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A new perspective on hexose transporter gene expression in Saccharomyces cerevisiae Greatrix, Bradley W. 2004

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A N E W P E R S P E C T I V E O N H E X O S E T R A N S P O R T E R G E N E E X P R E S S I O N IN  SACCHAROMYCES  CEREVISIAE  by  B R A D L E Y W. G R E A T R I X B. Sc. Hons. Biochemistry, Queen's University, 2000  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF S C I E N C E  in  THE F A C U L T Y OF G R A D U A T E STUDIES Genetics Graduate Program  W e accept this thesis as conforming to the required standards  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A June, 2004 © Bradley W . Greatrix, 2004  Library Authorization  In presenting this thesis in partial fulfillment 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.  Date (dd/mm/yyyy)  Name of Author (please print)  Title of Thesis:  A  AJf5o f f e g - S f g c . 7 ? u O u  Degree:  M/fe>Tm£  Department of  Of  /&Mgf\t£>  The University of British Columbia Vancouver, BC  Canada  SCtZteC^  ttExosB  TZAAj^^TBd  Year:  TPOfrfcA^  ABSTRACT Saccharomyces cerevisiae contains a family o f twenty hexose transporter (HXT) and ZiYT-related genes. Hexose transporters facilitate the uptake o f six-carbon sugars across the plasma membrane. O f the seventeen identified HXTs, only nine have assigned functions - some of which are still poorly defined. Despite extensive efforts to characterize the hexose transporters, the expression of HXT6 and HXT8-17 remains an enigma. In nature, S. cerevisiae finds itself under extreme nutritional conditions including sugars (both glucose and fructose) in excess o f 40 % (w/v), depletion o f nutrients and extremes of both temperature and p H . These conditions may affect the transcriptional activation of HXT genes for which no function has been assigned thus far. U s i n g 7iYTpromoter-/acZ fusions, we have identified novel conditions under which the HXT17 gene is expressed. HXT17 promoter activity is up-regulated i n media containing raffinose and galactose at p H 7.7 versus p H 4.7. W e demonstrated that HXT5, HXT 13 and to a lesser extent HXT 15 were all induced in the presence o f non-fermentable carbon sources. HXT1 encodes a low affinity transporter, and previous work b y other groups revealed that the HXT1 promoter activity peaked at 4 % sugar, and that expression o f this gene remained constant up to 8 % sugar (w/v). W e have confirmed these results, but also extended the range o f sugar concentrations tested up to 40 % (w/v). Initially, it appeared that HXT1 m R N A was upregulated 10-fold at 40 % vs 2 % glucose. However, in short-term osmotic shock experiments, HXT1 promoter activity was actually down-regulated by 40 % glucose. To reconcile these results we tested the half-life of HXT1 m R N A under osmotic pressure, and found the transcript to be stabilized in 40 % glucose. This stabilization is not dependent on HOG1. Furthermore, the stabilization of HXT1 m R N A does not appear to be gene specific because 30 minutes after transcriptional arrest there is five-fold more m R N A in osmotically stressed versus non-stressed yeast cells. The implication o f this observation is that a large portion o f S. cerevisiae m R N A molecules may have a decreased rate o f turnover during exposure to osmotic stress. ii  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF FIGURES  vii  LIST OF TABLES  ix  LIST OF ABBREVIATIONS  x  ACKNOWLEDGEMENTS  xi  CHAPTER I 1.0 INTRODUCTION 1.1 The HXT gene family  1 2  1.1.1 HXT1  2  1.1.2 HXT2 and HXT4  3  \.\3HXT3  4  \.\AHXT5  4  1.1.5 HXT6 and HXT7  5  IA.6HXT8-16  5  1.1.7 HXT17  6  1.1.8 SNF3, RGT2 and GAL2  6  1.2 Regulatory components of HXT gene expression: R g t l p , G r r l p , M t h l p , Stdlp  6  1.2.1 RGT1  6  1.2.2 GRR1  7  1.3 Regulation o f hexose transporters during wine fermentations  7  1.4 Glucose repression  8  1.4.1 SNF1  9 iii  1.4.2 MIG1  10  1.4.3 Hxk2p is involved in mediating glucose repression  11  1.5 Glucose sensing and signalling  12  1.5.1 The Rgt2p and S n D p glucose sensors  12  1.5.2 The R a s - c A M P pathway  13  1.6 Osmotic stress and the H O G pathway  14  1.6.1 S h o l p , S l n l p and Msb2p are the sensors of osmotic stress  15  1.6.2 Transcriptional response to osmotic stress  16  1.7 m R N A turnover and stabilization  17  1.7.1 Mechanism of m R N A degradation  19  1.7.2 Signal transduction and m R N A turnover  20  1.8 Scope and nature of this work  21  C H A P T E R II 2.0 M A T E R I A L S A N D M E T H O D S  23  2.1 Strains, plasmids and media  23  2.2 Yeast transformations and P-galactosidase assays  23  2.3 R N A isolation  24  2.4 Real-time P C R  24  2.5 Determination of water activity (a )  25  2.6 Osmotic shock assay  25  2.7 R N A stability assay  25  2.8 Statistical analyses  26  w  iv  C H A P T E R III 3.0 R E S U L T S  30  3.1 Effect o f glucose or fructose on HXT promoter activity at concentrations ranging between 0.2 % - 40 % (w/v)  30  3.1.1 Effect o f p H on HXT 17 promoter activity  31  3.1.2 HXT17 is expressed upon relief from anaerobiosis  32  3.1.3 HXT5, HXT13, and HXT15 are expressed during growth on ethanol, or glycerol and ethanol together 3.2 H i g h amounts o f glucose or fructose reduces water activity i n Y N B media  32 33  3.3 High glucose (40 % w/v) and 1.4 M N a C l increase HXT1 promoter activity to the same extent 3.4 The effect o f high glucose concentrations on HXT1 m R N A stability  33 34  3.5 Osmotic stress enhances retention of polyadenylated molecules after termination o f transcription in yeast cells  36  CHAPTER IV 4.0 D I S C U S S I O N  51  4.1 Effect o f glucose or fructose on HXT promoter activity at concentrations of0.2%-40%(w/v)  52  4.1.1 Effect o f p H on HXT17 