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Multiple mechanisms regulate the activity of Gal 4 Rohde, John Roy 2000

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"Multiple Mechanisms Regulate the Activity of Gal 4 By JOHN ROY ROHDE B.S. (Bacteriology) University of Idaho, 1992 M.S. (Bacteriology) University of Idaho, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE F A C U L T Y OF MEDICINE Department of Biochemistry We accept this thesis as confcu-hiiHig to f^he required standards THE UNIVERSITY OF BRITISH C O L U M B I A August 2000 © John Roy Rohde. 'XCD In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department of ^P^ocV-a^'s? v^-t; Cj^X H<I<WU/ DE-6 (2/88) ABSTRACT The GAL regulon of Saccharomyces cerevisiae provides a model for the study of eukaryotic gene regulation. Expression of the GAL genes is dependent on the transcriptional activator Gal4. Normally, Gal4 is inhibited by the negative regulator Gal80. Galactose binds to the inducer protein Gal3 which then becomes capable of interacting with Gal80. This causes a conformational change in the Gal4-Gal80 complex that allows Gal4 to activate transcription. Induction of the GAL genes is also associated with phosphorylation of the activator Gal4. Data presented in this thesis demonstrate that Gal4 phosphorylation is required for sensitive response to the inducer galactose. Phosphorylation of Gal4 is dispensible for induction in cells which possess fully functional alleles of GAL3; however, phosphorylation of Gal4 at S699 is absolutely required for the process of "long term adaptation" whereby yeast are able to induce the GAL genes in the absence of Gal3. S699 phosphorylation on Gal4 occurs independently of the Gal3-galactose signaling mechanism. The phosphorylation of Gal4 at S699 is mediated by the R N A polymerase II holoenzyme component SrblO and represents a genetically non-epistatic mechanism of induction to that mediated by Gal3-galactose. In contrast, SrblO and Gal4 phosphorylation at S699 are genetically epistatic with each other. These data suggest that induction of the GAL genes is controlled by two separate signaling mechanisms: one which is galactose specific and acts through Gal3, and a second signal which controls the activity of the transcriptional activator Gal4 through phosphorylation. The major implication of these observations is that multiple signaling pathways converge on a single transcriptional activator through holoenzyme-associated Cdks to regulate its activity, thereby allowing the appropriate level of transcription to match the cells environment and physiological status. ii t T A B L E OF-CONTENTS T A B L E OF CONTENTS hi LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii 1. INTRODUCTION 1 1.1 Rationale 1 1.2 THE Y E A S T Saccharomyces cerevisiae 2 1.3 General transcription factors 3 1.3.1 Promoter assembly and TFIID 4 1.3.2 TFIIB andTFIIF 6 1.3.3 TFIffiandTFIIH ' ' 7 1.4 R N A polymerase II holoenzyme 8 1.4.1 Carboxy-terrninal domain of the R N A polymerase II 9 1.4.2 The mediator sub-complex of the R N A polymerase II holoenzyme. 11 1.4.3 Transactivators and the recruitment model of gene activation 14 1.4.4 Signal transduction and transcription 19 1.5 Carbon metabolism in yeast 19 1.5.1 Glucose inactivation 20 1.5.2 Glucose repression 21 1.6 Protein phosphatases and gene regulation 25 1.7 cAMP 27 1.8 The GAL gene regulon 29 1.8.1 Gal4 29 1.8.1.1 DNA binding 30 1.8.1.2 Central region and inhibitory domains 33 1.8.1.3 Transcription activated by Gal4 33 1.8.2 Gal80 37 1.8.3 Gal3 37 1.8.4 G a l l - a bifunctional protein 38 "1.8.5 Respiration function in induction ' 42 1.8.6 Gal2 42 1.8.7 G a l l l 43 1.8.8 Other " G A L " genes - 44 1.8.9 Gal4 phosphorylation 44 1.8.10 Objectives 45 2. MATERIALS AND METHODS 47 2.1 D N A Manipulation 47 2.2 Growth curves 51 2.3 Thermotolerance 51 2.4 Strains 51 •2.5 Yeast Transformations 54 2.6 3-galactosidase activity 55 2.7 Mutagenesis . 5 6 2.8 Matings, Sporulation, Tetrad analysis 57 in 2.9 Sequencing - 58 2.10 Plasmid rescue 58 2.11 Phosphopeptide analysis 59 2.12 Immunoprecipitation 60 2.13 SDS P A G E and preparation of Tryptic Phosphopeptides 61 2.14 Two dimensional analysis'bf tryptic peptides 62 3. R E S U L T S 63 ' 3.1 S699 of Gal4 is required for proper induction in YT6G80-171 63 3.2 YT6::171 is Gal- due to a weak allele of GAL2 66 3.3 GALA S699 is not required for GAL gene induction in strains derived fromW303-lA 66 3.4 Gal4 phosphorylation at S699 is required for sensitive response to the inducer galactose 72 3.5 The W303-1A galactose signaling mechanism is dominant to YT6G80::171 72 3.6 Gal4 S699 phosphorylation is required for the long term adaptation (LTA) response to galactose 78 3.7 Gal4 is phosphorylated at S699 independently from the Gal3-galactose signaling pathway 81 3.8 SrblO is required for proper induction of the GAL genes 84 3.9 SrblO and Gal3 define two independent regulatory mechanisms for Gal4 87 3.10 SRB10 is epistatic to Gal4 699 phosphorylation for GAL induction 88 3.11 The GAL11P allele causes elevated basal activity in a gal3- background but does not fully bypass the requirement for GAL4 S699 phosphorylation 93 3.12 A phenotype for the S699A mutation in YT6::171 in gal80 cells 94 3.13 A genetic screen to isolate genes regulating Gal4 function 99 3.16 The swgl phenotype is due to a single mutation 101 3.15 SWG1 is allelic to GIP2 106 3.16 The swgl mutant is impaired in utilization of galactose and sucrose, but not raffinose or glucose 111 3.17 GIP2 does not affect Gal4 phosphorylation 111 4. D I S C U S S I O N 116 4.1 Differences between YT6 and W303 reveal the physiological purpose for S699 phosphorylation 116 4.2 Gal4 phosphorylation occurs independent of Gal3-galactose signaling pathway 118 4.3 ;Srbl0 acts independently from Gal3-galactose in GAL gene induction 118 4.4 Gal4 phosphorylation may Affect a step downstream of holoenzyme recruitment 119 4.5 Regulation of transactivators by holoenzyme kinases 124 4.6 Signals regulating the activity of Gal4 125 4.7 Future directions 125 4.8 Comparison with the lac operon of E. coli 128 N O M E N C L A T U R E 131 R E F E R E N C E S 132 Appendix A 148 A . l High basal level of GAL expression in srb'10 cells expressing Gal4 derivatives from ARS-CfiVplasmids 148 iv Appendix B - 151 B . l Novel phenotypes associated with the overexpression of Gal4 151 B.2 Cells overexpressing Gal4 exhibit an extended lag phase of growth 151 B.3 The extended lag phase associated with Gal4 overexpression is only seen in cultures taken from stationary phase 152 B.4 Cells overexpressing Gal4 exhibit enhanced thermotolerance 153 B.5 Summary and conclusions 153 v LIST OF TABLES Table I Components of the RNA polymerase II mediator 12 Table II Plasmids used in this study 49 Table III Yeast strains 53 vi LIST OF FIGURES Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 1. The recruitment model of gene activation. 2. Signaling mechanisms of glucose. 3. The metabolism of galactose. 4. Schematic representation of the functional domains of Gal4. 17 23 31 35 5. The basic strategy for regulation of the GAL genes in Saccharomyces cerevisiae. 40 6. Phosphorylation of S699 of Gal4 is required for efficient induction-ef the~GAL genes in yeast strain YT6G80:: 171. 64 7. Yeast strain YT6::171 is Gal- due to a weak allele of GAL2. 68 8. Phosphorylation of S699 of Gal4 is not required for the efficient induction of the GAL genes in yeast strain YJR10:: 131. 70 9. Phosphorylation at S699 is required for sensitive response to the inducer galactose. 74 Phosphorylation at S699 is required for efficient induction of the GAL genes in cells lacking a fully functional allele of GAL3. 76 Phosphorylation of S699 of Gal4 is required for long term adaptation. 79 Phosphorylation of S699 of Gal4 occurs independent of the Gal3-galactose signaling pathway. 82 The holoenzyme associated kinase SrblO plays a role in both repression and induction of the GAL genes. . 8 5 14. Gal3 and SrblO represent separate signaling mechanisms. 89 The Gal4 S699A mutation ise-pistatic to a mutation in srblO. 91 The GAL11P allele of GAL11 partially complements a gal3 mutation, but does not bypass the requirement for S699 phosphorylation of Gal4. 95 Phosphorylation at S699 of Gal4 is required for maximal induction in gal80 cells 10 11 12 13. 15. 16. 17. Figure 19. 20. 21. 22. Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure when grown in glucose-grown cells. 97 18. A genetic screen for isolation of genes involved in GAZJ-independent signaling. " 102 The swgl phenotype is due to a mutation in a single gene. 104 The swgl phenotype is complemented by GAL3. 107 The swgl phenotype is complemented by a plasmid containing GIP2. 109 The swgl mutation impairs growth on galactose and sucrose but does not effect growth on glucose or raffinose. 112 23. Phosphorylation of S699 of Gal4 is unaffected by mutations in gip2. 114 24. Possible mechanisms of action for the S699 phosphorylation of Gal4. 122 25. Two separate signals regulate the activity of Gal4. 129 26. The expression of GALA derivatives from ARS-CEN plasmids in a gal4, srblO strain results in extremely high basal-level of GAL expression. 149 27. Gal4 overexpression causes an extended lag phase of growth. 157 28. Gal4 overexpression does not causes an extended lag phase of growth when cells are taken from exponentially growing cells. 159 29. Yeast overexpressing Gal4 are more thermotolerant than gal4 cells. 161 30. Galactose protects GAL4 yeast but not gal4 yeast from heat shock. 163 vn LIST OF ABBREVIATIONS A absorbance Aa amino acid A D activation domain A R activating region ARS autonomously replicating sequence ATP adenosine triphosphate Bp base pair •• -B S A bovine serum albumin B A W P butanol/acetic acid/water/pyridine c A M P 3'-cyclic 5'-adenosine monophosphate CBP CREB (cAMP response element binding protein) binding protein Cdk cyclin dependent kinase C E N centromere Cpm counts per minute CRE cAMP response element CTD carboxy terminal domain CTP cytosine triphosphate dATP 2'-deoxyadenosine triphosphate dCTP 2'-deoxycytosine triphosphate dGTP 2'-deoxyguanosine triphosphate dNTP 2'-deoxynucleotide triphosphate dTTP 2' deoxythymadine triphosphate D B D D N A binding domain D H A T histone de acetyl transferase DMSO dimethylsulfoxide D N A deoxyribose nucleic acid DTT dithiothreitol EB ethidium bromide E D T A ethylenediaminetetraacetic acid GDP guanosine diphosphate GRD glucose response domain GTP guanosine triphosphate HAT histone acetyl transferase HIV human immuno defficiency virus ID inhibitory domain Kb kilobase L B Luria-Bertanni L T A long term adaptation M A P mitogen-activated protein MP micro Farad M W molecular weight NaOAc sodium acetate NP-40 nonidetP-40 OD optical density ONPG ortho-nitrophenylgalactoside viii P A G E polyacrylamide gel electrophoresis PBS 1 " phosphate buffered saline PCR polymerase chain reaction P D M phosphate depleted media PIC pre initiation complex PFA performic acid P K A protein kinase A PMSF phenyl methyl sulfonyl fluoride RIPA radio-labelled immunoprecipitation assay R N A ribonucleic acid R R N A ribosomal ribonucleic acid SD synthetic dropout SDS sodium dodecylsulfate SWG synthetic with GAL3 T A P TBP associated factor T B E " tris-borate-EDTA TBP T A T A binding protein TBS Tris-borate-saline T C A Trichloroacetic acid TE Tris-EDTA TFA triflouroacetic acid TFII transcription factor II Tris tris(hydroxymethylamino)methane Ts temperature sensitive U units UAS upstream activating sequence U V ultraviolet WB wash buffer WT wild type YEPD yeast extract peptone dextrose ix Introduction 1. INTRODUCTION 1.1 RATIONALE The primary goal of this project was to elucidate how signaling pathways converge upon the transcriptional activator Gal4 to regulate its activity. Transcriptional regulation is one of the most intensively studied areas of biology. This is due to the fact that many disease states, including cancer, are the result of improper control of transcription or certain signaling pathways which regulate transcriptional events. The GAL gene regulon of Saccharomyces cerevisiae has proven to be an excellent model for the study of eukaryotic gene regulation. Study of this system has yielded several hallmark discoveries, especially in deciphering how transcriptional activators work, these include: the first example of how activators bind D N A , how activation domains function, and the modular nature of activators which laid the groundwork for the wildly successful two hybrid system. As biochemical techniques advanced, the majority of work on Gal4 focused on defining the targets of this activator. Several targets within the transcription machinery are now known and will be discussed. While this exciting epoch of research provided a wealth of understanding as to how . activators function, the signaling events regulating them received proportionally less attention. Much of the pioneering work done in the laboratories of Ojvid Winge and Carl Lindegren focused on the induction of the enzymes needed for galactose metabolism (91), making this one of the oldest experimental systems in yeast genetics. Although research on induction had been relatively quiet in recent years, the past five years has seen a prodigious effort on this front. 1 Introduction Work from several laboratories has given us a detailed view of how yeast sense and respond to the ligand galactose. In addition to the recent advances in our understanding of galactose signal transduction, there have been concomitant advances in how signaling events regulate transcription. In 1994 the concept of the holoenzyme emerged and changed, and in some ways simplified, our view of transcription. It became clear that DNA-bound transcriptional activators could work simply by contacting this preassembled complex, thus recruiting the transcription machinery to the promoter. When these studies began, a detailed understanding of the galactose signaling events was not available; therefore, experiments were undertaken to understand the role of phosphorylation in the regulation of the activity of the transcription factor Gal4. Results presented here show that phosphorylation of Gal4 is required for full induction of Gal4-regulated genes. In addition, the signaling events responsible for Gal4 phosphorylation are independent of the galactose mediated signal transduction pathway. Instead, Gal4 phosphorylation is regulated by the holoenzyme -associated kinase SrblO independently of Gal3 or galactose. This finding demonstrates that multiple signaling pathways regulate the activity of the transcriptional activator Gal4. Finally, I have developed a genetic screen in an attempt to identify genes involved in Gal3-independent induction of Gal4 regulated genes. 1.2 THE YEAST Saccharomyces cerevisiae Owing to the ability of fermented liquids to "make glad the heart of man", the yeast Saccharomyces has been subjected to studies since the earliest recorded times (Genesis). The budding yeast Saccharomyces has several advantages which has placed it among the most useful 2 Introduction experimental systems. Its quick growth rate on simple media, the existence of stable haploid and diploid states, the similarity of techniques used in the study of bacteria, and the ease of genetic manipulation, all contribute to its popularity. The yeasts, which belong to the family Eumycotina, are grouped into three broad categories based on features of their life cycle. These include the Ascomycetes, the basidiomycetes, and the imperfect fungi. Ascomycetes, which contains Saccharomyces, all share the feature of enclosing their spores into a sac known as an ascus. The classification of yeasts has been difficult due to the few distinctive characteristics among yeast as well as their great number. Classification of the genus Saccharomyces is further complicated by the fact that many strains of this yeast used in fermentation are actually hybrids of one of three members: Saccharomyces cerevisiae is the species most commonly used in the yeast-based industries, Saccharomyces bayanus, which is associated with wine production, and Saccharomyces carlsbergensis which is used in brewing beer. Because these three species are capable of interbreeding it has been debated whether or not these species are separate or should all be considered different strains of Saccharomyces cerevisiae. D N A relatedness studies show however that these closely related species are indeed separate (135)., 1.3 GENERAL TRANSCRIPTION FACTORS Expression of genes controlled by R N A polymerase II is governed by proteins which can be divided into two major categories: the general transcription factors (GTFs) that assemble on the D N A at or near the site of initiation, and factors that bind D N A upstream of the sites of initiation and control the rate of transcription. The GTFs consist of at least 7 protein complexes 3 Introduction named TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ (TF for transcription factor, II for RNA polymerase II). Assembly of these factors at the initiation site results in formation of the preinitiation complex (PIC) which is capable of supporting transcription. There are two major rate-limiting steps which are required for the formation of the PIC. Both of these steps involve the recruitment of multiprotein complexes to the promoters of genes by DNA-bound transcriptional activators. 1.3.1 Promoter assembly and TFIID The major rate-limiting step to active transcription is the recruitment of TFIID to the core promoter elements. TFITD consists of the TATA-binding protein (TBP) as well as a set of TBP associated factors known as TAFs (although it should be noted that in the yeast literature TFIID is often interchangeable with TBP). TBP is required for transcription by all three eukaryotic polymerases (96). TBP's essential role in yeast transcription was identified genetically when it was isolated as SPT15 (58) and later biochemically (89). In addition to being required for the basal transcription, several lines of evidence suggest that TBP plays an important role in transcriptional activation as well. Many transcriptional activators, including Gal4, physically interact with TBP in vitro (166, 175). More importantly several genetic experiments in yeast demonstrate that the need for a transcriptional activator may be by-passed if TBP is artificially recruited to promoters (33, 127, 252). These experiments, and the observation that transcriptional activators increase the amount of TBP bound at promoters, has led to the idea that the primary function of activators is to recruit TBP to promoters. TBP recruitment appears to be a rate-limiting step in the initiation of transcription (128). Consistent with this model, several 4 Introduction TBP mutations were isolated which are capable of maintaining basal transcription but are defective for activation (reviewed in (92)). The TAFs have also been implicated in the activation of transcription, most notably in metazoan cells (34, 194, 195, 228). These studies demonstrated that TAFs could serve as coactivators for certain transcriptional activators, and the idea developed that the TAFs served to relay signals from DNA-bound activators to the general transcription machinery. In yeast, this does not appear to be the case because genetic studies have failed to identify any components of • TF1TD (other than TBP) required for activation of transcription. Based on homology to TAFs from metazoan cells, the yeast TAFs have been cloned and studies undertaken to determine their role in transcription (5, 168, 244). Surprisingly, TAFs do not seem to be essential for general transcription in yeast in vivo. Instead, it appears that the TAFs are required for transcription of a subset of genes,.perhaps most notably for certain genes involved in cell cycle progression (5). Binding of TFTfD to the site of initiation is thought to be stabilized by association with the general transcription factor TFIIA (107). TFIIA interacts directly with TBP and with certain transcriptional activators as well as the TAFs (258, 259). Initially TFIIA was found to be responsible for accurate transcription in vitro (162) but in more purified preparations, TFIIA appears to be dispensable (45, 211, 259). Recent experiments suggest that instead of acting as an activator per se, TFTJA may act as an anti-repressor. The most likely target of this activity is a protein identified as an inhibitor of TBP function, Mot l (6, 7). 