function of cardiolipin in mitochondria of Saccharomyces cerevisiae

Cardiolipin and its precursor phosphatidylglycerol, phospholipids found uniquely in membranes engaged in oxidative phosphorylation, play important roles in multimeric complexes of the energy transducing system (ETS) associated with the inner mitochondrial membrane. A combined molecular genetic and biochemical approach was used to more precisely define the role of cardiolipin in cell processes. ^ Strains of yeast Saccharomyces cerevisiae unable to synthesize cardiolipin because of the crd1Δ allele (encodes cardiolipin synthase) with different phenotypes were analyzed to determine which phenotypes are due to lack of cardiolipin. We concluded that many of the severe phenotypes ascribed to cells lacking cardiolipin, particularly when grown at 37°C, are because of the synergistic interaction of the crd1Δ mutation with the reduced expression of the PET56 gene which encodes a component essential for the formation of functional mitochondrial ribosomes. We also demonstrate that much of the reduced mitochondrial function in crd1Δ is because of reduced expression of ETS components at elevated temperature. ^ A crd1Δ mutant of S. cerevisiae has less severe physiological changes than strains lacking both phosphatidylglycerol and cardiolipin due to an increased level of phosphatidylglycerol, which might partially substitute for the cardiolipin-requiring functions. By varying the level of cardiolipin, we were able to correlate phenotypes in a dose-dependent manner with the level of cardiolipin to support more strongly an involvement of cardiolipin in a particular cellular process. There is almost complete lack of a supercomplex composed of cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) in extracts of cardiolipin-lacking mitochondria when compared to wild type cells and the level of supercomplex varies in proportion to the cardiolipin levels. Reduced cardiolipin levels also compromise the growth properties of yeast in a dose-dependent manner suggesting that the loss in growth efficiency is related to a role of cardiolipin that cannot be replaced by phosphatidylglycerol. An independent kinetic approach was performed to compare organization of the respiratory chain in wild-type and cardiolipin-lacking mitochondria. Cardiolipin-lacking mitochondria display kinetic properties for electron transfer between complexes III and IV via cytochrome c consistent with cytochrome c being a freely diffusible carrier, confirming complexes III and IV exist as individual complexes and not associated into a supercomplex in cardiolipin-lacking mitochondria. ^

Function and regulation of anionic phospholipids in mitochondria of Saccharomyces cerevisiae

As the major anionic phospholipids predominantly found in the mitochondrial inner membrane of eukaryotic cells, cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are of great importance in many critical mitochondrial processes. Pgs1Δ cells of Saccharomyces cerevisiae lacking both PG and CL display severe mitochondrial defects. Translation of several proteins including products of four mitochondrial DNA (mtDNA) encoded genes (COX1, COX2, COX3, and COB ) and one nuclear-encoded gene (COX4) is inhibited. The molecular basis of this phenotype was analyzed using a combined biochemical, molecular and genetic approach. ^ Using a mitochondrial targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5′ and 3′ untranslated regions (UTRs), lack of mtGFP expression independent of carbon source and strain background was confirmed to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but rather caused directly by the lack of PG/CL in the mitochondrial membrane. Re-introduction of a functional PGS1 gene restored PG synthesis and expression of the above mtGFP. Deletional analysis of the 5′ UTR of COX4 mRNA revealed the presence of a 50 nt sequence as a cis-acting element inhibiting COX4 translation. Using similar constructs with HIS3 and lacZ as reporter genes, extragenic spontaneous mutations that allowed expression of His3p and β-galactosidase were isolated, which appeared to be recessive and derived from loss-of-function mutations as determined by mating analysis. Using a tetracycline repressible plasmid-borne PGS1 expression system and an in vivo mitochondrial protein translation method, the translation of mtDNA encoded COX1 and COX3 mRNAs was shown to be significantly inhibited in parallel with reduced levels of PG/CL content. Therefore, the cytoplasmic translation machinery appears to be able to sense the level of PG/CL in mitochondria and regulate COX4 translation coordinately with the mtDNA encoded subunits. ^ The essential requirement of PG and CL in mitochondrial function was further demonstrated in the study of CL synthesis by factors affecting mitochondrial biogenesis such as carbon source, growth phase or mitochondrial mutations at the level of transcription. We have also demonstrated that CL synthesis is dependent on the level of PG and INO2/INO4 regulatory genes. ^

Investigations of stress responses in Saccharomyces cerevisiae: Transcriptional control and heat shock protein function

The baker's yeast, Saccharomyces cerevisiae responds to the cytotoxic effects of elevated temperature (37-42°C) by activating transcription of ∼150 genes, termed heat shock genes, collectively required to compensate for the abundance of misfolded and aggregated proteins and various physiological modifications necessary for the cell to survive and grow at heat shock temperatures. An intriguing facet of the yeast heat shock response is the remarkable similarity it shares with the global remodeling that occurs in mammalian cells in response to numerous pathophysiological conditions including cancer and cardiovascular disease and thus provides an ideal model system. I have therefore investigated several novel features of stress signaling, transcriptional regulation, and physiology. Initial work focused on the characterization of SYM1, a novel heat shock gene in yeast which was demonstrated to be required for growth on the nonfermentable carbon source ethanol at elevated temperature, and to be the functional ortholog of the mammalian kidney disease gene, Mpv17. Additional work addressed the role of two proteins, the Akt-related kinase, Sch9, and Sse1, the yeast Hsp110 protein chaperone homolog, in signaling by protein kinase A, establishing Sse1 as a critical negative regulator of this pathway. Furthermore, I have demonstrated a role for Sse1 in biogenesis and stability of the stress-response transcription factor, Msn2; a finding that has been extended to include a select subset of additional high molecular weight proteins, suggesting a more global role for this chaperone in stabilizing the cellular proteome. The final emphasis of my doctoral work has included the finding that celastrol, a compound isolated from the plant family Celasfraceae, a component of traditional Chinese herbal medicine, can activate heat shock transcription factor (Hsf1) in yeast and mammalian cells through an oxidative stress mechanism. Celastrol treatment simultaneously activates both heat shock and oxidative stress response pathways, resulting in increased cytoprotection. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.