promoter activity  54  4.1.2 HXT 17 is expressed upon relief from anaerobiosis  55  4.1.3 HXT5, HXT13 and HXT 15 are expressed during growth on ethanol, or glycerol and ethanol together 4.1.4 HXT1 m R N A is stabilized by glucose-induced osmotic stress 4.2 Conclusions  56 57 60 v  CHAPTER V 5.0 Future Directions  63  5.1 Identification of the full complement of m R N A molecules that are stabilized by osmotic stress  63  5.2 Identification of the regulatory components that mediate p H dependent HXT17 expression  LITERATURE CITED  64  65  vi  LIST OF FIGURES Figure 1. HXT4 promoter activity decreases in response to increasing extracellular sugar concentrations  37  Figure 2. HXT3 is constitutively expressed during growth in the presence o f glucose or fructose  38  Figure 3. HXT5 promoter activity is up-regulated i n response to high concentrations of extracellular glucose or fructose  39  Figure 4. HXT2 promoter activity up-regulated at low concentrations o f extracellular glucose or fructose  40  Figure 5. HXT1 promoter activity is increased in response to increasing extracellular sugar concentrations  41  Figure 6. HXT13 is expressed at low concentrations o f extracellular glucose or fructose  42  Figure 7. The complement o f expressed hexose transporters is distinct at low (2 % w/v) and high (40 % w/v) concentrations o f extracellular glucose  43  Figure 8. HXT17 is expressed higher at p H 7.7 versus 4.7 when grown i n the presence of raffinose and galactose  44  Figure 9. HXT17 is expressed in cells that are exposed to an aerobic environment after 80 hours o f anaerobic growth  45  Figure 10. HXT5, HXT13, and HXT15 are expressed in cells grown in Y N B media containing ethanol (2 % v/v), or ethanol and glycerol (2 % v/v each)  46  Figure 11. Osmotic stress-induced HXT1 expression requires glucose but decreases HXT1 promoter activity relative to non-stressed cells  ;  Figure 12. Several distinct transcripts are stabilized by osmotic stress  47 48  Figure 13. HXT1 m R N A is stabilized by osmotic stress after transcriptional arrest induced by thiolutin  49 vii  Figure 14. Global m R N A decay is reduced in osmotically stressed yeast cells  50  vm  LIST OF T A B L E S Table 1. Yeast strains used in this study  27  Table 2. Plasmids used in this study  28  Table 3. Primers used in this study  29  ix  LIST O F ABBREVIATIONS AMP AMPK ARE  P-gal cAMP cAPK cDNA CTD DEPC DNA elF EtOH Fru Glc Gly HOG HXT LSD MAP MAPK mRNA PCR Pi poly-A RNA RT  adenosine monophosphate AMP-activated protein kinase adenylate- and uridylate-rich beta galactosidase cyclic adenosine monophosphate cAMP-dependent protein kinase . complementary D N A carboxy-terminal domain diethyl pyrocarbonate deoxyribonucleic acid eukaryotic initiation factors ethanol fructose glucose glycerol high osmolality glycerol hexose transporter least significant difference mitogen activated protein mitogen activated protein kinase messenger ribonucleic acid polymerase chain reaction isoelectric point poly-adenylated ribonucleic acid reverse transcriptase  S. cerevisiae  Saccharomyces cerevisiae  SH3  STRE UTR v/v w/v X-gal YNB  sarcoma homology 3 stress responsive element untranslated region volume per volume (millilitres) weight (grams) per volume (millilitres) 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside yeast nitrogen base  YPD  yeast peptone dextrose  X  ACKNOWLEDGEMENTS First, I would like to thank Dr. Hennie van Vuuren for giving me a unique and exciting opportunity to work at the Wine Research Centre. I thank the members o f our lab for sharing ideas and motivating me to work hard. I also express my gratitude to Dr. M a r k Johnston for providing the HXT-lacZ plasmids, Dr. Richard Y o u n g for donating the Y 2 6 0 yeast strain and Pfizer Inc. for supplying the thiolutin compound. Finally I would like to thank my committee members, Dr. Christine Seaman and Dr. Jim Kronstad for providing input, guidance and their time to assist me with this project.  CHAPTER I 1.0 I N T R O D U C T I O N Glucose is the preferred carbon and energy source i n the baker's and wine yeast strains o f Saccharomyces cerevisiae. The primary role o f S. cerevisiae during wine fermentations is to convert the available hexoses (glucose and fructose) in grape must into ethanol, carbon dioxide and flavour compounds. Transport across the cell membrane proceeds via facilitated diffusion, and represents the first rate-limiting step in glucose metabolism, and thus alcoholic fermentation. H o w eukaryotic cells sense glucose, signal its presence, and ensure optimal utilization of this sugar remains a key fundamental question. Defects i n glucose signalling and uptake in S. cerevisiae can lead to severe and chronic problems with stuck or sluggish fermentations (10). In general, a wine fermentation is said to be complete when less than 0.4 % (w/v) o f the sugars (glucose and/or fructose) remain. While a typical fermentation may be completed in 7-10 days, a sluggish fermentation may take months. The problem is further compounded by the fact that while a vigorous fermentation has a protective layer o f carbon dioxide above the fermenting juice, a sluggish or stuck fermentation does not produce CO2 at high enough rate to have this protection and thus is susceptible to oxidation or contamination by spoilage microorganisms. Sluggish or stuck fermentations occupy valuable cellar space (during the harvest a fermentation tank may be used several times i n succession as different crops come in) and a spoiled wine (potentially > 10,000 L ) represents significant lost revenue for wineries. The causes o f stuck and sluggish fermentation are varied and are difficult to predict because o f a lack o f information regarding wine fermentations at the molecular level. Recently, several publications have become available, detailing various aspects o f wine fermentations using D N A microarrays (33, 83, 136, 152). Because uptake o f glucose and fructose represents the first critical step o f fermentation, we set out to characterize the transcriptional regulation o f  1  yeast hexose transporters in conditions that S. cerevisiae may encounter during wine fermentations.  