5 Introduction 1.3.2 TFIIB and TFIIF Two other GTFs, TFIIB and TFIIF, also play a role in stabilizing TFIID's binding to the promoter. TFIIB contacts TBP, TFIIF, as well as R N A polymerase II itself (11, 27, 28). TFIIB has been shown to interact with certain transcriptional activators and may thus play a role in activation of certain genes (148, 251). The notion that TFIIB is involved in activation is supported by the fact that one class of activation-defective TBP mutations are defective for their interaction with TFIIB (92). For some years it was known that TFIIB played a role in start site selection (11, 188, 189) although how this occurred was unclear. Recently, the crystal structure of human TFIIB complexed with TBP and D N A was determined and has shed light on the roles of TFIIB (234). The structure reveals that in addition to making D N A contacts immediately upstream of the T A T A box, TFIIB also interacts with the C-terminal "stirrup" of TBP and thereby determines the orientation of transcription. The third transcription factor complex that serves to stabilize the PIC is TFIIF (124, 198). Like TFIIB, TFIIF has also been implicated in proper start site selection (40). TFIIF carries out this function in a manner similar to bacterial sigma subunits by decreasing non-specific D N A binding by polymerase (40, 165). In fact human TFIIF can interact with E. coli polymerase and this interaction can be competed by bacterial sigma 70 (165). TFIIF may also act to bring in regulatory components of the transcription machinery. This notion is supported by the observation that one component of the yeast TFIIF, Tfg3, has also been isolated in larger complexes (125, 131) including the Swi/Snf chromatin remodeling machine (30). 6 Introduction 1.3.3 TFIIE and TFHH The TFIIE complex interacts with R N A polymerase II as well as with TFIIF (163). A l l of TFIIE's associated functions, including open complex formation and RNA polymerase II phosphorylation are linked to its interaction with the large complex known as TFHH (152, 163). There is some evidence that recruitment of TFIIH by TFIIE may serve as a type of transcriptional checkpoint in PIC formation (180). Structure-function studies of the G a l l l protein also support this view (207-209). TFIIH possesses enzymatic activities required for open complex formation, promoter clearance, and elongation. The formation of an open complex requires an ATP-dependent helicase activity (41, 111, 245). Evidence for this comes from experiments demonstrating that the requirement for TFIIH's D N A helicase activity may be bypassed when supercoiled or premelted templates are used (100, 184, 185, 238). A second enzymatic activity of TFIIH, the ability to phosphorylate the C-terminal domain of R N A polymerase, will be discussed later. R N A polymerase II exists in one of two forms: the unphosphorylated form known as HA, and the heavily phosphorylated form known as IIO. RNA polymerase II enters the PIC in the unphosphorylated form and is converted to the heavily phosphorylated form when transcription is initiated. In yeast, the catalytic subunit associated with CTD kinase activity is encoded by KIN28, which is homologous to human CDK7. In human cells, CDK7 also participates in cell cycle progression by serving as the Cdk-activating kinase. However, this is not the case in yeast cells where C A K activity is mediated by a seperate enzyme encoded by CAK1, Cakl which is not a component of TFIIH (119, 227). Examination of the subunits which comprise TFIIH revealed that it contains components involved in nucleotide excision repair (NER) (223). This observation helped to explain the 7 Introduction phenomenon of trancription-coupled-repair, where transcribed genes are preferentially repaired over non-transcribed ones (12). TFIIH's NER activity was formally demonstrated by the ability of purified TFIIH to rescue NER activity from yeast extracts prepared from mutants defective in this activity (245). This dual role of TFIIH in both D N A repair and transcription suggested the existence of a preassembled "repairasome", although evidence seems to favor a model where NER components are assembled when needed to establish TFIIH at the site of D N A damage (88). 1.4 RNA POLYMERASE II HOLOENZYME Once the general transcription factors TFIID and TFIIA have assembled at the promoter, the steps resulting in PIC formation are not yet resolved, though two predominant views have emerged. The more established view is known as the "step-wise" assembly model which dictates that the remaining GTFs are added sequentially to the DNA-bound, TFIID- TFIIA complex until a functional PIC is formed. This model was developed from biochemical studies using purified GTFs which indicated a prerequisite binding of certain GTFs before the binding of others (e.g. TFIIH may become part of the PIC only after TFTTF has bound) (27). Research based on this model was fruitful and has given us a detailed understanding of the specific roles of certain GTFs as well as their interactions with one another. In 1994 however, the stepwise assembly model of the PIC was challenged by the observation of a large preassembled complex consisting of most of the remaining GTFs, all of the core subunits of R N A polymerase II, and a subcomplex of proteins responsive to activators (referred to as the mediator). This complex comprises the eukaryotic RNA polymerase II holoenzyme (125, 131). 8 Introduction 1.4.1 Carboxy-terminal domain of the RNA polymerase II The carboxy-terminal domain (usually called the CTD) of the largest subunit of R N A polymerase II (Rpbl) consists of a repeating heptapeptide with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (repeated 27 times in yeast and 52 times in humans). In yeast, the CTD is essential for viability although truncations can be tolerated to some extent, resulting in a cold sensitive phenotype (131). Because phosphorylation of the CTD was associated with elongating (and presumably "activated") forms of the polymerase, this phenomenon has attracted intense investigation. In yeast, three kinases are known to be capable of phosphorylating the CTD. The first protein, Kin28, is the kinase subunit of TFIIH. TFIIH appears to play a role in promoter clearance, and Kin28 is associated with CTD phosphorylation after initiation of transcription (62). The second kinase is the cyclin dependent kinase SrblO (also known as ARE2, SSN8, GIG2, and UME5) (9, 146, 220, 243). SRB10 has been isolated in several screens employed to find factors involved in repression of inducible genes. The third CTD kinase complex CTDK-I , is encoded by C77c7(137), CTK2, and CTK3 (216). The major kinase activity of this complex encoded by CTK1 is closely related to CDK9 protein of metazoan cells (137). In mammalian cells, CTD phosphorylation is carried out by kinases similar to those in yeast cells. The best studied CTD kinase is the catalytic subunit of TFIIH, CDK7. The catalytic subunit of P-TEFb (positive elongation factor), first identified as a factor involved in elongating transcription complexes in Drosophila, has also been implicated in CTD phosphorylation (157). Finally, in mammalian cells there have been reports of the M A P kinases playing a role in 9 Introduction phosphorylating the CTD in response to serum in quiescent cells, as well as in response to heat stress (55, 241). Studies in yeast show that the specificity of phosphorylation within the CTD is subject to change dependent upon the stage of growth (186). Since the phosphorylation of different phosphoacceptor sites within the CTD are independently regulated, this implies that activity of the different CTD kinases may be independently regulated in response to environmental signals. Randy Strich's laboratory has shown that the stability of SrblO's cognate cyclin S rb l l , is regulated by environmental signals (42, 43). Cellular stresses such as heat shock, exposure to oxide radicals, or starvation for essential nutrients such as carbon, all result in the rapid degradation of S r b l l (42, 43). Additionally, it has been reported that the expression of SRB10 itself is regulated by the growth phase, as cells in stationary phase have substantially less SrblO protein (101). The activity of the other CTD kinases in response to environmental signals has not been investigated, however it appears that the activity of Kin28 (Cdk7) is also negatively regulated by heat shock (54). The function of regulated CTD phosphorylation has recently been clarified. Initially it was believed that this phosphorylation would be necessary for sending the polymerase on its way to active transcription, but this idea has not been formally demonstrated. A more established role for CTD phosphorylation has emerged in the coupling of transcription to R N A processing (38, 164). A family of proteins known as the SR proteins was shown to interact preferentially with phosphorylated CTD (259b). Subsequent work revealed that the machinery required for the 3' cleavage-polyadenylation factors also interacted with the phosphorylated CTD (164). Recent work from the laboratory of Phil Sharp has shown that phosphorylation of the CTD targets this 10 Introduction protein (Rpbl) for ubiquitination; the consequences of this are currently unknown, but it is postulated that this may target defective transcription complexes for destruction (167). 1.4.2 The mediator sub-complex of the RNA polymerase II holoenzyme Young and coworkers used a genetic approach to identify genes which could suppress the growth defect of a CTD truncation (131). This screen identified nine novel genes termed SRBs (suppressor of R N A polymerase B). Srb proteins were found-to copurify with many of the general transcription factors, as well as the RNA polymerase II core. The "holoenzyme" purified by Young and coworkers in 1994 consisted of R N A polymerase II, TFIIB, TFIIF, TFHH, Gal l 1 and the Srb proteins. In vitro experiments demonstrated that purified holoenzyme preparations could support transcription when supplemented with TFIID and TFIIE (125, 131). Most exciting was the finding that this preparation could respond to activators (130, 131). Earlier experiments had led to the postulate that a "mediator" complex must exist since purified GTFs alone could support basal transcription in vitro but were not responsive to transactivators (64, 123). Fractionation of this mediator activity in the Kornberg laboratory led to the identification of a complex associated with the CTD similar to that charactarized by Young and coworkers (125). The two holoenzyme preparations differ slightly in individual components but share the three functional properties that define the mediator: stimulation of TFIIH to phosphorylate the CTD; stimulation of basal transcription in vitro; and response to transcriptional activators in vitro (92, 125). The presence of certain transcription factors such as TBP (225) TFIIB (131), or the SWI/SNF chromatin remodeling complex (249) within the holoenzyme remains controversial. It is currently accepted that the mediator consists of the Srb proteins, Med proteins, and a 11 . V . . . -• Introduction subcomplex of G a l l l , Sin4, Rgrl , and Med3 (8, 92, 138, 142). Since it was first identified in yeast, there have been several mediator complexes described in metazoan cells including T R A P , . DRIP, A R C , and SMCC (reviewed in (21)). Recently it was determined that these different complexes share some of the same components, raising the possibility that many different mediator complexes may be formed by mixing and matching of a limited number of components (21, 83). Some mediator components had been identified previously as the products of genes involved in repression or activation of certain genes in response to environmental signals. This finding supports the notion that this complex is involved in relaying signals to the transcription machinery (214). Supporting biochemical evidence for this idea came when it was shown that some activators could be copurified with these mediator complexes (94, 201). It was recently demonstrated in mammalian cells that the Med proteins could serve as endpoints for signal transduction pathways (112). Table I summarizes the known components of the yeast mediator. TABLE I Components of the RNA polymerase II Mediator GENE ESSENTIAL CHARACTERISTICS REFERENCE SRB2 NO Provides functional link between CTD and TBP. Physically associates with Srb4, 5, 6. (125,131,225) SRB4 YES Target of Gal4, Physically associates with Srb 2, 5, 6. (125, 129, 131,225) SRB5 NO Binds TBP. Physically associates with Srb2, 5, 6. (125, 131,225) SRB6 YES Physically associates with Srb2,4, 5. (125,131,225) 12 Introduction SRB7 YES (94, 143) . SRB8, SSN5, ARE2 NO (94, 143) SRB9, SSN2, UME2 NO (94, 143) SRB10, SSN3, UME5, ARE1, GIG2 NO Cyclin dependent kinase with CTD kinase activity. Gal4 kinase. (98, 134, 146, 220, 243) SRB11, SSN8, UME3, GIG3 NO Cognate cyclin to SrblO. Degraded in response to environmental stress (42, 134, 146) GAL11, SPT13, SDS4, RAR3 NO Requires TELLE for function. In subcomplex with Sin4, Rgrl ,Med3. (10,61,125,209) SIN4, SSN4, TSF3, NO In subcomplex with Gal l 1, Rgrl,Med3. (36, 110, 142,214) RGR1 YES In subcomplex with Sin4, Ga l l l ,Med3 . (110,142) R0X3, SSN7 YES (87,203, 214) MED1 NO Mutations cause similar phenotypes to srblO/srbll (8, 169) MED2 NO (169) MED3, PGD1, HRS1 NO In subcomplex with Sin4, R g r l . G a l l l . (169) MED4 YES (169) MED6, MTT12 YES Human homolog is component of N A T complex (138, 169,219) MED7 YES (169) MED8 YES (169) 13 Introduction 1.4.3 Trans activators and the recruitment model of gene -activation While the RNA polymerase II holoenzyme is capable of supporting transcription in vitro, its activity and/or its recruitment to specific promoters is regulated by factors which bind to specific D N A sequences within the promoter elements upstream of the start site. These proteins, known as transcriptional activators (or transactivators), are responsible for providing the R N A polymerase II transcription machinery with its ability to transcribe specific genes. Given their importance in gene regulation, transactivators have been subject to intense biochemical study and these experiments have revealed a surprisingly conserved mechanism-of-action'throughout the evolution of eukaryotic cells (192, 193). This is underscored by the ability of many of these proteins to activate transcription in heterologous systems (118, 174). The ability to activate transcription is mediated through regions within the protein termed activation domains. Activation domains directly contact components of the general transcription complexes, which catalyzes recruitment to specific transcriptional start sites. Many activation domains share certain structural motifs or characteristics such as being rich in negatively charged or acidic amino acids (76, 77, 80); however, the presence of negatively chargedor acidic residues does not define an activation domain. The presence of these features in transactivators of diverse origin probably reflects that these proteins function through an evolutionarily conserved mechanism. Recent experiments, most notably those involving the holoenzyme component Gal l 1, demonstrate that any domain which contacts the transcription machinery of the R N A polymerase II holoenzyme can serve as an activation domain (10). Himmelfarb and coworkers described a dominant mutation in the putative "coactivator" G a l l l (97). This 14 Introduction dominant allele, termed GAL11P for "potentiator", was able to complement the activation defect of a weak allele of GAL4. Once it was learned that Gal l 1 was a component of the RNA polymerase II holoenzyme, a more detailed study of the Gal4-Gall lP interaction was undertaken. In these studies Barberis et al found that the GAL11P allele resulted in a protein which contacted the D N A binding domain of Gal4 (10). These investigators went on to show that the wild type Gal l 1 never contacted Gal4. The results of these studies suggested that gene activation could occur by simply recruiting the R N A polymerase II holoenzyme to a specific promoter. Indeed, many components of the polymerase II holoenzyme could serve as an activator when tethered to D N A by virtue of their contact with the holoenzyme. The prevailing view today is that transcriptional activators activate transcription by recruiting R N A polymerase II holoenzyme to the promoter, serving as "locator proteins" which tether the transcriptional machinery to the appropriate location (71, 174). There have been several reports which support the recruitment model of gene activation in vitro as well as in vivo (10, 70, 72, 121). It should be noted that recruitment is an ambiguous term which includes several modes by which transactivators might function. There have been studies which suggest transactivators can activate transcription by remodelling chromatin (26), inducing conformational changes within the transcription machinery (199), stabilizing formation of the PIC (102), or by enhancing the processivity of the transcribing complex (17, 256). Transactivators could accomplish any or all of these functions depending on the protein machine(s) it recruits to the specific gene. Recent experiments testing the recruitment model show that non-classical activators such as those composed of Gal4-Srb2 fusions do not activate transcription as well as bona-fide activators such as Gal4 (71, 174). The authors propose that 15 Introduction this may reflect a classical activator's broader range of targets within the transcription machinery. Figure 1 shows our current idea of how a typical R N A polymerase II transcribed gene is regulated in response to an activator. The specificity of DNA-bound activators lies in their ability to recognize its cognate site within a promoter. While the binding of a single transactivator can be sufficient to activate transcription, the promoters for most genes contain several sites available for the binding of tansactivators. These sequences are referred to as upstream activation sequences (UAS). This allows for transcriptional responses to different signals. As well, the presence of multiple binding sites for transactivators allows for cooperative D N A binding between the transactivators (and in turn more productive interactions with the transcription machinery) and thus leads to a synergistic increase in the rate of transcription (29). 16 Introduction Figure 1. The recruitment model of gene activation. (A.) Eukaryotic genes are normally silenced by the repressive effects of chromatin. (B.) D N A -bound transcriptional activators interact with components of the general transcription machinery ' which results in their recruitment to specific promoters. Most importantly, transcriptional activators are capable of recruiting TFIID which contains T B P , and the R N A polymerase II holoenzyme. Recruitment of these components to the promoter are rate-limiting steps required for transcription (C.) 17 Introduction 18 Introduction 1.4.4 Signal transduction and transcription How a cell can sense its surroundings and regulate its transcription accordingly is a fundamental question in biology. In most cases this is accomplished by controlling the activity of transactivators. Often, the extracellular cue remains outside the cell and its interaction with membrane bound factors sets off signaling cascades resulting in modulating the activity of transcription factors. These signaling cascades may result in the post-translational modification of transcription factors and the modulation of their activity. Probably the most common and certainly the best studied modification is phosphorylation. When it was discovered that transactivators were phosphorylated, it was postulated that this could enhance the activating potential of these proteins by introducing a negative charge to the transactivator. At the time, negative charge was correlated with activating function. This idea has never been tested. Instead, it appears that phosphorylation usually results in a change in the quartenary structure of the protein and this in turn, can favor or inhibit both intermolecular and intramolecular interactions. There have been examples of phosphorylation regulating the activity of transcription factors by a wide range of mechanisms. Some of these include: regulating the location of factors location within the cell (Pho4), (132) controlling the ability to bind D N A (Jun) (22), promoting the interaction with protein targets (CREB)(122), and favoring cooperative binding to D N A (NtrC) (35). 