1.1 The HXT gene family Glucose import into the yeast cell is facilitated by a group o f membrane-spanning proteins, termed hexose transporters ( H X T ) . Transport across the cell membrane represents the first rate-limiting step i n glucose metabolism. There are at least 20 members of the yeast hexose transporter family {HXT1 to HXT17, SNF3, RGT2 and GAL2), as identified by genetic studies and/or sequence homology (for reviews, see 11, 13, 63, 110). A l l o f the H X T gene products, with the exception o f H x t l 2 p are able to support growth on glucose when expressed individually in a strain deleted for all twenty transporter genes (178), indicating all o f the H X T genes encode functional glucose transporters. None o f the transporters is essential for growth on glucose however, indicating their functional redundancy. HXT1, HXT2, HXT3 and HXT4 are the best characterized members of the H X T family. The presence of multiple hexose transporters with differing affinities for glucose is reasonable given that S. cerevisiae is able to grow i n an extensive range o f sugar concentrations (0.1 % to > 40 % w/v).  1.1.1 HXT1 - HXT1 encodes a low affinity transporter that is maximally expressed in the presence o f high levels o f extracellular glucose (> 1 % w/v or 56 m M ) (111). HXT1 was originally isolated as a multi-copy suppressor o f a high-affinity glucose transport defect in snf3A cells (71), and later as a suppressor o f a potassium transport defect i n trklAtrk2A cells (62). Early studies revealed that HXT1 expression was maximal during lag and early-exponential phases o f growth (71). B y fusing the lacZ gene to the HXT1 promoter it was observed that HXT1 is expressed in the presence o f high (> 1 % w/v) glucose concentrations, reaching a maximum at 4 % w/v (111). HXT1 is not induced by glucose in grrlA cells, however normal 2  expression is restored in grrlA rgtlA mutants (see section 1.2 for a discussion o f GRR1 and RGT1). Interestingly, i n rgtlA mutants, basal HXT1 expression on galactose is higher than in wild-type cells, but is not fully induced on high glucose, indicating that i n addition to GRR1mediated derepression, a second activating signal is required for maximal HXT1 expression on high glucose (111). HXT1 expression is increased during exposure to osmotic stress, as caused by salt (1.0 M NaCI), sorbitol (1.5 M ) (46), or high sugar (40 % w/v) (33). Induction o f HXT1 by osmotic stress is dependent on the H O G (high osmolarity glycerol) pathway (128) (see section 1.6). It has been proposed that HOG1 -dependent HXT1 expression provides additional glucose for the synthesis o f glycerol, a compatible solute that accumulates during conditions o f osmotic stress (46).  1.1.2 HXT2 and HXT4 - HXT2 and HXT4 are high affinity transporters. Expression o f HXT2 and HXT4 is increased approximately five to twenty-fold in cells growing in the presence of low glucose (-0.1 % (w/v) or 5.6 m M ) versus cells grown either in the absence o f glucose, or in the presence o f high glucose (111). Like HXT1, HXT2 and HXT4 were cloned as multicopy suppressors o f the high-affinity glucose uptake defect i n snf3A mutants (64). HXT2 and HXT4 are maximally expressed at low concentrations (-0.1 % (w/v) or 5.6 m M ) o f glucose (111). The expression o f HXT2 and HXT4 is repressed in the absence o f glucose b y R g t l p . However, these genes have an additional level o f regulation by M i g l p and S n f l p that limits their expression to low concentrations o f glucose (discussed in section 1.4). HXT4 may have relaxed substrate specificity, as it is the only HXT gene that is able to complement a growth defect on galactose in a strain lacking the functional galactose transporter Gal2p (120). HXT4, along with HXT 11 is able to restore glucose transport activity to a glucose permease mutant (raglA) o f Kluyveromyces lactis (102, 120). Furthermore, although HXT4 is expressed only on low 3  glucose, it is unable to restore growth on 5 m M glucose, but only on higher concentrations (126). Indeed HXT4 has a K  m  for glucose o f 6.2 - 9.0 m M , whereas the corresponding K values m  reported for HXT2 are 1.5 m M - 2.9 m M (82, 125).  1.1.3  HXT3 - HXT3 is a low affinity glucose transporter that was originally identified along  with HXT1 as a suppressor o f a potassium transport defect i n trklAtrk2A cells (62). HXT3 is also a multicopy suppressor o f the snf3A growth defect on raffinose (62, 160). Raffinose is a trisaccharide composed o f galactose-glucose-fructose. Raffinose serves as a low source o f fermentable carbon as the glucose-fructose bond can be gradually hydrolyzed by invertase. HXT3 promoter activity is constitutive in the presence o f glucose but is independent o f sugar concentration (111). In the absence o f glucose HXT3 is repressed by R g t l p (111). There is also evidence that HXT3 expression reaches maximal levels upon entry into stationary phase (62).  