1.5 CARBON METABOLISM IN YEAST Yeast can use many different sources of carbon to meet demands for energy. While carbon sources such as glycerol, acetate, or ethanol may be used, S. cerevisiae is able to ferment 19 Introduction a wide variety of simple sugars resulting in the formation of ethanol as an end product (202). Glucose is the preferred sugar as it can enter glycolysis directly; other sugars must be converted to glucose by specific enzymes. Expression of the genes needed for conversion of alternative sugars to glucose is negatively regulated by the presence of glucose in a variety of ways (31). Glucose causes negative regulation by two major mechanisms: glucose inactivation where glucose inhibits the activities of proteins involved in the use of alternate carbon sources post-translationally, and glucose repression where the presence of glucose inhibits the transcription of genes. 1.5.1 Glucose inactivation Intensive study of the genes encoding glucose transporters has given us a great deal of information as to how the glucose signal is generated (reviewed in (31)). The yeast genome contains 20 genes within the glucose transporter family. These proteins differ in their affinities for glucose and in their expression patterns thus allowing yeast to respond to a wide range of glucose concentrations. Glucose transport is mediated by one of eight proteins encoded by HXT1-4, 6, 7, SNF3, and GAL2 and expression of any one allows yeast to grow on glucose (144). Interestingly it was determined that glucose signaling was still intact in cells bearing disruptions of all the glucose transporters, suggesting that the glucose signaling pathway does not require glucose transport (144). In support of this idea, dominant mutations within SNF3 and the glucose sensor RGT2 can cause glucose signaling in the absence of glucose (158, 182). These mutations occur within regions that are highly conserved among the glucose transporter family in yeast as well as those in mammalian cells (182). 20 Introduction A key regulator of the glucose transporter genes is the zinc finger-containing protein Rgtl (182, 183). Rgtl is a repressor of many of the glucose transporters whose expression is stimulated by the presence of glucose (183). In the presence of high levels of glucose, Rgtl is inactivated, allowing for the expression of the HXT genes. This inactivation requires the glucose sensors Rgt2 and Snf3, as well as the GRR1 gene product. Grr l has recently been identified as a member of the F-box family of proteins involved in targeting proteins for ubiquitin-mediated degradation (141). Thus it seems likely that the glucose signal generated by Snf3 and Rgt2 results in the targeted degradation of the repressor Rgtl (see Figure 2A). 1.5.2 Glucose repression Many glucose-repressible genes require the protein kinase Snfl for their expression (32). As a result, snfl cells can only grow on glucose as a source of carbon. Snfl is present in a complex of proteins that includes the activating subunit Snf4 as well as other proteins including Gal83, S ip l , and Sip2 (93). Snf4 interacts with a regulatory domain of Snfl when glucose levels are low (when Snfl is active) and this interaction is lost when glucose levels are high (when Snfl is inactive) (108). The Gal83/Sipl/Sip2 family of proteins interact with both Snfl as well as Snf4, and are thought to serve as scaffolds for the complex.in a similar fashion to the Ste5 protein in the pheromone response pathway (109, 254). It is also possible that these proteins play a role in substrate specificity of the kinase complex. A key substrate for Snfl appears to be the D N A binding protein M i g l , which binds to the promoters of several glucose repressed genes (173, 181). The subcellular location of M i g l is regulated by its phosphorylation state in response to glucose levels (47). When glucose levels 21 Introduction are high, M i g l is unphosphorylated and present in the nucleus. Upon glucose limitation (conditions which activate Snfl) M i g l is phosphorylated and transported to the cytoplasm. The phosphorylation state of M i g l is consistent with Snfl directly phosphorylating M i g l to inhibit its nuclear localization as demonstrated by the fact that in snfl cells M i g l is constitutively localized in the nucleus (47). Once M i g l binds to its target sites within a promoter, it recruits the general corepressors Ssn6 and Tupl (233b). At this point it is unclear how the general repressor Tupl acts to repress transcription, although several lines of evidence suggest that the mechanism involves the reorganization of chromatin to a more repressive state. Genetic evidence for this model comes from the observation that mutations within the tails of histones derepress Tupl-mediated repression (103). Biochemical studies demonstrate that Tupl is able to directly interact with histones H3 and H4 (57) and that Tupl mediated repression results in chromatin reorganization at the SUC2 promoter (73). Our current understanding of glucose inactivation and glucose repression is shown in Figure 2. 22 Introduction Figure 2. Signaling mechanisms of glucose. (A.) The repressor protein Rgtl represses the expression of genes activated by the presence of glucose. The presence of glucose is sensed by the membrane-bound glucose sensors Rgt2 and Snf3, which signal to the F-box protein Grr l . Grr l inactivates the repressor Rgtl , likely by targeting it for degradation by the 26S proteasome. (B.) In high glucose, the kinase Snfl is inactive and the M i g l protein is unphosphorylated and nuclear localized. In the nucleus, M i g l recruits the general repressors Ssn6 and Tupl to the promoters of genes subject to glucose repression. (C.) Upon glucose exhaustion, Snfl is activated, resulting in the phosphorylation of M i g l . Phosphorylated M i g l is localized to the cytoplasm and the genes are derepressed. 23 GLUCOSE Introduction Glucose Activated Gene REPRESSION G L U C O S E HIGH (SNF1 INACTIVE , U N P H O S P H O R Y L A T E D MIG! IS N U C L E A R ) B . DEREPRESSION G L U C O S E L O W (SNF1 A C T I V E , P H O S P H O R Y L A T E D MIG1 IS C Y T O P L A S M I C ) c . c y t o p l a s m 24 Introduction 1.6 PROTEIN PHOSPHATASES AND GENE REGULATION The activity of many proteins is controlled by theinterplay between protein kinases and protein phosphatases. The yeast genome encodes approximately 120 kinases. In contrast, there appear to be far fewer genes which are thought to encode protein phosphatases (105, 106). This raises the question as to how substrate specificity is achieved for phosphatases. Phosphatases are grouped into three families based on their substrate specificity, as well as their sensitivity to specific chemical inhibitors. The type I protein phosphatase (PP-1C) is one of the most conserved proteins in nature, showing greater than 80% identity between the yeast and mammalian proteins (63). In yeast, the type I phosphatase is ecoded by the GLC7 (DIS2S1) gene (63). As mentioned above, the identification of phosphatase substrates is a difficult problem. This is especially true for Glc7. First, purified Glc7 is capable of dephosphorylating a number of non physiological substrates in vitro (39). Second, Glc7 has been implicated in several different cellular processes including glycogen metabolism, glucose repression, sporulation, cell cycle progression and protein translation (19, 126, 215, 237). Third, null alleles of GLC7 are lethal in Saccharomyces (63). These difficulties have made it difficult to decipher the mechanisms by which Glc7 functions. Several years ago Cohen and coworkers proposed that the specificity of phosphatases could be controlled by targeting subunits (104). In this model interacting proteins would control either the cellular location and/or the activity of phosphatases, thereby controlling their substrate specificity. The phosphatase targeting model is now widely accepted, and both biochemical and genetic approaches have identified several proteins which interact and control the activity of the type I phosphatase (65, 218, 237). Regulation of the type I phosphatase (PP-1C) is best 25 Introduction understood in its role in glycogen metabolism in mammalian cells. Glycogen levels are regulated by two major enzymes: glycogen phosphorylase, which favors glycogen use; and glycogen synthase which catalyzes glycogen synthesis. Phosphorylation of glycogen phosphorylase enhances its activity, while phosphorylation of glycogen synthase decreases its activity. Both of these enzymes are good substrates for PP-1C. Thus, the activity of the phosphatase favors glycogen synthesis. PP-1C is targeted to glycogen by the regulatory protein G s u b . This protein interacts with glycogen and is in turn thought to be responsible for targeting PP-1C to its substrates. The association of G s u b with the catalytic subunit of PP-1C is controlled by phosphorylation. Protein kinase A phosphorylates G s u b which decreases its affinity for C s u b but not for its affinity for glycogen. This results in a release of free C s u b and decreases the synthesis of glycogen. The yeast protein GAC1 is the homolog of the mammalian G s u b (63). Overexpression of GAC1 results in hyperaccumulation of glycogen while gacl cells have constitutively low levels of glycogen. Genetic approaches in yeast have identified other putative targeting subunits for the type 1 phosphatases, including Regl which is involved in glucose repression (236), G ip l which is required for efficient sporulation (237), and other proteins which do not yet have a role ascribed to them (66, 237). Genetic evidence strongly suggests that the phosphatase Glc7 acts antagonistically to the i . • protein kinase Snfl (31, 66, 237). The Glc7 targeting subunit Regl is required for proper glucose repression (236). A closely related gene REG2 appears to play no role in glucose repression; however a regl, reg2, double mutant is inviable; this synthetic lethality is rescued by mutations in SNF1 (66). The most probable explanation for this phenotype is that the Glc7 acts 26 Introduction to dephosphorylate Snfl substrates or regulates the activity of the Snfl complex itself. Two hybrid experiments and recent biochemical studies favor a model whereby the phosphatase Glc7 regulates protein-protein interactions within the Snfl complex (49, 153). 1.7 cAMP In response to limiting nutrients, yeast cells exit the cell cycle and enter a quiescent state known as G 0 . As cells enter this state they acquire several characteristics such as thickening of the cell wall, increase in vacuole size, accumulation of the storage carbohydrate glycogen, and increased resistance to environmental insults such as heat shock (reviewed in (25, 224). In Saccharomyces, the c A M P pathway is involved in cell cycle progression and nutrient sensing. These roles are illustrated by phenotypes associated with mutations which either activate or diminish c A M P signaling. Mutants which have constitutively high levels of cAMP are extremely thermosensitive, fail to accumulate the storage carbohydrate glycogen, and fail to arrest in response to limiting nutrients (229, 231). Mutations which impair signaling of the c A M P pathway have the opposite phenotypes in that they sporulate even on rich media and accumulate glycogen (230). The cAMP pathway appears to repress genes necessary for proper adaptation to stress, such as the heat shock genes, and genes that are induced upon entry to stationary phase. Addition of rapidly fermentable sugars such as glucose to yeast growing on a poor carbon source causes a transient burst of cAMP, produced by adenylyl cyclase (224). In mammalian cells it has been well established that c A M P levels are controlled by the activity of G-protein coupled receptors (78). It was surprising then when it was discovered that in S. cerevisiae adenylyl cyclase was controlled by the activity of Rasl and Ras2 proteins (25, 231). 27 Introduction The sugar sensing mechanism which was presumed to be responsible for the activation of the Ras proteins remained clouded until recent work uncovered the existence of a G-protein coupled receptor system in yeast (133, 253). A G-protein coupled receptor encoded by GPR1 (253) is responsible for sensing glucose and activating the G-protein Gpa2 (151). Gpa2 in turn, activates adenylyl cyclase, similar to the Ras proteins. A rise in cAMP levels activates protein kinase A (PKA) which is comprised of the inhibitory subunit B c y l and one of three catalytic subunits Tpk l , Tpk2, and Tpk3 (229, 231). Although few downstream targets of the cAMP pathway have been identified, this pathway is clearly important in nutrient sensing. Mutations which constitutively activate the cAMP pathway resemble those of snfl cells in that they require glucose as a carbon source, are sensitive to heat shock, and fail to arrest their cell cycle in response to nutrient deprivation (226, 229). In spite of this correlation, cAMP does not appear to mediate glucose repression, as cells with constitutively low c A M P levels are still subject to glucose repression (13, 59, 226). Instead it seems that the sugar-stimulated cAMP signaling pathway is important in the adaptation to fermentable sugars from quiescent states such as sporulation (95). An additional role of the cAMP pathway is to negatively regulate the stress response. This is apparently controlled by the transcription factors Msn2 and Msn4 necessary for the activation of stress-induced genes (20, 160). Studies of Msn2 and Msn4 have also shed light on another function of the c A M P pathway: that of mediating cell cycle progression. In response to protein kinase A inactivation, cells arrest at G l phase of the cell cycle. This arrest is supressed by deletion of MSN2 and MSN4 implying that these transcription factors are responsible for the synthesis of a product which can inhibit cell growth (213). Additionally, it has been proposed that the protein kinase A pathway is responsible for translation of the CLN3 28 Introduction message (90). This could be a link between nutrient control and the proliferation of cells. Finally, a role for the cAMP pathway has recently been reported in the program of pseudohyphal development. In response to limiting nitrogen levels and in the presence of a fermentable sugar, diploid yeast will exhibit "foraging behavior" characterized by elongated cells growing in chains (79). Pseudohyphal growth requires components of the highly conserved M A P kinase cascade and it has been postulated that cross-talk between these two signaling pathways allows the cell to sense the availability of different nutrients (200). 1.8 THE GAL GENE REGTJLON S. cerevisiae is capable of growth on galactose as its sole source of carbon. Utilization of galactose is dependent upon the enzymes of the Leloir pathway that is responsible for converting galactose into glucose, making it available for glycolysis. A diagram of the reactions involved in galactose metabolism is shown in Figure 3. 1.8.1 Gal4 Expression of the GAL gene regulon is controlled by the transcriptional activator Gal4, the "gold standard" of transactivators. Studies that mapped the functional domains of this protein led to a general understanding of how transactivators control gene expression (192, 193). The different functional domains of Gal4 are shown in Figure 4. 29 Introduction 1.8.1.1 D N A binding Gal4 belongs to a family of transcription factors that includes Put3, Pdrlp, and Cha4 (46, 99, 145, 217). Members of this family share a common zinc cluster in their D N A binding domain; zinc has been demonstrated to be an essential cofactor for Gal4's ability to bind D N A (113). The D N A upstream of each GAL gene contains at least one binding site for Gal4 and the sum of all Gal4 binding sites within a promoter comprises the upstream activating sequences (UAS) (85, 86, 248). Gal4 binds D N A as a dimer that recognizes a-17 bp sequence with dyad symmetry. As with the entire family of zinc cluster D N A binding proteins, the signature for the Gal4 binding sites is the C G G triplets lying on the edges of the binding site (145, 197, 240). Most of the GAL genes contain more than one Gal4 binding site, to which Gal4 binds to cooperatively (120), resulting in synergistic activation of GAL genes with multiple Gal4 binding sites (29, 147). As is the case with the lambda repressor, cooperative binding of Gal4 provides a sensitive mechanism for controlling transcription. A small increase in the expression of Gal4 results in a dramatic increase in transcriptional activity (82). It should be noted that Gal4's ability to bind D N A cooperatively is not limited to interactions with itself; Veshee and coworkers demonstrated that binding of Gal4 is enhanced by its interactions with components of the transcription machinery, a prediction made years earlier (192). It has also been proposed that the multiple binding sites for Gal4 allow for more stringent repression as well. This comes from the observation that members of the GAL regulon with a single site for Gal4 binding (such as MEL1 or GAL2) are expressed constitutively whereas genes such as GAL1 or GAL7 are never expressed in the absence of galactose (114, 191). 30 Introduction Figure 3. The metabolism of galactose. Galactose enters the cell through the galactose-specific transporter encoded by GAL2. Once inside the cell, the Leilor pathway enzymes convert galactose into glucose which is able to enter glycolysis to meet the cells demands for carbon and energy. 31 Introduction CHjOH Gal (out) 1 MEL1 a-galactosidase GAL2 permease CKjOH HO J— O Gal (in) I GAL1 w kinase Gal-1-P GAL7 transferase UDP-Glu 3 ^ f ^ UDP-Gal Q - U D P GIu-1-P GAL5 mutase OCH2 HO J a OM Melibiose GAL10 epimerase Glycolysis Glu-6-P Introduction 1.8.1.2 Central region and inhibitory domains Detailed deletion analysis of Gal4 by Stone and Sadowski revealed that the central region contains domains which inhibit the ability of Gal4 to activate transcription (217). The inhibitory domains appear to be controlled by an adjacent region termed the glucose response domain (GRD). Full-length Gal4 is rapidly inactivated by the addition of glucose whereas Gal4 derivatives lacking the central region are insensitive to this inactivation (217). Mechanistically, this process is not well understood, although it appears to act independently from the glucose repression mechanisms mediated by Tupl acting through the URS (217). The inhibitory domains in Gal4 share homology to domains present in other regulatory proteins in yeast, including Put3 and Leu3 (68, 217). Removal of the inhibitory domain of Leu3 results in a constitutively active form of the protein. The GRD, which controls the activity of the inhibitory domains in Gal4, is not shared in these other proteins. 1.8.1.3 Transcription activated by Gal4 Gal4 activates transcription by recruiting components of the R N A polymerase II holoenzyme to the promoters of the GAL genes (192, 193). Gal4 has three domains that are capable of activating transcription when fused to a D N A binding doniain (116, 154, 155). Of these domains AR2, comprising the C-terminal amino acids 768-881, is both the strongest and best characterized. A variety of biochemical approaches have identified several targets of AR2 within the general transcriptional machinery, including TBP, Srb4, TFIIB, and SrblO (98, 129, 166). In vitro studies show that the ability of Gal4 derivatives to activate transcription correlates 33 Introduction well with their ability to interact with these targets (251). Mutations in either GALA or its target proteins which prevent interaction impair Gal4's ability to activate transcription (129, 251). The C-terminal 30 residues of Gal4 also interact with the negative regulator of the GAL gene regulon Gal80 (116, 154). 34 Introduction Figure 4. Schematic representation Gal4 functional domains. The N-terminal residues 1-65 contain the D N A binding domain. Gal4 has two activation domains; one located near the D N A binding domain and the strong activating region (AR2) located at the C-terminus. The central region of Gal4 contains domains responsible for inhibiting the activation function of Gal4. The inhibitory domains are regulated by the adjacent glucose-response domain (GRD). A cluster of phosphorylations reside within the glucose response domain (S691, S696, S699). Additional phosphorylation sites occur within the D N A binding domain ('site(s) unknown) as well as S837, within AR2. The C-terminal 30 amino acids are necessary and sufficient for interaction with the negative regulator Gal80. This domain also interacts with TBP. 35 Introduction Phosphorylations ) Q NH2 |ARI AR2 aal o o o o o o o o o o LO o o to o o COOH | aa881 DNA Binding (1-65) Inhibitory domains (320-585)and Glucose response domain (600-768) Inhibitory GRD GAL80/TBP interaction (851-881) 36 Introduction 1.8.2 GalSO Gal80 is a 45kD protein responsible for inhibiting the activity of Gal4 (232). Gal80 interacts with AR2 of Gal4 and is thought to mask the activator from its targets within the transcription machinery (140, 154). Indeed, Gal80 can block interaction between Gal4 and TBP as well as TFIIB in vitro (251). Interestingly, TBP and Gal80 interact with the same region of Gal4, but mutations have been made which differentially affect these interactions (4, 210). The authors of these studies conclude that the binding of Gal80 depends upon the formation of a precise secondary stucture while interactions with the general transcription machinery are not dependent upon sequence or structure. Mutations which inactivate Gal80 cause the GAL genes to be expressed constitutively. Conversely, there are dominant GAL80 alleles which cause insensitivity to galactose; yeast bearing these "super repressor" (GAL80s) alleles are incapable of expressing the GAL genes (51, 178). Inhibition by Gal80 is relieved upon induction by galactose. Clever genetic experiments using the GAL80s allele suggested that Gal80 dissociated from Gal4 upon galactose induction (187). However, more recent biochemical studies reveal that Gal80 may remain bound to Gal4 following induction, implying that galactose causes a conformational change in the Gal4-Gal80 complex rather than complete dissociation of Gal80 (140, 190). Relief of Gal80 inhibition is mediated by the product of GAL3. 1.8.3 Gal3 Induction of the GAL genes requires Gal3 (15, 233). Some of the earliest laboratory yeast strains were found to ferment galactose rapidly (within a day) while other strains required several days to begin fermenting galactose (204, 250). This phenomenon was termed "long term 37 Introduction adaptation" (LTA) and was among the first systems used to study gene expression in eukaryotic cells. The difference in the response to galactose was found to be caused by naturally occurring mutations in the GAL3 gene. Early work on induction suggested that Gal3 was responsible for modifying galactose in some way, and that the galactose derivative served as an inducer for the GAL genes, analogous to the conversion of lactose to the inducer allolactose for the E. coli lac operon (24, 233). Experiments by Nogi examining the kinetics of GAL induction using a temperature sensitive allele of GAL5 suggested that it was more likely that Gal3 itself was the inducer of the system (177). This notion was supported by the observation that overexpression of GAL3 resulted in expression of the GAL genes even in the absence of galactose (14, 16). Recent work by Blank and Hopper also supports this view: mutagenesis of GAL3 resulted in the isolation of several alleles which cause constitutive expression in the absence of galactose (18). Fukasawa and coworkers have demonstrated that Gal3 directly interacts with Gal80 in a galactose-dependent manner (222, 257). This interaction is stabilized by ATP, but does not require ATP hydrolysis (190, 260). Piatt and Reece have reconstituted induction of the GAL regulon in vitro using purified Gal3, Gal80, and a Gal4 derivative comprised of the DNA-binding domain 1-147 fused to the C-terminal 30 amino acids. This work demonstrates that a Gal4C-30-Gal80-Gal3 ternary complex is capable of activating transcription in vitro (190). 1.8.4 Gall-a bifunctional protein Early studies into the process of galactose induction identified a role of the galactokinase Ga l l . It was found that yeast which were gal3, and bearing mutations in any of the GAL1J, or 38 Introduction 10 genes were unable to respond to galactose (233). Sequence analysis of GAL1 and GAL3 genes show that they are closely related (15, 222). Studies in the Hopper laboratory demonstrated that overexpression of GAL1 could partially supress the loss of Gal3 function (14, 16). In the closely related yeast Kluveromyces lactis, a single protein related to Gall and Gal3 serves both the inducer and galactokinase functions (260). Recent work by Hollenberg show that these are separable functions, as mutations of K. lactis GAL1 can be isolated which abolish galactokinase function but do not affect inducer function and vice versa (242). The biochemical studies conducted by Piatt and Reece suggest that the Gal l protein is a poor inducer in comparison to Gal3 (190). A model of the current understanding of induction is shown in Figure 5. 39 Introduction Figure 5. The basic strategy for regulation of the GAL genes i n S. cerevisiae. S. cerevisiae. (A.) In the absence of galactose the negative regulator Gal80 binds directly to Gal4 and masks its interaction with its targets within the transcription machinery. (B.) Galactose enters the cell through the galactose-specific permease encoded by GAL2. Galactose: binds to the inducer protein Gal3 (or the closely related Gall), which then interacts with the negative regulator Gal80. The Gal3-Gal80 interaction is dependent upon both galactose and ATP. Gal3-galactose causes a conformational shift in the Gal80-Gal4 interaction which allows Gal4 to make contact with components of the transcription machinery. Recruitment of the GTFs to GAL promoters results in transcription. 40 A. Unlnduced-galactos© absent Introduction Introduction 1.8.5 Respiration function in induction The observation that ATP is necessary for the stabilization of the tripartite complex of Gal4-Gal80-Gal3 (190, 222, 257, 260) may explain a long standing mystery in induction of the GAL genes. Douglas and Pelroy noticed that yeast with deficient mitochondrial function induce the GAL genes very poorly (52). Cells bearing a gal3 mutation are known to grow on galactose-containing media with low efficiency, although this phenotype can be supressed by deletions of the negative regulator Gal80 (50, 52). Investigations which sought to explore, this link resulted in the identification of an allele of GAL2 (called IMP2) (239). Thus, when cells can take in enough galactose, the function of the mitochondria becomes less important for induction. 1.8.6 Gall The permease responsible for galactose transport is encoded by GAL2 (172). Gal2 posesses both high and low affinity functions where galactose enters through a process of facilitated diffusion (196). Interestingly, Gal2 is capable of transporting glucose as well as galactose (196). As one might expect, the ability of Gal2 to transport glucose results in competitive inhibition of galactose entry, and thus provides another mechanism of glucose repression of the GAL genes (that of inducer exclusion) (161). As discussed above, many naturally occuring isolates of yeast have mutations within GAL2 which impair their ability to transport galactose, even though these strains are able to induce GAL genes quite effectively with high concentrations of galactose.(239). 42 Introduction 1.8.7 Galll GAL11 was originally cloned by Suzuki and Fukasawa as a gene required for galactose metabolism (221). Subsequently, GAL11 has been cloned in many different screens which implicate it in a diverse range of cellular processes from telomere maintenance to vacuolar inheritance and lowered expression of a subset of genes including Ty and MATa (60, 176). Most, if not all of these phenotypes appeared to result from improper transcription in response to transactivators such as Gal4 or Rapl (60, 97). galll mutants display many of the same phenotypes as mutations in SIN4 or RGR1. The pleitropic nature of GAL11 mutations, as well as the similarity of phenotypes associated with galll, sin4, and rgrl mutations, was explained when it was shown that G a l l l is a component of the R N A polymerase II holoenzyme mediator and exists in a subcomplex containing Sin4, as well as Med3 (142). The G a l l l mediator complex has been shown to enhance the response to many activators in vitro. A role for relaying signals from activators in vivo comes from the observation that G a l l l is required for the proper phosphorylation of Gal4 (150) as well as the observation that some acidic transactivators such as VP16 interact directly with G a l l l (139). Interestingly, G a l l l function appears to be specific to promoters which contain a typical T A T A element, as promoters with TATA-less promoters are unaffected by GAL11 mutations (209). Rigorous biochemical studies have shed light on the function of G a l l l . While TFIIH and G a l l l are always present in holoenzyme preparations, TFIIE is usually absent (92). G a l l l interacts both genetically and biochemically with TFHE and TFIIH, and mutations of these components that specifically abolish their interaction with G a l l l results in phenotypes strikingly similar to galll mutations (207, 208). Also, disrupting the Gal l l -TFIIE interaction diminishes 43 Introduction the ability of the CTD kinases to properly phosphorylate the CTD of the polymerase. These observations suggest that Gal l 1 may be involved in the activation of holoenzyme-associated CTD kinases. 1.8.8 Other "GAL" genes Mutations in many other genes cause defects in GAL transcription, but whose role is as yet unknown. Some, such as components of the Swi/Snf chromatin-remodeling machinery, or R N A polymerase II holoenzyme components, are likely to reflect general defects in transcription while others, such as mutations in BCY1 or RAS2 (229, 231) result in misregulation of signaling pathways which are poorly understood. It should be noted that the GAL1-10 promoter, which is the most commonly studied promoter of the GAL regulon, contains both a strong UAS element as well as URS elements which result in glucose repression. Mutations causing misregulation through either element could lead to very similar phenotypes. 1.8.9 Gal4 phosphorylation Hopper and coworkers were the first to report that Gal4 is a phosphoprotein as observed by the appearance of more slowly migrating species in SDS polyacrylamide gels (170). Using phosphoamino acid analysis, Sadowski and coworkers determined that Gal4 is phosphorylated primarily on serine residues (205). Further work identified a phosphorylation site within the activation region II at S837 (205). Phosphorylation at this site was shown to correlate precisely with activation and fit well with the growing body .of evidence suggesting that Gal4 phosphorylation and activation of transcription were tightly linked events (170, 171, 205). These 44 Introduction included the observations that only Gal4 derivatives that are able to enter the nucleus, are able to bind DNA, and are able to activate transcription, are capable of becoming phosphorylated (205). It was also noted that the putative Gal4 coactivator Gal l 1 (later identified as a component of the R N A polymerase II mediator) was required for proper Gal4 phosphorylation (150). Based on these observations it was proposed that Gal4 phosphorylation occurred as a consequence of its ability to activate transcription, rather than being a prerequisite for transcriptional activation function. Despite these correlations, the role of Gal4 phosphorylation remained elusive since an S837A mutation resulted in no discernable phenotype in induction by galactose (205). Another puzzling observation was that Gal4 phosphorylation could be stimulated by sugars other than galactose in gal80 cells (206). In 1996 a cluster of phosphorylation sites.residing in the GRD were identified (206). One of these at S699 was shown to be necessary for Gal4 to activate transcription in strains expressing Gal80, thus setting the stage for genetic and biochemical studies to elucidate the role of Gal4 phosphorylation in GAL gene activity. 1.8.10 Objectives When I began the present study it had been shown that S699 was required for GAL induction in wild type strains, but not for activation by Gal4 in gal80 cells. This suggested that phosphorylation of Gal4 at S699 was not simply controlling the stability of Gal4 protein. Two major questions required addressing: /) what are the signaling events that control phosphorylation of S699 and ii) how does S699 phosphorylation control the activity of Gal4? To address the first question it seemed reasonable to investigate the connection between S699 phosphorylation and the known signaling components of the GAL gene's. Additionally, 45 Introduction establishing the identity of the Gal4 S699 kinase could perhaps yield insight to signals controlling GAL gene induction. 46 Materials and Methods 2. MATERIALS AND METHODS 2.1 DNA MANIPULATION Most recombinant D N A procedures were performed as described in Maniatis et al (156). Purification of restriction enzyme-digested D N A was as follows. After complete digestion with the appropriate enzymes, the reaction mixture was fractionated by electrophoresis in I X TBE buffer on agarose gels containing ethidium bromide. The fragments were visualized with long wave U V light and excised from the gel. The gel slices were then placed in a 0.5 ml tube with a needle sized hole in the bottom on top of a small piece of glass wool. This tube was placed inside a 1.5 ml tube and then centrifuged for 20 seconds. The collected sample was then adjusted to 400 ul with TE (pH 8.0) and the D N A was precipitated by adding 1 ml of 95% ethanol and 50 ul of 3M NaOAc (pH 5.2). The D N A was dried and resuspended in TE (pH 8.0) before use. Ligations were performed at 16°C for 12 hours using NEB T4 ligase in 50 mM Tris-HCl (pH 7.5), 10 mM M g C l 2 , 10 mM DTT, 1 m M ATP, and 25 mg/ml B S A in a final volume of 10 ul. Propagation and manipulation of most plasmids was in E. coli DH5a strain (F-, 80, lacZMIS (lacZYA-argF) U169, deoR, recAl, endAl, hsdR17 (rK, mK+) SupE44 thi-1, gyrA96, relA). Stratagene SURE cells were used for electroporation (el4-(McrA-) A(mcrCB-hsdSMR-mrr)171 endAl supE44 thi-1 gyrA96 relAl lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [F' proAB laqcPZAM15 TnlO (Ter~)]). 47 Materials and Methods The following oligonucleotides were used for amplification of GAL3: 5' -G G G G G G G A A T T C T G T T A C C A C A T T G A C A A C C C C - 3 ' (forward) and C C C C C C T C G G A G T C A T G C T G C C G C C G C G A A G G G A (reverse). Oligonucleotides for the amplification of G1P2 were 5' - C C C C T C T A G A A T G T A T A T A A A G G C A G A A C A G - 3 ' (forward) and 5 ' -GACCGGTCGACCTATCAAGGCTGGGGCTG-3 ' (reverse). The following media were prepared as described in Kaiser (117) L B 1% Bacto-tryptone 5% Bacto-yeast extract l % N a C l 2 X Y T 1.6 % Bacto-tryptone -1 % Bacto-yeast extract 0.5% NaCl Ampicillin was added to 100 u.g/ml when appropriate. Plates contained an identical formulation plus 2% agar. A rubidium chloride technique was used to prepare competent E. col (156). Competent cells were aliquoted and stored at -70°C. For each transformation, 200 \x\ of cells were thawed on ice and incubated with D N A on ice for at least 30 minutes. Cells were heat shocked in a 37°C water bath for 2 minutes and then added to 1 ml of L B . Cells were incubated for 1 hour before plating on L B containing ampicillin. Plasmids are listed in Table II. 48 Materials and Methods T A B L E II Plasmids Used in This Study Plasmid Use Construction/source YCplac22 YCpG4 YCptriple YCp699 pJRG2 pTLG2 pIS120 TRP1 marker; single copy (CEN) yeast plasmid. TRP1 marker; single expresses full length TRP1 marker; single expresses full length S837A TRP1 marker; single expresses full length copy plasmid (CEN); wild type G A L 4 copy plasmid (CEN); GAL4 S691A, S696A, copy plasmid (CEN); GAL4S699A Expresses GAL2 from its own promoter; HIS3 marker; single copy (CEN). GAL2 sequence in URA3 vector. Single copy (CEN). Two-step URA3 disruption plasmid for GALL (75) (206) (206) (206) Contains EcoRI fragment from pTLG2. (235) removes nucleotides +34-+1141 (gall-120) pKW002 Expresses GAL4A683 derivative from ADH1 promoter; TRP1 marker; single copy (CEN). pJR006 Expresses GAL4A683 derivative from ADH1 promoter; URA3 marker; single copy (CEN). pJR008 Expresses GAL4A683 derivative from ADH1 promoter; LEU2 marker; single copy (CEN). YEplacl 12 TRP1 marker; multicopy plasmid (2jx). pDN3 Expresses full length GAL4 from ADH1 promoter; TRP1 marker; high copy (2\x). pDN3/Y14 Expresses full length GAL4 C14Yfrom ADH1 promoter; TRP1 marker; multicopy plasmid (2fx). (217) Bam/Hindlll fragment of pKW002 in pRS316. Bani/HindTII fragment of pKW002 in pRS315. (75) (205) (205) pJR015 pRS314 library HIS3 marker; ARS-CEN plasmid; Expresses GAL3 from its own promoter. TRP1 marker; single copy yeast plasmid; single copy (CEN) genomic library. PCR amplified GAL3 product digested with EcoRI/EcoRV and cloned into pRS313. (212) 49 Materials and Methods pK0G4 Disruption plasmid for GAL4. pRS306 , URA3 marker; yeast integrating plasmid p306G3 URA3 marker; yeast integrating plasmid carrying GAL3. pJR013 gal3::ADE2 disruption plasmid pKOG3 gal3: :LEU2 disruption plasmid pLKS57 GAL80 sequence cloned into URA3 vector; single copy (CEN). (212) Ffindlll/BamHI fragment of GAL3 in pRS306. Bg l l l fragment containing ADE2 in BglU digested p306G3. Bgl l l fragment containing LEU2 in Bgl l l digested p306G3. pKOG80 Gal80::LEU2 disruption plasmid Gall4 Contains Bglll/Bamffl hisG-URA3-hisG (2) deletion fragment from pNKY51 cloned blunt into the SphI/ Xhol sites of YCpG4. pRS313 HIS3 marker; single copy yeast vector single copy ( C E N ) . (212) pRS314 TRP1 marker; single copy yeast vector single copy ( C E N ) . (212) pRS316 URA3 marker; single copy yeast vector single copy ( C E N ) . (212) pRS303 HIS3 marker; yeast integrating plasmid. (212) pIS027 TRP1 marker; high copy yeast vector (2u.). p J R 0 1 4 TRP1 marker; Expresses GIP2 from ADH1 promoter; high copy plasmid (2u.). pRS313 HIS3 marker; single copy yeast vector single copy ( C E N ) . (212) pRS314 TRP1 marker; single copy yeast vector single copy ( C E N ) . (212) pRS316 URA3 marker; single copy yeast vector single copy ( C E N ) . (212) pRS303 H1S3 marker; yeast integrating plasmid. (212) pIS027 TRP1 marker; high copy yeast vector (2u.). p J R 0 1 4 TRP1 marker; Expresses GIP2 from ADH1 promoter; high copy plasmid (2tx). Bgl l l fragment containing LEU2 in pLKS57 PCR amplified GIP2 digested with Xbal/Xhol and ligated into pIS027. 50 Materials and Methods 2.2 GROWTH CURVES Growth curves were performed by growing cultures in 5 ml of liquid SD media to saturation at 30°C. Cultures which maintained an A 6 0 0 fo r one day were considered to be in stationary phase. Cultures were then diluted into 30 ml of fresh SD media in triplicate to equivalent A 6 0 0 . Growth was monitored by A 6 0 0 (100 u.1 samples in 900 u,l of sterile SD media). Results presented are averages of the three independent cultures and vary less than 15% for each point. 2.3 THERMOTOLERANCE Thermotolerance of cells was determined as follows. Cultures were grown in liquid SD media to log phase as determined by monitoring A 6 0 0 . Samples (100 u.1) were diluted.in YEPD, and subjected to heat shock at 55°C for 30 minutes. Cells were plated on YEPD media and grown at 30°C to determine survival in comparison with non heat shocked cells. Survival is presented as the average of 3 independent heat shocked samples. 