1.1.4  HXT5 - Overexpression o(HXT5 is sufficient to restore growth on glucose to an hxtl-17A  gal2A strain (178). HXT5 encodes a functional hexose transporter with moderate affinity for glucose ( K = 1 0 m M ) that is maximally expressed under conditions that cause slow growth (31, m  172). For example, in batch cultures an increase in temperature or osmolarity, as well as growth in the presence o f ethanol or glycerol or a depletion o f glucose all induce the expression o f HXT5 (172). Microarray data have also identified HXT5 as inducible by increased temperature (37) or osmolarity (33, 37, 117, 128, 182). The induction of HXT5 when glucose is depleted is a function o f growth rate and is independent o f glucose derepression because in exponentially growing hxk2A cells i n the presence o f glucose HXT2 and HXT4 are derepressed, while HXT5 expression is not detected (116). HXT5 promoter analysis revealed two stress-responsive elements ( S T R E ) , two Hap2/3/5p binding sites and one P D S (post-diauxic shift) element (14,  4  172). Interestingly, Verwaal et al. also reported that the HXT5 promoter appears to be homologous to that o f GSY2, which encodes an enzyme involved i n glycogen synthesis, and noted that HXT5 and GSY2 exhibit a similar expression pattern (172).  1.1.5 HXT6 and HXT7 - HXT6 and HXT7 exist in tandem on chromosome I V , separated by approximately 3.5 kb, and are 1.5 kb downstream o f HXT3. Hxt6p and Hxt7p are highly related, differing b y only two amino acids over the entire 570 amino acid sequence (126). Neither o f the differing amino acids appears conserved within the hexose transporter family (13). O f the characterized transporters from this family (HXT1-7), HXT6 and HXT7 have the highest affinity for glucose, with a K  m  value o f approximately 1.0 m M (82, 125). In wild-type strains, the  expression of HXT7 is repressed i n the presence o f high concentrations o f glucose, but increases as glucose reaches depletion (184). Interestingly, despite their high sequence similarity, HXT6 and HXT7 appear to be regulated independently. The expression o f HXT7 is much higher than that o f HXT6 in wild-type strains under similar growth conditions (30, 125). Furthermore, HXT6, in addition to being regulated by the general glucose repression pathway (see section 1.4) responds to a novel signal transduction pathway involving Snf3p (73). M o r e specifically, the maintenance o f HXT6 glucose repression is dependent on SNF3 expression even when glucose is abundant.  1.1.6 HXT8-16 - To date, very little is known about the regulation o f HXT8-16. A s mentioned, HXT11 is capable o f restoring glucose uptake in a raglA strain o f K. lactis (102). HXT 11 and HXT9 were also identified as targets for the transcriptional activator Pdr3p (for pleiotropic drug resistance) (102). Deletion o f HXT11 and/or HXT9 confers resistance to cycloheximide (protein synthesis inhibitor), sulfomethuron methyl (aceto-lactate synthase inhibitor) or 4-nitroquinoline-  TV-oxide (mutagen). This is interesting given that when expressed individually Hxt9p and H x t l l p are functional glucose carriers (178), and yet their expression is entirely independent o f extracellular glucose. Rather, their expression is linked to a transcription activator that also regulates proteins that confer drug resistance.  1.1.7 HXT17 - HXT17was  identified by a microarray experiment as a target o f a constitutively  active form o f the M a c l p transcription factor (40). M a c l p regulates the expression of high affinity copper uptake genes under copper-deficient conditions (58). However, when cells were treated with a copper-specific chelator to mimic copper limitation, HXT17 was not induced, indicating the effect may have been due to a property of the specific MAC1 mutant allele.  1.1.8 SNF3, RGT2 and GAL2 - SNF3 and RGT2 gene products do not transport glucose per se, but rather serve as sensors o f extracellular glucose concentrations (discussed in section 1.5.1) (109). Gal2p serves as a galactose transporter (100).  1.2 Regulatory Components of / / A T Gene Expression: R g t l p , G r r l p , M t h l p and S t d l p  1.2.1 RGT1 - RGT1 encodes a D N A - b i n d i n g protein that serves as both an activator and a repressor of HXT gene expression. In the absence o f glucose, R g t l p represses HXT1 - HXT4; addition o f glucose to the media causes inhibition o f R g t l p activity, and subsequent derepression of various HXT genes (111). Repression of transcription by R g t l p requires the general transcriptional repressors Ssn6p and T u p l p (112). Glucose-mediated inhibition of R g t l p activity requires G r r l p (see next section). Interestingly, R g t l p is required for both repression and activation of HXT1 gene expression. In rgtlA cells, HXT1 is expressed in the absence o f 6  glucose, but does not reach maximum expression levels i n the presence o f high amounts o f glucose (2 % w/v) (111). Recently it was shown that R g t l p becomes hyperphosphorylated in response to high concentrations o f glucose (4 % w/v) (93). Hyperphosphorylation is required for converting the protein to an activator because phosphorylation was abolished i n snfiA, rgt2A and grrlA mutants, and induction o f HXT1 expression is also lacking in these strains.  1.2.2 GRR1 - GRR1 (Glucose Repression Resistant) encodes an F-box protein associated with the ubiquitin proteolysis machinery (72). GRR1 expression is required for HXT gene expression, and this is due to the requirement o f G r r l p for R g t l p inactivation. The glucose-repression defect of grrl A strains is therefore indirect, as cells are unable to transport any amount o f glucose (111, 169). It has been proposed that G r r l p regulates R g t l p activity b y targeting the protein for degradation (110). However a recent report suggests an intermediate, M t h l p links these two proteins (34). MTH1, and the closely related STD1 are two genes that are important for the proper regulation o f i i Y T g e n e expression (139, 143); mthlAstdlA  cells express HXT1-4 even in  the absence o f glucose (139). Both M t h l p and Stdlp are able to interact with the membrane bound glucose sensors Rgt2p and Snf3p (67, 139), and both proteins localize to the membrane and the nucleus, making them good candidates as transducers o f the glucose signal that activates or derepresses transcription (139). Indeed, it was recently established that the phosphorylation and dissociation o f R g t l p from HXT promoters is mediated b y Grrlp-dependent degradation o f M t h l p (34).  