2.4 STRAINS Genotypes of the strains used in these studies are listed in Table III. The YT6::171 and the YT6G80::171 are derived from S288C. All'other strains are derived from W303-1A. Gene disruptions were performed as either one step, or two step disruptions as described in Kaiser (117). Strains were maintained on YEPD media. For long term storage, cultures were grown in .51 Materials and Methods Y E P D to saturation and 0.5 ml of culture was added to. 0.5 ml of sterile 50% glycerol and frozen at -70°C. 52 Materials and Methods TABLE III Yeast Strains STRAIN GENOTYPE SOURCE YT6G80::171 MATcc, ade2, canl, his3, lys2, leu2, trpl, ura3, gal4::hisG, gal80 del, LEU2::GAL80, URA3::GALl-lacZ (205) YT6::171 MATa, ade2, canl, his3, lys2, leu2, trpl, ura3, gal4::hisG, gal80 del, URA3::GALl-lacZ (205) W303-1A MATa, ade2, canl, his3, leu2, trpl, ura3 H. Ronne YJR5 MATa, ade2, canl, his3, leu2, trpl, ura3, URA3::GAL1-lacZ This study YJR7 MATa, ade2, canl, his3, leu2, trpl, ura3, gal3::LEU2 This study YJR7::131 MATa, ade2, canl, his3, leu2, trpl, ura3, gal3::LEU2, URA3::GALl-lacZ This study YJR10 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG This study YJR10::131 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, URA3::GALl-lacZ This study YJR11::131 MATa, ade2, canl, his3, leu2, trpl, ura3, gal80::LEU2, URA3::GALl-lacZ This study YJR14 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, gal3::LEU2 This study YJR14::131 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, gal3::LEU2, URA3::GALl-lacZ This study YJR14a MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, gal3::LEU2, URA3::GALl-lacZ This study H617 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3 H. Ronne YJR20 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3, gal4::hisG, URA3::GALl-lacZ This study YJR21 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3, This study YJR21::131 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3, gal4::hisG, URA3::GALl-lacZ This study YJR47 MATa, ade2, canl, his3, leu2, trpl, ura3, srblO::HIS3, gal3::LEU2 This study YJR48 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, gal3::ADE2, LEU2::GAL11P, URA3::GALl-lacZ This study YJR52 MATa, ade2, canl, leu2, trpl, ura3, gal3::LEU2 This study 53 Materials and Methods YJR58 MATa, ade2, canl, his3, leu2, trpl, ura3, gal3::LEU2, gall-120 This study YJR59 MATa, ade2, canl, his3, leu2, trpl, ura3, gal4::hisG, gal3::LEU2, srb 10::HIS3, URA3::GALl-lacZ This study YJR60 MATa, ade2, canl, his3, leu2, trpl, ura3, gal3::LEU2,~ swgl-1 This study BY4742 MATa, his3, leu2, lys2, ura3 Research Genetics BY4741 MATa, his3, leu2, metl, ura3 Research Genetics R.G.10189 MATa, his3, leu2, lys2, ura3, gip2::Hyg Research Genetics R.G. 189 MATa, his3, leu2, metl, ura3, gip2::Hyg Research Genetics 2.5 YEAST TRANSFORMATIONS Most yeast media was prepared as described by Kaiser (117). Synthetic phosphate-depleted media was made by dissolving 7g of yeast nitrogen base without amino acids, 1 g of amino acid supplement, and the required carbon sources in 1L dH20. 10 ml of concentrated ammonium hydroxide and 10 ml of 1M M g S 0 4 were added and the solution was stirred vigorously, and then allowed to stand for 30 minutes at room temperature. The precipitate was filtered through Whatman 3M paper, adjusted to pH 5.8 with HC1, and autoclaved. YEPD 1% yeast extract 2% Bacto-peptone 2% glucose SD 0.7% yeast nitrogen base (without amino acids) supplemented with the following: 20mg/l each of adenine, uracil, tryptophan, histidine, arginine, methionine; 30 mg/1 each of tyrosine, 54 Materials and Methods leucine, isoleucine, lysine; 50 mg/1 of phenylalanine; 100 mg/1 each of glutamic acid and aspartic acid; 150 mg/1 of threonine; 375 mg/1 of serine. One or more of the amino acids listed above were omitted when indicated. Carbon sources were added to the following concentrations when indicated: glucose 2%, rafinose 2%, galactose 2%, lactic acid 10%, glycerol 5%, ethanol 2%. For plates, agar was added to 2%. Sporulation medium: Potassium acetate 1% Bacto-agar 2% For most transformations of plasmid D N A into yeast, a modified lithium acetate transformation procedure was used as described in Kaiser (117). Briefly, a large loop of cells was resuspended in an eppendorf containing 300uL of P L A T E (45% PEG 4000, 100 mM lithium acetate, 10 m M Tris-Cl (pH7.5), 5 mM EDTA), and 50 uL of DMSO was added. Usually about 1 \xg of plasmid D N A (or 5 uJ of miniprep DNA) was added and the mixture was vortexed and allowed to incubate at room temperature for 2-20 hours. The mixture was then heat shocked in a 42°C water bath for 15 minutes and the entire mixture was plated onto selective media and incubated at 30°C. The method of Gietz and Sugino was used when transforming with a plasmid library (74). 2.6 p-GALACTOSDDASE ACTIVITY (3-galactosidase activity measured in two ways. In cases where the GALl-lacZ activity was low (for example when using theYT6G80::171 and YT6::171 strains), enzymatic activity 55 Materials and Methods was measured from lysed cells and protein activity was normalized to the amount of protein in the cell as described (205). In most experiments using W303-1A derived strains, P-galactosidase activity was measured in permeablized cells and the activity is expressed in Miller units according to Guarente (84). Briefly, the optical density of each culture was determined at 600 nm just prior to the assay. A 100 ul sample was added to 500 u.1 Z buffer (100 mM sodium phosphate pH 7.0, 10 mM potassium chloride, 1 mM MgS04, 0.27% 2-mercaptoethanol). The cells were permeablized by adding 50 ul each of 0.1% SDS and chloroform and vortexing for 30 seconds. One hundred sixty u,l of ONPG substrate (0.4% ONPG in Z-buffer) was added and the reaction was incubated at 30°C until a yellow color developed. The length of the reaction was recorded and the reaction was stopped by the addition of 500 ul of 1 M sodium carbonate (pH 11.0). The (3-galactosidase activity was determined by the following formula: P-galactosidase units = A ^ X 1000 OD, n n X vol. X time oOO Where vol. is the volume of sample assayed in mis, and time is the length of the assay in minutes. In all time course experiments, the data points are the average of three separate cultures grown from independent colonies. A l l data points are within 15% of one another and repeated at least twice. 2.7 MUTAGENESIS Yeast strain YJR7::131 was grown in Y E P D to an O D 6 0 0 of 1.0. Cells were aliquotted by adding 0.5 ml of culture to 0.5 ml of sterile 50% glycerol and then quickly frozen at -70°C. An 56 Materials and Methods aliquot of cells was thawed on ice and serial dilutions were made in sterile dH2Q. To determine survival rates of the cells, 100 jLtl of the unmutagenized cells were plated on SD media and incubated at 30°C. Two hundred \x\ of cells for mutagenesis were plated onto SD media containing glycerol and lactic acid as the carbon sources and allowed to dry. Cells were mutagenized by exposure to 3 mJ ultraviolet light in a Stratagene crosslinker and then incubated at 30°C. By comparing the number of colonies on the unmutagenized cultures to the mutagenized cultures, an optimal dilution giving a survival rate of approximately 10% was determined. After colonies had formed they were replica plated onto EB-galactose plates and incubated at 30°C. The master plates were wrapped with parafilm and stored at 4°C. After 5 days, the EB-galactose plates were compared with the master plates. Putative mutants, unable to grow on EB-galactose plates, were recovered and grown on YEPD. 2.8 MATINGS, SPORTJLATION, TETRAD ANALYSIS Matings were performed as follows: yeast to be mated were grown overnight on YEPD medium as patches at 30°C. A large loopful of one of the strains was resuspended in 1 ml of YEPD and 250 \x\ was spread onto a YEPD plate and allowed'to dry. The other strain was replica plated onto the lawn of cells and the plate was incubated at 30°C for 2 hours. The plate was then replica plated onto selective media and incubated at 30°C. Sporulation was performed as descrbed by Kaiser (117). Briefly, diploid yeast were grown overnight in 5 ml of YEPD and 1 ml was pelleted by centrifugation for 20 seconds. The pellet was resuspended in 1 ml of dFTiO and 300 |0.1 was plated onto sporulation media. After 2 57 Materials and Methods days, and when asci had developed, a loopful of spores were resuspended in 300 u.1 of TE sorbitol and 25 u.1 of glusulase (Dupont) was added and allowed to digest at room temperature for 20 minutes. Digested asci were streaked onto YEPD and dissected using a Zeiss micromanipulator. 2.9 SEQUENCING Sequencing was performed using a ABI 310 genetic analyzer by the following method. l-300ng of plasmid (usually 1 ul of miniprep DNA) 1-3 pmol of primer (in a volume of 2 ul, and 2ul of Perkin Elmer Big Dye termination mix were brought to a final volume of 5ul in a 250 ul thin-walled PCR tube and subjected to PCR at the following conditions: 96°C 5 minutes (denaturation) 96°C 5 seconds 50°C 10 seconds 60°C 4 minutes cycle was repeated 25 times. The D N A was precipitated as described above, dried and resuspended in 5 ul of Perkin Elmer Template Supression Reagent, heated to 95°C for 2 minutes in a heating block and loaded on to the ABI 310 Genetic Analyzer. 2.10 PLASMID RESCUE Rescue of library plasmids was performed by the following method. Yeast were grown on selective media at 30°C. A loopful was suspended in 300 ul of lysis buffer (2% Triton X -100, 1% SDS, lOOmM NaCl, lOmM Tris pH 8.0, 1 mM EDTA) and 200 pi of 58 Materials and Methods phenol:chlorofdrm:isoamyl alcohol (25:24:1) was added. Two scoops of glass beads (approvimately 200uJ per scoop) were added and the mixture vortexed at 4°C for 10 minutes. Samples" were centrifuged for 5 minutes and the aqueous layer was collected into a new tube. D N A precipitated as described previously, and resuspended in 20 [x\ of dH20. The Stratagene SURE cells were used for electroporation. Electrocompetent SURE cells were prepared as follows. Cells were grown in 500 ml SOB medium at 37°C to a OD420 of 0.5. Cells were collected by centrifugation in a GSA rotor spun at 7,000 rpm for 20 minutes. The cell pellet was resuspended in 500 ml of ice cold wash buffer (10% glycerol). Cells were centrifuged again and resuspended in the residual wash buffer (about 10 ml) and aliquoted into sterile tubes. Cells were then quickly frozen in liquid nitrogen and stored at -70°C until needed. Cells were thawed on ice and 20 ul were mixed with 1 or 2 u.1 of D N A to be electroporated. Cells were electroporated at 330mF (capacitance) and 4 kV (voltage) using a B R L Cell-Porator. Electroporated cells were added to 1 ml of L B and incubated at 37°C for 1 hour before plating, on to L B containing ampicillin. Plasmid D N A was isolated from these transformants for sequence analysis. Sequencing of all plasmids rescued from the pRS314 library was performed as.described above using the M13/pUC sequencing primer (New England Biolabs). 2.1.1 PHOSPHOPEPTTDE ANALYSIS Cells bearing GAL4 expression plasmids were grown in 50 ml of SD glycerol at 30°C until they reached a A 6 0 0 o f approximately 1.0. The culture was then harvested, washed 2 times with phosphate depleted media and resuspended in 50 mis of phosphate depleted media. The 59 Materials and Methods cultures were starved for phosphate for 2 hours, pelleted, then resuspended in 0.5 ml of phosphate depleted media. Next, 5 mCi of H 3 3 2 P 0 4 (in 0.02 N HC1, ICN Biochemicals) was added as well as galactose and raffinose to a final concentration of 2% each. Cells were incubated at room temperature for 1 hour. 2.12 IMMUNOPRECEPITATION Labeled cells were washed 3 times in ice cold lysis buffer (50mM Tris, pH 8.0, 5 mM MgCl 2 , 150 m M NaCl, 5 m M NaF, 2 mM ZnS0 4) containing the protease inhibitors 1 mM PMSF, 10 ug/ml aprotinin, and 1- u M leupeptin. The cells were then lysed in 400 ul of the same buffer with 200 ul of glasss beads (Sigma Chemical Co., acid-washed, equal volumes of 150-200 micron and 450-600 micron sizes) by vigorous vortexing at 4°C for 30 minutes. At this time 400 u.1 of 2X RIPA buffer (20mM Tris, pH 8.0, 200 m M EDTA, 2% NP40, 1% sodium deoxycholate, 2% SDS, and protease inhibitors) was added, and the samples were vortexed for an additional 20 minutes. Samples were clarified by centrifugation at 4°C at 17000 g for 30 minutes, and the supernatant transferred to a new tube containing 50ul of 10% formalin-fixed Staphylococcus aureus (Zymed Labs). The samples were incubated at 4°C for 30 min with constant agitation, and then centrifuged. The supernatant was removed to a new tube, and 5 ui of rabbit anti-Gal4 B D polyclonal antibody was added (205). The mixture was incubated on ice for 1 hour, and then the immunocomplexes were precipitated by the addition of 50 ul of 10% S. aureus and incubation at 4°C for 1 hour with constant agitation. Immunoprecipitates were recovered by centrifugation and successively washed with Wash 1 (lOmM Tris, pH8.0, 1M NaCL 0.1% NP40), Wash 2 (10 mM Tns,j>H 8.0, 0.1 M NaCl, 0.1% NP40, 0.1% SDS), Wash 3 60 Materials and Methods (10 m M Tris, pH8.0, 0.1% NP40), and then 1XRIPA buffer (each containing protease inhibitors). The immunoprecipitates were then resuspended in I X SDS sample buffer (156), incubated at 37°C for 10 minutes, and the S. aureus removed by centrifugation. 2.13 SDS PAGE AND PREPARATION OF TRYPTIC PHOSPHOPEPTTDES SDS P A G E was'performed as described by Laemmeli (136). Gels were exposed to X-ray film (Kodak Co. Biomax) to identify the location of the labelled proteins. Gel slices were then excised and dehydrated by the addition of 500 \xl of acetonitrile. The acetonitrile was removed after 10 min, and 200 yd of ammonium carbonate containing 10 m M DTT was added and incubated at 56°C for 1 hour. The DTT solution was removed and 200 | i l of 55mM iodoacetamide in 100 mM ammonium carbonate was added and incubated in the dark at room temperature for 45 minutes. The gel slice was washed with 100 u,l of 100 m M ammonium carbonate and then dehydrated by the addition of 200 jo.1 of acetonitrile and incubation for 10 minutes. The wash and dehydration were then repeated and the gel slice was then dried in a speedvac. The gel slice was resuspended in 100 | i l of 50 m M ammonium carbonate and 10 ul of 1 mg/ml trypsin (Boeringer-Manheim) was added and incubated at 4°C for 45 minutes. Another 10 \x\ of trypsin was added and incubated at 37°C for 15 hours. The mixture was centrifuged and washed 3 times with 200 (il of 20 m M ammonium carbonate. The supernatants from the 3 washes were combined and 200 u.1 of 5% formic acid in 50% acetonitrile was added and incubated for 20 minutes at room temperature. This was repeated twice more for a total of 60 minutes and resulting in a final volume of 600 |J,1 formic acid iodoacetamide. The samples were 61 Materials and Methods Finally the dried sample was 2.14 TWO DIMENSIONAL ANALYSIS OF TRYPTIC PEPTIDES Dried phosphopeptides were dissolved in 15 ul of pH 2.1 buffer (2% formic acid, 8% acetic acid), and equivalent cpm were spotted for comparable samples on the right hand corner (two cm up and 5 cm from the bottom) of a thin-layer cellulose plate (Whatman). The plate was then moistened evenly with pH 2.1 buffer and mounted on a flat bed electrophoresis apparatus. Electrophoresis was performed in pH 2.1 buffer at 1000 V for 50 minutes. After electrophoresis, the plate was dried and ascending thin-layer chromatography was performed in the second dimension in B A W P buffer (60 ml dH 2 0, 75 ml 1-butanol, 50 ml pyridine, 15 ml acetic acid) for 8-24 hours until the solvent front reached 2cm from the top of the plate. The plates were dried and exposed to X-ray film or phosphor-imager screens (Kodak). then lyophilized and resuspended in 300 (al of dH 2 0 3 times, analyzed by Cerenkov counting. 62 Results 3. R E S U L T S 3.1 S699 OF GAL4 IS REQUIRED FOR PROPER INDUCTION IN YT6G80::171 The main objective of this work was to gain insight to the signaling mechanisms that regulate Gal4 activity. Earlier work in our laboratory had mapped a number of phosphorylation sites on Gal4. These include a cluster of serine residues located within the glucose response domain (S691, S696, S699) as well'as one serine residue within the activating region 2 at S837 (205). Of these, only S699 appeared to be required for efficient GAL gene induction as determined by P-galactosidase assays comparing Gal4 WT to Gal4 S699A mutations. The phenotype associated with GAL4 S699A mutation was observed only in GAL80 yeast. In other words, gal80 cells bearing the S699A mutation were able to activate GAL gene activity at levels similar to wild type Gal4 (206). In order to examine this more closely we examined the kinetics of GAL gene induction over time (Figure 6). After addition of galactose, the S288C-derived strain YT6G80 (gal4, GAL80) bearing WT Gal4.induced GAL gene expression within 5 hours, reaching a maximum level at 10-15 hours; however, cells harboring Gal4 S699A did not induce GAL gene expression, even after 25 hours. 63 Results Figure 6. Phosphorylation of S699 of Gal4 is required for efficient induction of the GAL genes in yeast strain YT6G80::171. Yeast strain YT6G80::171 was transformed with YCplac22 {gal4-){U), YCpG4 (WT) (•), YCptriple (triple)(0), or YCpS699A (S699A)(«). Yeast cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to early log phase. Galactose was added to a final concentration of 2% from a separately sterilized 40% stock and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples at the indicated time points. LVgalactosidase was measured in cell lysates as described in the Materials and Methods. 64 Results 30-1 o o v~\ o o — r- cs en Hours 65 Results 3.2 YT6::171 IS Gal DUE TO A WEAK ALLELE OF GAL2 The major gene products required for GAL induction are the galactose permease Gal2 and the inducer protein Gal3. It was found that the strain YT6::171 (gal4, gal80) expressing WT Gal4 from a centromeric plasmid (YCpG4) was unable to form colonies on media containing galactose as the sole source of carbon (EB-galactose). This is not surprising, as most S288C derived strains bear mutations in gal2 which makes mitochondrial function essential for use of galactose (3). This strain is gal80 and reporter gene constructs demonstrate that the GAL genes are expressed maximally (206). Figure 7 shows that this Gal- phenotype could be complemented by a plasmid bearing GAL2. This result is reminicent of a report from the Jaenning laboratory which showed that GAL gene induction could occur in strains bearing mutations in GAL2, that prevent growth on galactose media (239). 3.3 GAL4 S699 IS NOT REQUIRED FOR GAL GENE INDUCTION IN STRAINS DERIVED FROM W303-1A The Gal4 S699A mutation was also found to impair GAL induction in YM707 {gal4, GAL80) which is derived from the same S288C background as YT6G80:: 171. The YT6G80::171 strain induces GAL gene expression fairly slowly (Figure 6) and also grows more slowly in comparison to another yeast strain W303-1A. Most laboratory strains derived from S288C are known to contain mutations in the GAL2 locus (239). W303-1A is a commonly used laboratory strain that has been shown to rapidly induce the GAL genes and bears a fully functional GAL2 allele (3, 172). Surprisingly, I found that in contrast to the S288C-derived strains, the GALA S699A mutation had no effect on GAL gene induction in W303-1A (Figure 8). 66 Results Such differences between strains are seen on occasion and in some cases have given insight to cellular processes (a recent example is in determining the requirement of the transcription factor FI08 in pseudohyphal development (149)). In comparing the data in Figure 6 to Figure 8 it is evident that the W303-lA-derived strain YJR10::131 shows more rapid induction of the GAL genes than the YT6G80::171 and that serine to alanine mutations of Gal4 do not impair GAL gene expression, including S699A (Figure 8). In contrast, this mutation displays a strong defect in GAL induction in the YT6G80:: 171 strain (Figure 6). 67 Results Figure 7. Yeast strain YT6::171 is Gal- due to a weak allele of GAL2. Yeast strain YT6::171 is Gal- due to a defective allele of GAL2. Yeast strain YT6::171 was transformed with combinations of the following plasmids: pRS313 is an ARS-CEN HIS3 vector control, pJRG2 is pRS313 containing a genomic GAL2 fragment. YCplac22 is an ARS-CEN TRP1 vector control, YCpG4 is YCplac22 containing a GALA genomic D N A fragment. Transformed strains were streaked onto EB-galactose plates, incubated at 30°C for 5 days and then photographed. 68 Results pJRG2, YCpG4 69 Results Figure 8. Phosphorylation of Gal4 S699 is not required for efficient induction of the GAL genes in yeast strain YJR10::131. Yeast strain YJR10::131 was transformed with YCplac22 ( vector control gal4-), YCpG4 (WT Gal4), YCptriple (Gal4 S691A, S696A, S837A), or YCpS699A ( Gal4 S699A). Cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to early log phase. Galactose was added to a final concentration of 2% from a separately sterilized 40% stock, and expression of a GALl-lacZ reporter gene was monitored by measuring (3-galactosidase activity from culture samples after 2.5 hours. $-galactosidase activity was measured in permeablized cells as described in the Materials and Methods. 70 71 Results 3.4 Gal4 PHOSPHORYLATION AT S699 IS REQUIRED FOR SENSITIVE RESPONSE TO THE INDUCER GALACTOSE Typically, GAL expression is induced experimentally by adding galactose to a final concentration of 2%. Because YT6G80::171 appeared to be defective in the GAL2-encoded permease (Figure 7), it appeared that the effect of the Gal4 S699A mutation might be overcome by the high level of galactose used in these experiments. To test this idea, the induction of GAL genes was examined in YJR10::131 bearing either wild type Gal4 or the Gal4 S699A mutation. Figure 9 panel A shows that the Gal4 S699A mutation does not affect induction when galactose is added to a concentration of 2%. However, when cells are induced with limiting amounts of galactose (0.02%), induction of the GAL genes was impaired by the Gal4 S699A mutation, indicating that phosphorylation of this site is required for sensitive response to the inducer (Figure 9, panel B). 3.5 THE W303-1A GALACTOSE SIGNALING MECHANISM IS DOMINANT TO YT6G80::171 The differential effect of the GALA S699A mutation in the S288C and W303-1A yeast backgrounds, combined with the fact that some S288C-derived strains respond more slowly to galactose (not shown and compare (Figures 6 and 8)), suggested that there may be differences in the signaling mechanisms between these strain backgrounds. The observations which demonstrate that various S288C-derived strains bear weak GAL2 and/or GAL5 alleles are consistent with this notion (239, 250) and Figure 7. To directly examine the role of GAL3 in GAL gene induction of these two strains, diploid yeast strains were constructed by crossing 72 Results YT6G80::171 with either YJR10::131 (GAL3) or YJR14::1'31 (gal3). The Gal4 S699A mutation did not alter induction of GAL genes by 2% galactose in diploid yeast produced by crosses produced between YT6G80::171 and YJR10::131 (GAL3) haploids (Figure 10), demonstrating that the W303-1A phenotype is dominant over that of YT6G80::171. However, in contrast, the GALA S699A mutation prevented efficient induction in diploids produced from crosses between YT6G80::171 and W303-1A haploids bearing a disruption in ga/3 (YJR14::131). These results demonstrate that the Gal4 S699 phosphorylation is necessary for full Gal4 activity in response to limiting inducer (galactose), or when the inducer signaling mechanism is impaired. One implication of this observation is that induction of Gal4 activity must involve two different mechanisms, represented by Gal3-galactose, and the S699 phosphorylation. The requirement for the latter mechanism may be bypassed by concentrations of galactose in excess of that which is required for efficient induction in strains with fully functional GAL3 alleles. 73 Results Figure 9. Phosphorylation at S699 is required for sensitive response to the inducer galactose. Yeast strain YJR10::131 was transformed with YCplac22, vector control ( • ) , YCpG4, WT Gal4 (•), or YCpS699A, Gal4 S699A (•). Cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to early log phase. Galactose was added to a final concentration of 2% (A.) or 0.02% (B.) from a separately sterilized 40% stock and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples taken at the indicated times. (3-galactosidase activity was measured in permeablized cells as described in the Materials and Methods. 74 Results 75 Results Figure 1 0. Phosphorylation at S699 is required for efficient induction of the GAL genes in cells lacking a fully functional allele of GAL3. Diploid yeast produced by crossing YJR10::131 (GAL3) or YJR14::131 (gal3) with YT6G80::171 were transformed with YCplac22 ( vector control, gal4-), YCpG4 (WT Gal4), or YCpS699A ( Gal4 S699A). Cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to early log phase. Galactose was added to a final concentration of 2% from a separately sterilized 40% stock and expression of a GALl-lacZ , reporter gene was monitored by measuring the (3-galactosidase activity from culture samples after 2.5 hours. |3-galactosidase activity was measured in permeablized cells as described in the Materials and Methods. 76 77 Results 3.6 Gal4 S699 PHOSPHORYLATION IS REQUIRED FOR THE LONG TERM ADAPTATION (LTA) RESPONSE TO GALACTOSE Yeast bearing a gal3 disruption induce GAL gene transcription after several hours or even days in response to galactose (204, 250). This L T A response to galactose was observed in some of the earliest laboratory strains of Saccharomyces, and suggests that the GAL genes are regulated by a second mechanism independently of Gal3 (204, 250). Because mutations to GALA S699 have-a more severe effect in the S288C background such as YT6G80::171, which appear to have a weak GALS allele, it was important to examine whether S699 phosphorylation was necessary for L T A in yeast completely lacking GAL3. The experiment shown in Figure 11 revealed that GAL gene induction occurred 24 hours post-galactose addition in W303-1A yeast bearing a gal3 disruption and expressing wild type GAL4, but that gal3 yeast expressing the GAL4 S699A mutation never expressed the GAL genes, even after 70 hours. This result clearly demonstrates that S699 phosphorylation is absolutely required for GAL induction in the absence of Gal3, and supports the argument that this modification represents a Gal3-independent regulatory mechanism for Gal4 activity. 78 Results Figure 1 1. Phosphorylation of Gal4 S699 of is required for long term adaptation. Yeast strain YJR14:: 131 (gal3) was transformed with either YCpG4 or YCpS699A. Yeast cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to very early log phase (A600=0.2). Cultures bearing the YCpG4 plasmid (•) and another bearing the YCpS699A plasmid (•) were induced with galactose at a final concentration of 2% from a separately sterilized 40% stock. Parallel cultures bearing YCpG4 ( • ) and YCpS699A (O) were left uninduced. In each case the expression of a GALl-lacZ reporter gene was monitored by measuring the p-galactosidase activity from culture samples taken at the indicated times. (3-galactosidase activity was measured, in permeablized cells as described in the Materials and Methods. 79 Results Hours 80 Results 3.7 Gal4 IS PHOSPHORYLATED AT S699 INDEPENDENTLY FROM THE Ga 13-GALACTOSE SIGNALING PATHWAY "This is purely a biochemical problem." Ojvid Winge (1948) GAL3 shares significant homology to the galactokinase encoded by GAL1 (14, 222). In fact it has been demonstrated that GAL1, when overexpressed, is able to substitute for Gal3 in the role of inducer (16). Additionally, Reece and Piatt recently demonstrated that Gal l can fuctionally interact with Gal80p in a galactose- and ATP-dependent manner, similar to Gal3 (190). The genetic data presented here suggests that Gal4 is phosphorylated at S699 by a mechanism independent of the Gal3-galactose signaling pathway and it was of interest to know if this was also independent of Gal l activity. To determine this, Gal4 was subjected to phosphopeptide analysis from wild type cells and cells bearing disruptions in both GAL1 and GAL3. Figure 12 shows that the phosphorylation pattern of Gal4 in both wild type cells as well as that of the gall gal3 mutant are nearly identical. Most important is the fact that peptide 1 (indicated by the arrow) is equally present in both cases. This peptide has been shown to contain S699, and is dependent upon the R N A polymerase II holoenzyme-associated kinase SrblO (98). It should be noted that the Gal4 derivative used to map phosphorylation sites is comprised of residues 1-147, fused to 768-881. This derivative causes a slightly higher basal level of expression of GAL genes compared to full-length Gal4 but is regulated in a similar manner and contains all sites of phosphorylation. Despite repeated attempts, we have been unable to recover sufficient amounts of labelled full-length Gal4 for phosphopeptide analysis. 81 Results Figure 12. Phosphorylation of Gal4 S699 occurs independently of the Gal3-galactose signaling pathway. Yeast strain YJR58 (gall, gal3) in panel A , or W303-1A in panel B, bearing the plasmid pJR006 expressing Gal4 A683 (1-147, 768-881) were grown in liquid media containing glycerol and lactic acid as the carbon source to early log phase. Cells were washed in phosphate depleted media and labeled with [j2P]-orthophosphate at the same time that galactose and raffinose were added to a final concentration of 2% from separately sterilized stocks. After 2 hours, Gal4 protein was isolated by immunoprecipitation and analyzed by tryptic phosphopeptide mapping. Approximately equal cpm for each sample was analyzed. In both panel A and panel B the peptide indicated by the arrow corresponds to tryptic peptide 1 as described by Hirst (98),which contains S699. 82 Results WT gall-, gal3-83 Results 3.8 SRB10 IS REQUIRED FOR PROPER INDUCTION OF THE GAL GENES Once a phenotype associated with the Gal4 S699 phosphorylation was identified, it was of interest to determine the nature of the kinase. Earlier work in our laboratory suggested that the kinase was likely to be a component of the general transcription machinery, as Gal4 phosphorylation appeared to occur as a consequence of activation (205). There are two kinases in the R N A polymerase II holoenzyme: Kin28 (the catalytic component of TFIIH) and the recently identified SrblO. Both are cyclin-dependent kinases capable of phosphorylating the CTD of R N A polymerase II. Null mutations in kin28 are lethal and loss of Kin28 function results in loss of polymerase II transcription. Mutations in srblO are viable and are associated with the derepression of a subset of genes (100, 134, 243). Additionally, SrblO has been implicated in the activation of genes (9, 134, 146).. Young and coworkers observed that SrblO activity was required for the RNA polymerase II holoenzyme to be responsive to activators such as Gal4 (146). Additionally, very similar screens carried out in the laboratories of Marian Carlson and Hans Ronne identified a role for SrblO in the activation of the GAL genes (9, 134). These observations prompted examination of the role of SRB10 in the induction of the GAL genes. Figure 13 shows that in strains containing wild type Gal4 there is a slight (but reproducible) derepression of the GAL genes in srblO- cells growing in non-inducing conditions. Upon induction by galactose however, srblO cells are impaired for expression of the GAL genes. These results are very similar to those obtained in the Carlson laboratory (134). 84 Results Figure 13. The holoenzyme-associated kinase SrblO plays a role in both repression and induction of the GAL genes. (A.) Yeast strain YJR5 (WT) or H617::131 (srb 10-) were grown to log phase in selective liquid media containing glycerol and lactic acid as the carbon source and expression of a GALl-lacZ reporter gene was assayed by measuring the (3-galactosidase activity from culture samples. (B.) Galactose was added to a final concentration of 2% from a separately sterilized 40% stock and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples after 2.5 hours. (3-galactosidase activity was measured in permeablized cells as described in the Materials and Methods. 85 Uninduced Results WT srbW Induced T X WT srbW 86 Results 3.9 SrblO AND Gal3 DEFINE TWO INDEPENDENT REGULATORY MECHANISMS FOR Gal4 Gal4 phosphorylation has been shown to occur independently of Gal3 (171) or Gal l (Figure 12), and the results presented above demonstrate that the Gal4 699A mutation affects a regulatory mechanism that occurs independently of Gal3. Furthermore, our laboratory has previously shown that WT Gal4 phosphorylation does not necessarily require galactose, because phosphorylation can be stimulated by either galactose or glucose in gal80 strains where Gal4 activity is constitutive (205, 206). Taken together with the finding that S699 phosphorylation is mediated by the R N A polymerase II holoenzyme component SrblO (98), it seemed likely that the regulatory effect of SrblO on GAL transcription must occur independently of Gal3. This possibility was examined by testing whether gal3, srblO, and gal3 srblO strains were capable of growth on galactose as the sole source of carbon, as a measure of their ability to induce GAL transcription. For this purpose I used Y E P galactose containing ethidium bromide (EB-galactose). I found that disruption of gal3 reduces growth on EB-galactose compared to wild type yeast (Figure 14). Similarly, strains bearing srblO disruptions grow slightly more slowly on EB-galactose than do wild type yeast. However, in contrast, yeast bearing a mutation in both srblO and gal3 are incapable of growth on EB-galactose, demonstrating that both Gal3 and SrblO are required for full GAL gene induction. The additive effects of gal3 and srblO mutations support the notion that Gal3 and SrblO are involved in independent or parallel mechanisms for GAL gene regulation. 87 Results 3.10 SRB10 IS EPISTATIC TO GAL4 699 PHOSPHORYLATION FOR GAL INDUCTION As shown above, and in agreement with other published data (9, 134, 146) an srblO mutation caused a decrease in the induction of the GAL genes. Martin Hirst in our laboratory has demonstrated that SrblO is capable of phosphorylating Gal4 at S699 in vitro, and phosphorylates this site on recombinant Gal4 in vitro (98). I found that the Gal4 S699A mutation did not cause an additional defect in GAL transcription in an srblO mutant, indicating that SRBW and Gal4 S699 are genetically epistatic to one another and act in equivalent, instead of parallel regulatory mechanisms (Figure 15). This result is consistent with the results presented above, which show that gal3 and srblO or Gal4 S699A mutations have additive effects on GAL induction. A reservation to this experiment is explained in Appendix A (Figure 26) which demonstrates that the srblO mutation causes an elevated level of GAL transcription when Gal4 is expressed from its own promoter on an ARS-CEN plasmid. The experiment presented in Figure 15 is in a gal3 mutant and the galactose induction seen probably reflects the redundant activity of Gal l . 88 Results Figure 14. Gal3 and SrblO represent separate signaling mechanisms. Yeast strains W303-1A (WT), H617 (srblO-), YJR7 (gal3-), and YJR47 (gal3, srblO-) were streaked on EB-galactose plates as described in materials and methods. Plates were incubated at 30° C for 5 days and photographed. • 89 Results WT srbior gal3~srb10~ 90 Results Figure 15. The Gal4 S699A mutation is epistatic to a mutation in srblO. Yeast strain YJR59 (gal3 srblO) was transformed with either YCpG4 or YCpS699A. Yeast cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to A 6 0 0=0.5. Cultures bearing YCpG4 plasmid (•) or YCpS699A plasmid (•) were induced by adding galactose to a final concentration of 2% from a separately sterilized 40% stock, or YCpG4 ( • ) and YCpS699A (O) cultures were left uninduced. In each case the expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples taken at the indicated times. (3-galactosidase activity was measured in permeablized cells as described in the Materials and Methods. 91 Results 250-1 N O T - CM CO T f U3 (O Hours 92 Results 3.11 THE GAL11P ALLELE CAUSES ELEVATED BASAL ACTIVITY IN Agal3- BACKGROUND BUT DOES NOT FULLY BYPASS THE REQUIREMENT FOR GAL4 S699 PHOSPHORYLATION A major question to address was how Gal4 S699 phosphorylation was controlling activity of the protein. To answer this question, it would be useful to dissect at which step in the transcription cycle Gal4 S699 phosphorylation had its effect. Three possibilities seemed plausible: i) Gal4 S699 phosphorylation regulates the Gal4-Gal80 interaction,, ii) Gal4 S699 phosphorylation favors the recruitment of factor(s) that results in more efficient transcription, or iii) a combination of the two processes whereby Gal4 phosphorylation causes more productive recuitment of factor(s) for transcription which results in a concominant weakening of the Gal4-Gal80 interaction. The dominant allele of GAL11, GAL11P, represents a tool to potentially provide insight in to how the Gal4 S699 phosphorylation functions. GAL11P mutant protein, but not WT GAL11 protein, has been shown to interact with the D N A binding domain of Gal4 (10). It is thought that this interaction artifactually causes activation through artificial recruitment of the holoenzyme. If phosphorylaton of S699 acts at a step in the transcription cycle before recruitment, mutations of this residue in cells expressing GAL11P should bypass the requirement for S699 phosphorylation. If, instead, Gal4 S699 phosphorylation is required for a step downstream of recruitment, one might expect that the Gal4 S699A mutation should inhibit activation in cells expressing GAL11P. Figure 16 shows the results of this experiment. It was found that the GAL11P allele caused elevated basal GAL transcription in glycerol-grown cells. I also found that the Gal4 S699A mutation impaired induction of the GAL genes in GAL11P cells 93 Results (compared to Gal4 WT) by about two fold in both glycerol and galactose grown cells. I interpret these results to mean that the S699A mutation affects Gal4 function in a way that is not fully ' bypassed by aritficial recruitment of the R N A PolII holoenzyme. 3.12 A PHENOTYPE FOR THE S699A MUTATION IN YT6::171 IN gal8 0 CELLS The results obtained in Figure 16 using the dominant allele of GAL11 (GAL11P) imply that Gal80 is capable of inhibiting the artificial recruitment of Gal l IP. In addition, the elevated level of activation seen in non-inducing conditions (without galactose) suggested that Gal4 S699 phosphorylation may affect transcription independent from regulating the Gal4-Gal80 interaction. If this were true, the Gal4 S699A mutation should affect transcription in gal80 yeast, as well as wild-type yeast. It might be that the high level of activation seen in gal80 cells could mask the phenotype of the S699A mutation (similar to the situation with the strong GAL3 allele in high galactose). To limit the level of activation by Gal4 in gal80 yeast, cells were grown in medium containing 2% glucose. Glucose acts through multiple mechanisms to inhibit the activity of the GAL1-10 promoter used in this study (115); however the slightly higher expression from the plasmid encoded GALA allows for some activity in gal80 cells (Sadowski, unpublished). Surprisingly it was found that cells bearing Gal4 S699A showed a two-fold reduction in the level of GAL gene expression compared to cells expressing Gal4 WT (Figure 17). This result demonstrates that the effect of the GAL4 S699A mutation cannot act solely by regulating the interaction between Gal4 and Gal80. 94 Results Figure 16. The GAL11P allele of GAL11 causes elevated levels of GAL expression in gal3 cells, but does not fully bypass the requirement for Gal4 S699 phosphorylation. (A.) Yeast strain YJR48 was transformed with YCplac22 (gal4-), YCpG4 (WT), or YCpS699A (S699A). (A.) Yeast were grown in selective liquid media containing glycerol and lactic acid as the carbon source to log phase and expression of a GALl-lacZ reporter gene was assayed by measuring the (3-galactosidase activity from culture samples. (B.) Galactose was added to a final concentration of 2% from a separately sterilized 40% stock and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples after 2.5 hours. (3-galactosidase was measured in cell lysates produced by lysing cells with glass beads as described in the Materials and Methods. 95 A. Glycerol Results 12.5 10 J 7.5 4 54 2.5 -I 0 J T 1 ga/4- WT S699A B. Galactose 300 100 0 ga/4- WT S699A 96 Results Figure 17. Phosphorylation at Gal4 S699 is required for maximal induction in gal80 cells grown in the presence of glucose. Yeast strain YT6::171 (gal80) was transformed with YCplac22 (gal4-), YCpG4 (WT), or YCpS699A (S699A). Cells were grown in liquid selective media containing glycerol, lactic acid, and 2% glucose to log phase and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples. P-galactosidase was measured in cell lysates produced as described in the Materials and Methods. 