1.3 Regulation of hexose transporters during wine fermentations Deletion o f the genes HXT1-HXT7 abolishes hexose transport in S. cerevisiae and prevents growth on glucose (125, 126). It is therefore not surprising that HXT gene expression is required for wine fermentations. Because o f the broad range o f extracellular sugar 7  concentrations during wine fermentations (decreasing from >20 % w/v to less than 0.5 % w/v), the coordinated expression o f different hexose transporters is a dynamic process. Evidence for the role o f specific hexose transporters during wine fermentations was provided when Luyten et al. expressed HXT1-HXT7 individually in a haploid industrial strain o f S. cerevisiae (78). The high-affinity transporters Hxt2p, Hxt6p and Hxt7p were all required for normal fermentation kinetics, although Hxt2p was required for growth initiation, whereas Hxt6p and Hxt7p were required at the end o f the fermentation. The low affinity transporters H x t l p and Hxt3p were able to complete the fermentation when expressed individually. Hxt3p had fermentation kinetics very similar to that o f the wild-type strain, whereas the H x t l p - o n l y strain was less efficient once the cells reached stationary phase. The conclusion o f the authors was that at least four or five hexose carriers are involved in successful alcoholic fermentations.  1.4 Glucose repression S. cerevisiae utilizes a wide variety o f carbon sources for energy and biosynthesis (7). Glucose and fructose are able to enter glycolysis directly, while other mono-, di- and trisaccharrides require cleavage or modification by enzymes prior to glycolysis. A s a result, glucose (or fructose) is the preferred carbon source and serves as an important regulator o f metabolism in S. cerevisiae. When glucose is present in the surrounding environment, genes encoding enzymes that are required for metabolism o f alternative carbon sources are repressed. In addition, genes involved i n gluconeogenesis and in respiration are also repressed in the presence o f glucose. This phenomenon is known as "carbon catabolite repression", or glucose repression ( G R ) . While in some cases glucose can affect enzyme levels through changes in the a b  translation or protein turnover rate, or by inactivating the protein, the major mode o f metabolic Because no known catabolites of glucose have been shown to be involved in this phenomenon, we will use the term "glucose repression". It is generally accepted that both glucose and fructose are able to illicit the "glucose repression" response. Therefore, in this context only glucose will be discussed. a  b  8  regulation for glucose is at the m R N A level. More specifically, a change in the rate of transcription in response to glucose is the primary output of signal transduction pathways involved in G R (for a review see 35). The identification of proteins involved in glucose repression has largely been accomplished through isolation of mutants that have either constitutively repressed G R genes, or express these genes even when glucose is present in the media. The core elements of glucose repression are the S n f l kinase complex and M i g l p .  1.4.1 SNF1 - SNF1 encodes the a subunit of a heterotrimeric serine/threonine protein kinase complex (termed the S n f l kinase in yeast). The S n f l protein kinase is highly conserved among eukaryotic organisms (AMP-activated protein kinase ( A M P K ) in mammals). In yeast, the S n f l kinase is required for derepression of many genes when glucose is limiting, including genes involved in gluconeogenesis, glycogen storage and alternative carbon utilization (reviewed by (43)). Additionally the S n f l kinase complex is required during meiosis (50), haploid invasive growth (26), diploid pseudohyphal growth (65), and the aging of yeast cells (5). S n f l p is associated with several other proteins in yeast, including the y subunit of the complex Snf4p, and one of three possible P subunits S i p l p , Sip2p or Gal83p (for reviews see 20, 35). S n f l p has an amino-terminal catalytic domain and a carboxy-terminal auto-inhibitory regulatory domain. A t " h i g h " concentrations of glucose (2 % w/v) the S n f l p regulatory domain inhibits catalytic activity of the kinase, whereas at low concentrations of glucose (< 0.5 % w/v) Snf4p limits this r  interaction and enables kinase activity (54). The three P subunits associate with S n f l p to form distinct kinase complexes, and may be involved in specifying a particular Snf kinase function (183). For example, cells expressing SIP I as the sole p subunit are unable to grow aerobically in the presence of glycerol and ethanol as carbon sources (138). Sip2p and Gal83p appear to be  9  involved in regulating S n f l kinase activity during invasive growth o f haploid strains of S. cerevisiae (174). Finally, Sip2p may be involved in regulating the aging process o f yeast cells by limiting the chromatin modifying activity o f the S n f l kinase in young yeast cells (generation Oor 1) (74). The S n f l kinase complex is itself regulated by phosphorylation, and biochemical experiments have revealed at least three upstream kinases (49, 97, 156). When glucose is limiting, S n f l p is phosphorylated on Thr 210, thereby activating the kinase. When glucose is returned to the media, S n f l p is deactivated by the stimulation o f dephosphorylation by protein phosphatase 1 (PP1). The catalytic subunit o f P P 1 , Glc7p, together with the regulatory subunit R e g l p reverts S n f l p back to the autoinhibited state in the presence o f glucose (137).  1.4.2 MIG1 - MIG1 was originally identified in a screen for genes that mediate repression of the GAL1 promoter i n S. cerevisiae (99). MIG1 is identical to SSN1 (168) and CAT4 (142). M i g l p is a D N A - b i n d i n g protein with a C2H2 zinc-finger domain capable o f binding the consensus ( G / C ) ( C / T ) G G ( G / A ) G sequence i n the promoters o f glucose repressible genes (77). Furthermore, an A T - r i c h region is required 5 ' to the G C box, presumably to allow flexibility in the D N A (77). M i g l p binds to the promoters o f several glucose-repressible genes, including SUC2 and GAL1 (98, 99). Deletion mapping o f the MIG1 gene revealed that a small carboxyterminal domain comprising the last 24 amino acids is sufficient to support MIG1 -dependent repression when fused to a D N A binding domain (107). Furthermore, two internal elements in the protein are responsible for the inactivation of M i g l p i n the absence o f glucose. Miglp-dependent repression seems to be related to the sub-cellular localization o f the protein. When cells are grown in the absence o f glucose, or with only trace amounts o f glucose (< 0.6 m M ) M i g l p is primarily located in the cytoplasm. U p o n exposure to higher levels o f glucose M i g l p translocates to the nucleus within 180 seconds (28). Several lines o f evidence 10  suggest that the nuclear-cytoplasmic localization of M i g l p is regulated by Snflp-dependent phosphorylation. First, M i g l p is differentially phosphorylated in response to glucose, and changes in the phosphorylation status of the protein are concurrent with its cellular relocalization (28, 165). Second S n f l p phoshorylates M i g l p in vitro (166), and in snfl A mutants, M i g l p is constitutively nuclear and dephosphorylated (28). The consensus model of Miglp-dependent glucose repression states that M i g l p represses transcription by recruiting the general transcriptional repressor Ssn6p-Tuplp (59, 165). Upon phosphorylation, presumably by S n f l p , M i g l p is transferred to the cytoplasm, thereby allowing dissociation o f Ssn6p-Tuplp from the target promoter. Recent evidence necessitates a slight modification of this model, although the key proteins involved have remained unchanged. MSN5 encodes a nuclear transport receptor that is responsible for the nuclear export of M i g l p upon phosphorylation, however in msn5A strains M i g l p is phosphorylated normally, but remains in the nucleus (29). A l s o , at the GAL1 promoter, which is the classic glucose-repressible promoter, Ssn6p-Tuplp is constitutively tethered to the D N A under both activating and repressing conditions (114). Finally, a very recent report demonstrated by coimmunoprecipitation experiments that Snflp-dependent phosphorylation of M i g l p disrupts the interaction with Ssn6p-Tuplp and allows transcription (at least oiGALl),  despite the repressor  complex being retained at the promoter (113). Therefore, although phosphorylation mediates the interaction between M i g l p and Ssn6p-Tuplp, it is not responsible for tethering the repressor complex to target promoters. The authors pointed out that the physiological relevance o f localizing M i g l p to the cytoplasm remains unresolved.  1.4.3 H x k 2 p is involved i n mediating glucose repression - In addition to S n f l p and M i g l p , glucose repression involves the glucose-phosphorylating enzyme hexokinase 2 (Hxk2p). This kinase is encoded by the HXK2 gene and acts in the glycolytic pathway to phosphorylate glucose 11  (or fructose) at carbon-6 (for a review see (89)). Although hxk2A strains are defective for glucose repression, this defect is not related to the glucose phosphorylating activity o f the enzyme, because overexpressing GLK1 in an hxklAhxk2A background does not restore glucose repression (134). H x k 2 p may play a direct role in repressing transcription because the protein at least partially localizes to the nucleus (124), and this nuclear localization is required for glucose repression o f SUC2, GLK1 and HXK1 (44, 131). V e r y recently it was reported that Hxk2p interacts with M i g l p as part o f a D N A binding complex at glucose repressible promoters (1).  1.5 Glucose sensing and signalling Glucose is the preferred carbon and energy source for S. cerevisiae (and most eukaryotic organisms). Although much is known about the metabolism o f glucose, how cells are able to sense and respond to glucose is still only partially understood. Because defects in glucose sensing and uptake can lead to serious metabolic disorders such as diabetes, an understanding o f the glucose sensing and signalling pathway has profound importance.  1.5.1 The Rgt2p and Snf3p glucose sensors - Two members o f the HXT gene family appear to serve as glucose sensors in S. cerevisiae. SNF3 and RGT2 are the most divergent members o f the glucose transporter family, with only 25 % similarity (11, 13, 63). Work from several laboratories indicates that Snf3p and Rgt2p serve a regulatory rather than metabolic role. First, analysis o f the transport kinetics i n a snf3A mutant suggested that the defect in high-affinity glucose uptake is due to the loss of more than a single transporter (24). Second, overexpression of SNF3 or RGT2 i n a strain deleted for seven HXT genes (hxtlA-hxt7A) (126) does not restore growth capability on glucose (73, 108). Finally, dominant mutations i n SNF3 and RGT2 were identified that caused glucose-independent expression o f several HXT genes (109). A distinguishing feature o f these two proteins is an unusually long C-terminal tail (> 200 amino  12  acids) that is predicted to reside i n the cytoplasm, whereas the C-terminal tails o f other glucose transporters are