97 Results 60 H 50 -I gal4- WT S699A 98 Results 3.13 A GENETIC SCREEN TO ISOLATE GENES REGULATING Gal4 FUNCTION Biochemical and genetic data suggest that Gal4 is regulated by a mechanism independent from Gal3-galactose which involves the holoenzyme Cdk, SrblO (reference (98) and Figures 11-15). I undertook a genetic approach to identify components which are involved in Gal3-independent Gal4 regulation. The screen that I used was similar to a synthetic lethal screen. Synthetic lethal screens are useful for identifying genes which are involved in a process but which act independently from a known component. Here, the goal was to identify genes involved in induction by galactose, but which operated independently from Gal3. Based on the observation that gal3, srblO yeast are unable to grow on EB-galactose plates, it seemed reasonable to predict that mutations affecting upstream regulatory components of SrblO should cause a Gal- phenotype in combination with gal3. The screen is outlined in Figure 18 and was carried out as follows: gal3 cells were mutagenized with 4mJ of U V irrradiation (to give approximately 90% killing) and then allowed to form colonies on media containing glycerol and lactic acid as the carbon source. Once colonies had formed, they were replica-plated onto medium containing galactose as the sole source of carbon (EB-galactose). Allowing the mutagenized cells to form colonies on glycerol-lactic acid media before replica plating to EB-galactose served two purposes. First, gal3 yeast grow poorly on EB-galactose plates (50, 204) and allowing colonies to form ensured that a sufficient amount of cells, would be transferred to the EB-galactose plates. Second, growing yeast on the non-fermentable carbon sources of glycerol and lactic acid eliminated mutations affecting respiration which would give a Gal- phenotype (3). After 5 days, growth on the E B -99 / Results galactose plates was compared to growth on the glycerol-lactic acid master plates. Colonies which could not grow on EB-galactose plates were isolated and subjected to further analysis. The mutants were mated to a strain of the opposite mating type, and the resulting diploids were examined for growth on EB-galactose to determine if the Gal- phenotype was dominant or recessi,ve. Recessive mutants were transformed with a plasmid bearing GAL3 to determine if the Gal- phenotype was caused by a mutation in GALA or one of the known Gal4-regulated genes. Mutants that were Gal- when containing a GAL3 plasmid were discarded. Approximately 700,000 colonies were screened and roughly 250 colonies were initially picked. Mutagenesis was performed as according to Materials and Methods. Most colonies showed only a slight Gal- phenotype when tested again and were discarded. These 38 mutants with a strongly reproducible Gal- phenotype were transformed with a plasmid bearing a genomic copy of GAL3 on an ARS-CEN plasmid and tested for growth on EB-galactose plates. Cells which were Gal- when containing the GAL3 plasmid were discarded as they likely contained mutations in known GAL genes. Only three mutants fell into this class, which reflects that the screen was not saturated. The remaining mutants were mated to a strain of the opposite mating type to test if the mutation was dominant or recessive. Only one appeared to be dominant. In the end, 34 mutants were collected which were recessive and had a strong, reproducible Gal-phenotype which was not complemented by GAL3. One of these mutants showing an especially strong phenotype was selected for further characterization. 100 Results 3.14 THE swgl PHENOTYPE IS DUE TO A SINGLE MUTATION One mutant with a strong Gal- phenotype in a gal3 background was designated swgl (for synthetic with GAL3). Tetrad analysis indicated that the Gal- phenotype segregated 2:2; indicating that the phenotype was due to a mutation in a single genetic locus. This mutant was successively backcrossed to ensure that the Gal- phenotype was not influenced by unlinked genes (Figure 21). The Gal- phenotype was complemented in a backcrossed, haploid swgl strain using a genomic library carried on the ARS-CEN plasmid pRS314. The complementing library plasmid was retransformed into the swgl mutant to make sure that the Gal-i- phenotype was dependent upon the plasmid. 101 Results Figure 18. A genetic screen for isolation of genes involved in Gal3-independent signaling. Yeast strain YJR7::131 (gal3) was mutagenized by exposure to U V irradiation and surviving cells were allowed to form colonies on selective media containing glycerol and lactic acid as the carbon source. Colonies were replica plated to EB-galactose plates and allowed to form colonies. The EB-galactose replicas were compared to the glycerol lactic acid master plates to identify putative Gal- colonies. Putative mutants were transformed with pGAL3 which is an ARS-CEN plasmid expressing GAL3 from its own promoter. Mutants in which the Gal-phenotype could be complemented by pGAL3 were subjected to further analysis. Genes responsible for the Gal- phenotype were identified by transforming the mutants with a pRS314-derived plasmid library and selecting for cells that could form colonies on EB-galactose plates. 102 Results UV Mutagenesis of ga!3 cells o ° o o Compare to master and select Gal-Complement back to wild type (Gal+) using plasmid library Rescue plasmid and sequence 103 Results Figure 19. The swgl phenotype is due to a m u t a t i o n in a s i n g l e gene . The original mutation displaying the swgl phenotype was successively backcrossed to either the parent strain (YJR7::131) or an isogenic strain of the opposite mating type YJR51). Tetrads were dissected on YEPD media and allowed to form colonies for 3 days. These segregated haploids were replica plated to EB-galactose plates, incubated for 6 days and photographed. 104 Results YEPD EB GAL 105 Results 3.15 SWG1 IS ALLELIC TO GIP2 The swg mutations display a Gal- phenotype when combined with a gal3 mutation. Consistent with this, several of the plasmids that conferred a Gal+ phenotype to swgl gal3 contained GAL3. Five plasmids contained inserts of a region from chromosome V. The smallest insert of the complementing plasmids directed attention to the GIP2 gene. GIP2 was identified in a two-hybrid screen using the type I phosphatase Glc7 as bait (237). GIP2 shares homology to Gael, the regulatory subunit of Glc7, which is involved in glycogen metabolism. Roach and coworkers found that GIP2 is not directly involved in glycogen metabolism (.37). The observation that mutations in gip2 cause a Gal- phenotype in combination with gal3 represents the first phenotype caused by mutations in this gene. This is shown in Figure 20; swgl gal3 mutants are incapable of growth on EB-galactose, but growth can be restored by transformation with a plasmid expressing GAL3. A swgl gal3 mutant is capable of growing on EB-galactose when harboring an ADH-GIP2 expression plasmid, but not a vector control (Figure 21), supporting the conclusion that the swgl phenotype is caused by a mutation in GIP2. 106 Results Figure 20. The swgl mu ta t ion only causes a G a l - phenotype i n c o m b i n a t i o n wi th gal3. Yeast strain YJR60 (swgl, gal3) was transformed with the plasmid pJR015 which is an ARS-CEN plasmid expressing a genomic copy of GAL3 (swgl, gal3 CEN-GAL3), or pRS313 (swgl, gal3 vector). These transformants were streaked with W303-1A (GAL3) and YJR7 (gal3) onto EB-galactose plates, incubated at 30°C for 5 days and photographed. 107 Results Results Figure 21. The swgl phenotype is complemented by a plasmid expressing GIP2. Yeast strain YJR60 (swgl; gal3) was transformed with plasmid pJR014 which expresses GIP2 from the ADH1 promoter, or pIS027 the vector control. Strain YJR7 (gal3) was transformed with pIS027. Transformants were streaked onto EB-galactose plates, incubated for 5 days at 30°C and then photographed. 109 Results 110 Results 3.16 THE SWG1 MUTANT IS IMPAIRED IN UTILIZATION OF GALACTOSE AND SUCROSE, BUT NOT RAFFINOSE OR GLUCOSE Because Gal4 phosphorylation is mediated by components of the R N A polymerase II holoenzyme (SRB10, SRB11, GAL11 (98, 150)), it was expected that some mutations identified in the synthetic gal3 genetic screen might display pleitropic phenotypes. Figure 22 shows that while swgl gal3 cells are equally capable of growth on EB-glucose or EB-raffinose, they grow more slowly on media containing sucrose or galactose as the sole carbon source. This is important because the effect of the swgl mutation on sucrose utilization implies that its Gal defect is likely due to a defect in the derepression mechanism (common to expression of both the GAL genes as well as invertase) rather than the induction mechanism, which is specific to the GAL gene regulon. 3.17 GIP2 DOES NOT AFFECT Gal4 PHOSPHORYLATION Since the effect of the swgl/gip2 mutation on the GAL genes is not specific, one would expect that there would be no change in the phosphorylation status of Gal4 in either gip2 cells or wild type cells. Indeed there appears to be no change in the phosphorylation profile from immunoprecipitated Gal4 isolated from either wild type cells or gip2 cells (Figure 25). Importantly, spot 1 (indicated by the arrow) is present in both peptide maps, indicating that S699 is phosphorylated in both cases. Additionally, it should be noted that the gip2 mutation did not appear to affect the overall level of phosphorylation of Gal4 as determined by comparing the amount of radiolabeled Gal4 isolated from the two different strains (not shown). I l l Results Figure 22. The swgl mutation impairs growth on galactose and sucrose but does not effect growth on glucose or raffinose. Yeast strains YJR7 or YJR60 were streaked on media containing galactose, sucrose, or glucose; incubated at 30°C for 4 days and then photographed. 112 Results 113 Results Figure 23. Phosphorylation of S699 of Gal4 is unaffected by mutations in gip2. Yeast strain BY4742 in panel A , or 10189 in panel B, bearing the plasmid pJR008 (A683 Gal4 expression plasmid) were grown in liquid media containing glycerol and lactic acid as the carbon source to early log phase. Cells were washed in phosphate depleted media and labeled with 32 [ P]-orthophosphate at the same time that galactose and raffinose were added to a final concentration of 2% from separately sterilized stocks. After 2 hours, Gal4 protein was isolated by immunoprecipitation and analyzed by tryptic phosphopeptide mapping. Each sample contains approximately equal cpm for each sample was analyzed. In both panel A and panel B the peptide indicated by the arrow corresponds to tryptic peptide 1 as described by Hirst which contains S699 (98). ' ,.' . 114 Results WT gip2-115 Discussion 4. D I S C U S S I O N The GAL gene regulon in 5. cerevisiae is an excellent model for the study of eukaryotic transcription. The signaling mechanisms regulating the expression of the GAL genes have received a great deal of attention in recent years and we now have a good idea of how galactose is sensed and the signal for induction generated through Gal3. The results presented here demonstrate that the problem of GAL induction is far from solved. Multiple signaling pathways exist which regulate the activity of the transactivator Gal4. One signal acts through the Gal3 protein and is specific for galactose. A second signal acts through phosphorylation of the transactivator Gal4. This signal is mediated'bythe R N A polymerase II holoenzyme associated kinase SrblO and appears to be more general in nature. 4.1 DIFFERENCES BETWEEN YT6 AND W303 REVEAL THE PHYSIOLOGICAL PURPOSE FOR S699 PHOSPHORYLATION The yeast strain YT6G80::171 was initially used to assay different Gal4 derivatives bearing serine to alanine changes at potential phosphorylation sites. This proved to be fortunate because when studies were initiated using the W303-1A background, it was not possible to replicate the results obtained in the YT6G80::171 strain. In other words, the S699A mutation had no effect upon galactose induction in the W303 background, calling into question the relevance of Gal4 phosphorylation in induction. This was alarming at first until the two strains were crossed and it was determined that there was a genetic difference between these two strains. These experiments led to a closer examination of the components of the galactose signaling 116 Discussion mechanism in YT6G80::171. When attempts were made to grow YT6::171 on EB-galactose plates, it was observed that this strain was Gal-. This was surprising because the strain was clearly capable of inducing the transcription of Gal4 regulated genes as measured by (3-galactosidase assays (205). The Gal- defect could be complemented by a functional copy of the galactose specific permease GAL2. This observation was in agreement with earlier studies of galactose fermentation in S288C-derived strains (239). A supporting and even more satisfying result was the observation that the S699A mutation was required for full induction in the W303-1A background when induction employed extremely low levels of galactose. Normally, galactose is employed at a final concentration of 2% to study induction of Gal4 regulated genes. This does not likely reflect a common environment of yeast growing in the wild. Recently, a report called into question the significance of Gal4 phosphorylation in the process of induction. Ding and coworkers isolated suppressors of an inactive allele of Gal4 and found that the activating function of Gal4 could be restored in GALA alleles which had acquired deletions in the inhibitory region of GALA (48). The results of this study were interpreted to mean that all of Gal4's regulatory signals could be mediated by the D N A binding domain and the activating region 2 (a conclusion reached nearly 10 years earlier by Ma and Ptashne (154)). Some of these deletions also removed the region containing S699 and it was argued that Gal4 phosphorylation does not play a role in induction. The results presented here show that under certain conditions of high galactose concentrations it is possible to bypass the need for phosphorylation. Instead, phosphorylation of Gal4 may represent a way for the cell to fine tune its expression level and this may be overlooked in a crude "on/off measure of transcriptional activation. It is probably true that there is more often a need for cells to be able to respond to 117 Discussion low levels of galactose. For this reason, it is likely that phosphorylation of Gal4 at S699 reflects an important regulatory mechanism which allows the cell to be extremely sensitive to the amount of the inducer (galactose). 4.2 Gal4 PHOSPHORYLATION OCCURS INDEPENDENT OF Gal3-GALACTOSE SIGNALING PATHWAY Probably, the most significant results presented in this thesis were those that adressed the role of phosphorylation at S699 in conjunction with the inducer protein Gal3. Both genetic and . biochemical experiments demonstrated that phosphorylation at S699 is independent of the Gal3-galactose signal. Additionally, these experiments shed light on one of the oldest problems in yeast genetics: long term adaptation (250). The results demonstrate that phosphorylation of S699 is absolutely required for long term adaptation. Again, these results imply that the S699 phosphorylation acts in concert with the Gal3-galactose signal because it does not appear that S699 phosphorylation alone can suffice for activation of the GAL genes. 4.3 SrblO ACTS INDEPENDENTLY FROM Ga 13-GALACTOSE IN GAL GENE INDUCTION Earlier work had led to the notion that Gal4 was phosphorylated as a consequence of its interaction with some component of the general transcription machinery. Once our laboratory had identified the S699 site as being critical for Gal4 activity it was important to learn the nature of the Gal4 kinase. Because Gal4 phosphorylation appeared to be coupled to activation of transcription, the components of the RNA polymerase II holoenzyme seemed to be likely 118 . Discussion candidates. There are two kinases that are stably associated with the R N A polymerase II holoenzyme: Kin28, which is responsible for the kinase activity of TFIIH, and SrblO, which is a component of the mediator complex. Both of these proteins are cyclin dependent kinases that are capable of phosphorylating the CTD of R N A polymerase II. Martin Hirst in our laboratory undertook a biochemical study to examine which of these kinases may be phosphorylating Gal4. His results demonstrate that while both kinases are capable of phosphorylating Gal4, only SrblO is capable of phosphorylating S699 (98). This is congruent with the available genetic data as well. First, the laboratories of Hans Ronne and Marian Carlson had cloned srblO in a genetic screen identifying it as a gene necessary for glucose repression as well as induction of the GAL genes (9, 134). Additionally, Young and coworkers had noticed a Gal defect in srblO cells (146). Finally, the experiments presented here and in the work of Martin Hirst (98), tested the effect of the srb 10 mutation in a gal3 background and revealed that these two signaling events are genetically non-epistatic and represent two independent regulatory mechanisms. In contrast, it was found that Gal4 S699 and SrblO are genetically epistatic. 4.4 G a l 4 PHOSPHORYLATION M A Y AFFECT A STEP DOWNSTREAM OF HOLOENZYME RECRUITMENT With the identification of the Gal4 kinase as SrblO, an important question which remains is how does S699 control Gal4 function? In the initial report of this phosphorylation site, it was suggested that this phosphorylation may control the Gal4-Gal80 interaction. This interpretation was based on the observation that there was no difference in the level of GAL induction in gal80 cells. ,Gal4 phosphorylation could simply "lock" the Gal80-Gal4 complex into a conformation ( 119 Discussion more favorable for active transcription. Another possibility is that the Gal4 phosphorylation may affect the interaction with some other factor(s) which results in a complex more favorable for transcription and that the observed effect on the Gal4-Gal80 interaction could be indirect. In this model, Gal4 phosphorylation would behave similarly to that of S133 phosphorylation of the mammalian transactivator CREB (122). Phosphorylation of CREB at S133 results in a productive interaction with the coactivator CBP. CBP is a histone deacetylase as well as being a component of the R N A polymerase II holoenzyme. These different possibilities are outlined in Figure 24.. None of these models addresses which stage of the transcription cycle is affected by Gal4 phosphorylation at S699. Productive transcription requires: i) recruitment of the RNA polymerase II holoenzyme to a promoter, ii) promoter clearance, Hi) elongation of the polymerase complex, and iv) reinitiation to the promoter. One can envision regulation at any of these steps. The results presented here do not distinguish at which step the Gal4 phosphorylation acts. However, that the Gal4 S699A mutation results in a reduction of GAL gene transcription in gal80 glucose grown cells implies that the role of the S699 phosphorylation cannot be due solely to a loss of the Gal4-Gal80 interaction. If the phosphorylation affected recruitment of the R N A polymerase II holoenzyme, the GAL11P allele should have bypassed the need for this / phosphorylation and there would be equal transcription from both wild type and S699A Gal4. Instead the cells bearing the Gal4 S699A mutation are defective for transcription compared to cells bearing WT Gal4. This experiment is difficult to interpret but suggests that while Gal80 is able to partially block the artificial recruitment of Gal l IP, artificial recruitment does not completely bypass the requirement for 699 phosphorylation. Work from the laboratory of David 120 Discussion Bentley is worth noting here. Detailed biochemical studies by these authors identify that transactivators control the transition between "activated" and "non activated" forms of RNA polymerase II holoenzymes (1, 256). In studies of GAL transcription, activated (elongating) forms of the R N A polymerase II holoenzymes occur only after addition of galactose even though Gal4 is able to associate with the R N A polymerase II holoenzyme in these conditions (1). The results of the phosphopeptide analysis in gall, gal3 cells are consistent with this data. Even more encouraging is Bentley's observation that the holoenzyme-associated kinase SrblO is also required for activated forms of the RNA polymerase II holoenzyme (1). Work from other systems, most notably the studies of the HIV transactivator tat imply that transactivators play an important role in the formation of productively elongating forms of transcription (255). 121 Discussion Figure 24. Possible mechanisms of action for the S699 phosphorylation of Gal4. (A.) Gal4 activates transcription by contacting targets within the transcription machinery even when unphosphorylated. (B.) Phosphorylation of Gal4 could cause an alteration in the interaction between Gal4 and its negative regulator Gal80 which could "lock" this complex into a conformation more favorable for induction. (C.) Phosphorylation of Gal4 could result in the ability of Gal4 to contact additional targets which unphosphorylated Gal4 is unable to do. A third possible mechanism could be a combination of the two models where the contact with the additional target results in a weakening of the Gal4-Gal80 interaction. 