only approximately 50 amino acids (84). A s mentioned previously, M t h l p and Stdlp interact with these C-terminal tails, as well as with R g t l p , and both M t h l p and Stdlp are required for Rgtlp-mediated repression o f transcription i n the absence o f glucose (69). Stdlp can localize to the nucleus or the cytoplasmic membrane making it a good candidate for transducing a glucose signal, and genetic evidence indicates S t d l p and M t h l p antagonize the /^XT-induction signal from Rgt2p and Snf3p (139). However, the exact nature o f the signal that is transmitted via these proteins is unclear. A n additional component o f the Snf3p-Rgt2p signalling pathway may be Y c k l p (90). YCK1 (and its paralogue YCK2) encodes a membrane-associated casein kinase that interacts with Rgt2p and phosphorylates M t h l p and Stdlp in vitro (90). Furthermore, overexpression o f YCK1 leads to constitutive HXT1 expression, and conversely HXT1 expression is abolished in ycklAyck2A mutants. Therefore, based on existing evidence the current model o f Rgt2p (and likely Snf3p) mediated glucose signalling is as follows: Glucose is sensed by binding to an extracellular domain o f Rgt2p and generates an intracellular signal leading to Ycklp-mediated phosphorylation o f M t h l p . Phosphorylated M t h l p is subsequently targeted for degradation by G r r l p , which limits the interaction with R g t l p and therefore derepresses HXT gene expression.  1.5.2 T h e R a s - c A M P pathway - Cyclic A M P ( c A M P ) is a major second messenger in eukaryotes. In S. cerevisiae, c A M P signalling is central to the control o f metabolism, stress resistance and proliferation. Synthesis o f c A M P from A T P is catalyzed b y the enzyme adenylate cyclase (encoded in yeast b y CYR1). C y r l p is in turn regulated by two signals, from Ras proteins (Raslp, Ras2p) or the G-protein coupled receptor ( G p r l p ) (for a review see 133). R a s l p and Ras2p appear to be involved in regulating basal adenylate cyclase activity, whereas G p r l p mediates the glucose-induced activation o f c A M P synthesis (23). The key effector of the 13  R a s - c A M P pathway is the cAMP-dependent protein kinase ( c A P K ) (162, 163). c A P K exerts many pleotropic effects on yeast cells, some o f which can be attributed to cAPK-mediated regulation o f the general stress response transcription factors Msn2p and Msn4p (39, 85, 140). Glucose sensing b y the R a s - c A M P pathway involves G p r l p and Gpa2p, the yeast homolog o f a mammalian G-protein (96). Glucose induction o f c A M P synthesis in yeast requires both the G-protein coupled receptor system and a separate glucose phosphorylation process (by G l k l p , H x k l p or Hxk2p) that renders adenylate cyclase responsive to Gprlp-Gpa2p activation (132). This c A M P signal is apparently required primarily for the transition from respirative growth on a non-fermentable carbon source to growth on glucose (23).  1.6 Osmotic stress and the H O G pathway For unicellular organisms, the ability to withstand a sudden osmotic challenge is critical for survival. The natural environment o f S. cerevisiae includes both hyper- and hypo-osmotic conditions, as quantified by water activity (or the chemical potential o f free water in solution). High sugar or high salt environments reduce water activity and thus illicit an osmotic stress response from yeast (33, 46). When exposed to osmotic stress yeast cells decrease in size and rapidly begin to synthesize glycerol as a compatible solute (17, 91) (for a review o f early work see 12). The transcriptional response to osmotic stress in S. cerevisiae is mediated by the H O G (high osmolarity glycerol) signal transduction pathway and the key effector o f this pathway is H o g l p . HOG1 was identified, along with PBS2 in a screen for osmosensitive mutants; hoglA andpbs2A strains are unable to grow on high-osmolarity medium (16). Together HOG1 and PBS2 form part o f an osmotic stress-responsive mitogen-activated protein kinase ( M A P K ) signal transduction cascade ( H o g l p is phosphorylated in vivo by Pbs2p during osmotic stress). M A P kinase cascades are common signalling modules i n eukaryotes and each pathway mediates the response to one or more extracellular stimuli, including sensing nutrient availability, hormones  14  or cytokines (reviewed b y 130). Because yeast cells are readily amenable to genetic experiments they have been instrumental i n understanding the architecture and signalling mechanisms o f M A P kinase cascades. Indeed, the best characterized M A P K cascade is the yeast pheromone response pathway (66). A defining feature o f M A P kinase cascades is the presence o f three levels o f protein kinases that activate each other in a step-wise manner. These kinases are classed as one of: A M A P kinase ( M A P K - i.e. HOG1), a M A P kinase kinase ( M A P K K ) and a M A P K K kinase (or M A P K K K ) . In the H O G pathway there are three M A P K K K s (Ssk2p, Ssk22p and Stel l p ) , one M A P K K (Pbs2p) and a M A P K (Hoglp).  1.6.1 Sholp, Slnlp and Msb2p are the sensors of osmotic stress - Increases in external osmotic pressure appear to be sensed by two proteins in S. cerevisiae, S h o l p and S l n l p (79, 80). Signalling by S h o l p and S l n l p during osmotic stress converges on Pbs2p, but the two pathways are not entirely redundant.  