122 Discussion A. 123 Discussion 4.5 REGULATION OF TRANSACTIVATORS BY HOLOENZYME KINASES It is becoming apparent that one function of holoenzyme-associated kinases is to regulate the activity of transactivators (98, 201). The work presented here as well as that of Martin Hirst in our laboratory contribute to of this growing view. In the case of the retinoic acid receptor, R A R a , phosphorylation by TFIIH leads to an increase in transcription although it is not yet known mechanistically how this occurs (201). The transactivator Tat which is responsible for the upregulation of HIV transcription is also phosphorylated by the holoenzyme associated kinase pTEFB/CDK9 (261, 262). Phosphorylation by CDK9 results in enhanced elongation of transcription. Teleologically, it makes sense for holoenzyme-associated kinases to regulate the activity of transactivators. This scenario would provide the cell with a means to fine-tune its level of transcription to match its physiological needs. In other words, making the activity of the transactivator dependent on components of the transcription machinery ensures that the cell does not make transcripts it does not need. This idea is very similar to that of a checkpoint in the cell cycle. The analogy is especially good when one considers all the different ways a cell has to keep from making macromolecules in adverse conditions. Usually, cells grown in the laboratory are supplied with an excess of all components necessary for growth so that this idea has not been rigorously investigated. Work from the laboratory of Johan Thevelein is beginning to address this question in yeast. Preliminary reports suggest that yeast cells indeedhave mechanisms which can control their levels of transcription in response to a complete set of nutrients rather than only a specific cue (44). 124 Discussion 4.6 SIGNALS REGULATING THE ACTIVITY OF Gal4 How might the GIP2 gene product regulate GAL gene activity? Upon the original cloning of this gene, it was discovered that G1P2 shared homology to genes whose products regulate the activity of the type I phosphatase Glc7. In fact, GIP2 was cloned by Tu and coworkers based on its ability to interact with the Glc7 protein in a two-hybrid assay (237). G/P2 contains a sequence (GVNK) which is contained in the yeast proteins Gael, Pigl , and Pig2, as well as other genes involved in glycogen metabolism in both mammals and plants (37). Based on this finding, Roach and coworkers examined the role of GIP2 in glycogen metabolism i -and found that GIP2 does not play a role in this process (37). The activities of the Glc7 phosphatase appear to act antagonistically with that of the protein kinase Snfl in all cases studied thus far (31, 66). The fact that a gene identified here as playing a role in the induction of the GAL genes was associated with Glc7 phosphatase (known to act antagonistically to the activities of Snfl) was initially disturbing. Snfl is implicated in the derepression of the GAL genes. Sequence alignment with GIP2 shows that GIP2 is most closely related to mammalian proteins which appear to be inhibitory subunits of the type I phosphatase in mammalian cells. If Gip2 is an inhibitory subunit of Glc7 this would bring it back in line with the role of Glc7 and Snfl playing opposing roles in the process of derepression (induction) of Gal4-regulated genes. This is a particularly exciting possibility as there has been no inhibitory subunit of Glc7 yet identified. 4.7 FUTURE DIRECTIONS The work presented here has provided insights into the role of phosphorylation of Gal4 in induction of Gal4 regulated genes. Towards achieving this, many new questions have been 125 Discussion revealed as well. These can be roughly divided into two categories: what is the mechanism by which S699 phosphorylation controls Gal4's activity, and what proteins are involved in the phosphorylation and dephosphorylation of Gal4? The answer to the first question will require rigorous biochemical approaches similar to those already being undertaken and which have identified affinities for activators and their targets (251). Now that many targets of Gal4 are known, it will be necessary to test the relative affinities of these targets for phosphorylated and uphosphorylated forms of Gal4. As the transcription field moves toward reconstitution experiments using purified components this question can be addressed and it will become clear where phosphorylation of Gal4 acts in the transcription cycle. To date, most of transcriptional studies have looked at proteins that control aspects of initiation. Studies in our laboratory have not clearly revealed if this is where S699 is functioning. It is equally likely that the Gal4 phosphorylation is controlling some post-initiation, event such as promoting elongation by the R N A polymerase II holoenzyme. Yeast genetics could also be employed to address this question. One likely scenario which could result in the GALA S699A phenotype would be that unphosphorylated Gal4 is unable to effectively recruit some component(s) needed for effective transcription. Our laboratory currently has versions of yeast bearing a single copy of GALA S699A expressed from its own promoter and this construct could be useful here. Exploiting the gal3 phenotype in conjunction with the GALA S699A mutation (no growth on EB-galactose), multicopy libraries could be transformed into yeast and genes that rescue growth identified. If, for example, some component needed for full activation of R N A polymerase II were not being recruited to the transcription reaction, this approach should identify such a factor. 126 Discussion To identify components that regulate the phosphoryation state of Gal4, a genetic approach will likely be most useful. The identification of more of the swg genes should help here. It is promising that the first gene identified in this screen, GIP2, appears to have a role in signaling. The classification of the swg genes into complementation groups is currently being pursued. Once accomplished, it will be useful to test the genetic interactions of these mutations with SRBW. One prediction of my model is that signals that regulate SrblO activity will control the activity of Gal4. It is hoped that the swg mutations will reveal how environmental signals are transduced to the general transcription machinery. 127 - Discussion 4.8 COMPARISON WITH THE lac OPERON OF E. coli "Is there a thing of which it is said, "See, this is new"? It has been already, in the ages before us. There is no remembrance of former things..." -Ecclesiastes 1:10 "What works survives; what does not work becomes extinct. Elegant solutions are used again and again." -Benno Mutter-Hill, in The lac Operon The classic work of Douglas and Hawthorne framed the understanding of the GAL regulon in comparison with the lac operon in E. coli (50) and this message is equally relevant today. Expression of the lac operon, like that of the GAL genes in Saccharomyces cerevisiae, is subject to both positive and negative control. In both cases, a ligand-specific signal (lactose or galactose) acts to inactivate a repressor (LacI or Gal80). The transactivator in the lac operon, Cap, is regulated by a separate signal (cAMP levels). Similarly, the results presented in this thesis show that the activity of the transactivator Gal4 is controlled by a separate signal which acts through phosphorylation of Gal4. The two separate signals allow the cell to fine tune its level of expression in order to match its environment and physiological state. A model consistent with results of this thesis is presented in Figure 25. 128 Discussion Figure 25. Two separate signals regulate the activity of Gal4. A galactose-specific signal acts through Gal3 (or Gall) to relieve the repression of the negative regulator Gal80. A second signal acts through the holoenzyme-associated kinase SrblO and controls the phosphorylation of Gal4 at S699. Signals known to regulate the activity of SrblO (such as the growth phase and environmental stress) in turn regulate the activity of Gal4. 129 Discussion galactose-specific signal environmental stresses carbon limitation heat shock oxidative stress 130 Nomenclature NOMENCT ATTIRE Wildtype (dominant) alleles^of genes in Saccharomyces are represented by italicized capital. letters, such as GAL1 and STE5. The corresponding recessive mutation is represented by lower case italicized letters such as gall and ste5. 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Genes Dev. 11:2622-2632. 147 Appendix A APPENDIX A A . l HIGH BASAL L E V E L OF GAL EXPRESSION IN srblO CELLS EXPRESSING Gal4 DERTVATTVES F R O M ARS-CEN PLASMIDS In an attempt to determine if Gal4 S699A was epistatic to srblO, I measured 3-galactosidase activity in gal4- yeast expressing Gal4 derivatives from ARS-CEN plasmids. I observed an extremely high basal level of expression in these cells. Additionally, this strain background was GAL3 which I later found was capable of masking the S699A phenotype. This may be due to the higher expression of Gal4 from the centromeric plasmid in the srblO mutant. 148 Appendix A Figure 26. The expression of Gal4 derivatives from ARS-CEN plasmids in a gal4, srb 10 strain results in extremely high basal level of GAL expression. Yeast strain YJR21::131 was transformed with YCplac22 (•) , YCpG4 (©), YCptriple (O), or YCpS699A (•). Yeast cultures were grown in liquid selective media containing glycerol and lactic acid as the carbon source to early log phase. Galactose was added to a final concentration of 2% from a separately sterilized 40% stock and expression of a GALl-lacZ reporter gene was monitored by measuring the (3-galactosidase activity from culture samples from samples taken at the indicated times. (3-galactosidase activity was measured in permeablized cells as described in Materials and Methods. ' 149 Appendix o m t- in <n. in m d • t- oi Hours 150 Appendix B A P P E N D I X B B . l NOVEL PHENOTYPES ASSOCIATED WITH THE OVEREXPRESSION OF GAL4 A growing population of yeast is characterized by three stages: an initial lag phase, logarithmic growth, and finally stationary phase as the cells deplete the available nutrients. In response to nutrient limitation, yeast cells will exit the cell cycle and enter a state very similar to G 0 associated with quiescent mammalian cells (53, 247). Indeed thisis a separate developmental phase, not simply an arrest at one point of the cell cycle. This point is best demonstrated by the isolation of a mutant in the laboratory of Richard Singer termed GCS1 (53). This mutant is able to enter stationary phase properly but is unable to resume growth again when supplied with proper nutrients. B.2 CELLS OVEREXPRESSING GAL4 EXHIBIT A N EXTENDED LAG PHASE OF GROWTH When I first entered the laboratory, I examined a phenotype that had been noticed by earlier investigators in the course of their studies with Gal4. It was continually noted that cells bearing high copy expression plasmids producing WT Gal4 (such as pDN3) grew much slower than did the control cells that did not express Gal4. I performed a series of growth curves comparing the growth of cells that contained no Gal4 and cells harboring Gal4 expressed from high copy plasmids from a strong promoter. To ensure that the cultures being compared were at a similar stage of growth, I began by growing cultures to saturation for two days and would 151 Appendix B -monitor their growth by optical density (OD 6 0 0) to make sure that each culture had ceased proliferation. Cultures were then be diluted into fresh medium and their growth rates were compared by monitoring their optical densities. As a control for the growth.curve experiments I also monitored the growth of cells harboring pDN3Y14. This plasmid produces Gal4 that is impaired in its ability to bind D N A (205). The results of the growth curve experiments allowed me to quantitate the growth defect of the cells that were overexpressing Gal4. Interestingly the growth profiles of the Gal4 overexpressing cells was characterized by an extended lag phase (Figure 27). Once these cells began to proliferate, they showed a doubling time similar to the cells that were expressing the defective derivative of Gal4. Cells bearing the pDN3Y14 derivative of Gal4 do not exhibit the extended lag phase associated with cells expressing Gal4 WT. I interpret this to mean that the ability of Gal4 to bind D N A is required for the extended lag phase. In agreement with this is the observation that galactose exacerbates the effect of Gal4 overexpression (see below). B.3 THE EXTENDED L A G PHASE ASSOCIATED WITH GAL4 OVEREXPRESSION IS ONLY SEEN IN CULTURES T A K E N FROM STATIONARY PHASE One explanation for the extended lag phase seen in cells overexpressing Gal4 is that these cells are having trouble exiting stationary phase. In order to synchronize the cultures I had used cultures that were in stationary phase. I decided to test if cells overexpressing Gal4 taken from exponentially growing cultures also exhibited an extended lag phase compared to gal4 cells. Figure 28 shows that the cells overexpressing Gal4 do not exhibit an extended lag phase compared to the gal4 control culture. One interpretation of this result is that cells overexpressing 152 Appendix B Gal4 have trouble exiting stationary phase but once they have done so, are able to grow j normally. B.4 CELLS OVEREXPRESSING GAL4 EXHIBIT ENHANCED THERMOTOLERANCE As cells enter stationary phase they acquire certain characteristics generally associated with enhanced resistance to environmental stresses. One of the best studied is the acquisition of thermotolerance. Cells in stationary phase are more resistant to heat stress (for example 55°C for 30 minutes). If the cells overexpressing Gal4 were having trouble exiting stationary phase then I reasoned that these cells would also be more thermotolerant. Figure 29 shows that this is indeed the case; cells overexpressing Gal4 are protected from heat stress compared to gal4 cells by approximately 2 orders of magnitude. A report had earlier noticed that treating cells with galactose could protect yeast cells from heat stress (246). Because this was similar to data I had acquired, I tested if this protective effect of galactose was dependent on GALA. I treated both wild type and gal4 cells with galactose for 2 hours and then subjected them to heat shock. As Figure 30 shows, galactose treatment is only able to protect cells that contain Gal4. B.5 SUMMARY AND CONCLUSIONS The experiments described here were performed in order to better understand the poor growth associated with the overexpression of Gal4. I learned that this poor growth is due to an extended lag phase in cells which overexpress Gal4. This extended lag phase requires Gal4's ability to bind D N A and can be exacerbated by galactose. The growth profile associated with 153 Appendix B Gal4 overexpressing cells lead me to investigate the link between Gal4 overexpression and stationary phase. To address this I examined if Gal4 overexpression caused these cells to become more thermotolerant (a characteristic of stationary phase cells). This proved to be the case; Gal4 overexpression causes cells to become more resistant to heat stress by about 2 orders of magnitude. In addition to the extended lag phase and the increased thermotolerance I noticed that Gal4 overexpression seemed to effect purine metabolism. The strains of yeast I was using were adel which cause an accumulation of a red pigment associated with the synthesis of adenine. I noticed that adel cells that overexpressed Gal4 did not accumulate the red pigment which is another characteristic associated with stationary phase cells (81). Finally, I also noticed that Gal4 overexpression causes aberrant cell morphology as reported previously (159). It is worth noting that the closely related protein L A C 9 from K. lactis is not tolerated on multicopy plasmids (23). Mechanistically, there are two likely explanations for the toxic effects of Gal4 overexpression. The first is that the toxic effect is due to "squelching", an ambiguous term usually defined by the ability of a transcription factor to interfere with the transcription of unlinked genes by competing for limiting components of the transcription machinery (192). The data presented here are not favorable to this hypothesis. First, the Y14 derivative that does not bind to D N A does not cause the toxicity, even though its activation domain is presumably . capable of interacting with the transcription machinery. Second, the Gal4 overexpression results in distinct phenotypes (such as the inhibition of purine bio-synthesis and aberrant morphology) not associated with the overexpression of other transactivators such as Rapl (67). Finally, if it 154 Appendix B were the case that overexpression resulted in the limitation of some component essential for the transcription of all genes, then one would expect a growth profile reflecting constant slow growth. In other words, we would expect to see slow growth throughout the growth curve, not only at specific stages of growth. Another possibility is that Gal4 overexpression could cause the expression of genes dependent on Gal4 for their expression. The genes involved in galactose metabolism are likely not responsible for the toxicity. Although a buildup of galactose 1-phosphate is toxic to cells, this only occurs in yeast which bear a mutation in the gal7 gene. Since the yeast strain used in these experiments is capable of growth on galactose (when complemented with a functional allele of GAL2), we can assume that these cells are GALL Additionally, a toxic effect of Gal4 overexpression is seen in all carbon sources tested (not only on galactose). The question that begs then is: are there other genes regulated by Gal4? The answer is yes; the gene GCY1 has already been identified although there is very little known about it (179). There have been at least two other reports which point to a role of Gal4 in regulating genes independently from galactose metabolism (56, 69). The first report observed that post-translational modifications of certain proteins involved in cell wall synthesis are under the control of the Gal4-Gal80 system (69). Similarly, the second report noticed that an isoform of UDPG-pyrophosphorylase, a protein involved in storage carbohydrate metabolism is absent in cells carrying a mutation in the inducer gal3 (56). Both reports speculate that there are gene products responsible for the modification of these enzymes which are under the control of the Gal4 protein. One could imagine that overexpression of gene products involved in cell wall synthesis or storage carbohydrate metabolism (both processes drastically effected by entry to stationary phase) may 155 Appendix B result in aberrant growth characteristics. Presumably the identity of all Gal4 regulated genes will soon be known as genome wide analysis becomes more common. Perhaps once all of the Gal4 regulated genes are identified it will be possible to more closely test the hypothesis that Gal4 overexpression causes the expression of genes involved in the maintenance of stationary phase. 156 Appendix B Figure 27. Ga l4 overexpress ion causes an extended lag phase o f g r o w t h . Yeast strain YT6:: 171 was transformed with either pDN3 or pDN3Y14. Transformants were grown in liquid SD media to saturation and then diluted into fresh media containing glucose or galactose to a final concentration of 2% and their growth was measured by monitoring their optical density (A 6 0 0 ) . Cultures containing pDN3 were grown in either glucose ( • ) or galactose(B). Cultures bearing the pDN3Y14 plasmid were grown in glucose (•). 157 Appendix B Appendix B Figure 28. Ga l4 overexpress ion does not cause an extended l a g phase of g rowth when cells are taken f rom e x p o n e n t i a l l y g rowing c e l l s . Yeast strain YT6::171 was transformed with either pDN3 (•) or pDN3Y14 (• ) . Transformants were grown in liquid SD media to mid-log phase as determined by monitoring the A 6 0 0 and then diluted into fresh media containing glucose to a final concentration of 2% and their growth was measured by monitoring their optical density (A 6 0 0 ) . 159 Appendix B o It} o o Hours 160 Appendix B Figure 29. Yeast overexpressing Gal4 are more thermotolerant than gal4 cells. Yeast strain YT6:: 171 was transformed with either YEplac'l 12 (gal4-) or pDN3 (GAL4). Transformants were grown in liquid SD media to mid log phase as determined by monitoring optical density A 6 0 0 and samples were subjected to a 55°C heat shock as described in materials and methods. 161 Appendix Appendix B Figure 30. Galactose protects GAL4 yeast but not gal4 yeast from heat shock. Yeast strain W303-1A (GAL4) or YJR10::131 (gal4) was grown in liquid SD media to mid log phase as measured by optical density A 6 0 0 and treated with either galactose or glucose to 2% for 2 hours. Samples were then subjected to a 55°C heat shock for 30 minutes as described in materials and methods. 163 Appendix B glucose + + + galactose + + 164 

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