S h o l p is a membrane-spanning protein that contains an intracellular  Src homology 3 (SH3) domain that binds Pbs2p (79). During osmotic stress, S h o l p uses Cdc42p, Ste20p and Ste50p to activate the M A P K K K Stel l p which in turn activates Pbs2p by phosphorylation (for reviews see 48, 106). Activation by Stel l p leads to Pbs2p-mediated phosphorylation o f H o g l p and causes H o g l p to migrate from the cytoplasm into the nucleus to activate transcription (16, 80). S l n l p is also a membrane spanning protein but is distinct from S h o l p in that it possesses an intracellular histidine kinase domain (119). The S l n l p branch o f osmo-sensing involves a three-component phosphorelay system from S l n l p to Y p d l p and then S s k l p (119). S l n l p is actually a negative regulator o f osmotic stress signalling because it is activated under hypo-osmotic conditions leading to phosphorylation (and deactivation) o f S s k l p . Upon exposure to high osmolarity, S l n l p is inhibited, leading to dephosphorylation of S s k l p and thereby facilitates activation o f the M A P K K K Ssk2p (or the redundant Ssk22p) by S s k l p (80, 118). It is worth mentioning that a third osmo-sensor, Msb2p has recently been identified (104). 15  MSB2 encodes a protein that was originally discovered as a multicopy suppressor o f CDC24 (8). Because components o f the S h o l p branch o f the H O G pathway are shared by other M A P kinase pathways, hoglA (or pbs2A) cells exposed to osmotic stress stimulate transcription o f genes bearing the pheromone-response promoter element (103). This cross-talk is abolished in stellAhoglA  cells, but only partially blocked i n sholAhoglA  cells suggesting that there is an  additional osmotic stress responsive activator o f the H O G pathway upstream o f Stel l p . Only in sholAmsb2AhoglA  cells is the cross talk ablated indicating that MSB2 encodes a weak but  physiologically relevant osmosensing protein that operates i n parallel to the S h o l p branch (104).  1.6.2 T r a n s c r i p t i o n a l response to osmotic stress - The induction o f the H O G pathway by osmotic stress is rapid and transient. Indeed nuclear accumulation o f H o g l p is evident within one minute o f exposure to osmotic stress, and the induction o f osmotic stress-responsive genes occurs within 10 minutes (127, 129). There are several transcriptional regulators involved in the osmotic stress response including S k o l p (122), Msn2p/Msn4p (128, 141), H o t l p (129), M s n l p (129), S g d l p (3), Gcn4p (3) S m p l p (27) and Skn7p (3) (reviewed b y 48). These transcriptional regulators mediate transcription o f distinct subsets o f the osmo-responsive genes in a H o g l p dependent manner albeit the specific mechanism can vary. For example H o t l p activates transcription by interacting with H o g l p which in turn recruits R N A polymerase II to target promoters (4). S m p l p is phosphorylated by the H o g l p kinase and this phosphorylation is essential for Smplp-mediated gene expression (27). A third mechanism involves S k o l p , which is a repressor that inhibits transcription o f several osmo-inducible genes b y recruiting the Cyc8pT u p l p general repressor complex but this repression is reversible b y phosphorylation o f S k o l p by H o g l p (121). Further, H o g l p phosphorylation o f S k o l p converts the repressor into an activator that recruits the S A G A histone acetylase and S W I / S N F nucleosome remodelling complexes (123). 16  A s mentioned above, one of the first responses of yeast to osmotic stress is the synthesis of glycerol as a compatible solute to counter-balance the osmolyte gradient across the plasma membrane. Glycerol synthesis is mediated in yeast by the glycerol-3-phosphate dehydrogenase (GPD1) gene, and GPD1 is highly induced in response to osmotic stress (46). Although GPD1 is considered the paradigm of the transcriptional response to osmotic stress, D N A microarray data have revealed up to 2177 genes that have altered expression during osmotic stress (at least two-fold) depending on the osmolyte and concentration thereof (33, 105, 117, 128). A significant portion of these genes belong to a group of transcripts that have altered expression in response to a wide variety of stressors. Depending on the study, there are 499 "environmental stress response" (21) or 900 "common environmental response" (37) genes in the S. cerevisiae genome.  1.7 m R N A turnover and stabilization Nearly all aspects of cell biology and physiology are dependent on proper regulation of gene expression. The titration of m R N A molecules inside a cell requires coordination of both synthesis and turnover rates of various transcripts at all times. U n t i l recently however, gene expression studies had focused mainly on transcription initiation as the only control point for changing the expression level for a given gene. However, m R N A turnover appears to play an important role in the proper regulation of gene expression (135). Rates of turnover for individual m R N A s can vary by more than two orders of magnitude (18, 135, 149, 151). While the decay rate for many genes is constant, there are numerous m R N A s for which the stability is altered in response to environmental factors (for reviews see 41, 179). In yeast and higher eukaryotes, m R N A degradation is initiated by a shortening of the poly-adenylated (poly-A) tail at the 3' end of the transcript (95, 148). Shortening of the p o l y - A tail leads to removal of the 5' 7-methylguanosine cap structure by decapping enzyme(s) (167) 17  which then exposes the transcript to cleavage by the 5'-3' exonuclease X r n l p (51) (reviewed in 53). Alternatively, following deadenylation m R N A degradation can proceed from 3' to 5' by the cytoplasmic exosome complex (52). Since the cap structure and p o l y - A tail both play a role in the stabilization of m R N A molecules it is not surprising that their formation is highly coordinated to the transcription process. Indeed the 5' cap is added to nascent m R N A molecules that are only 20-30 nucleotides long (25, 57). Poly-adenylation is carried out by the p o l y - A polymerase (Paplp) (75, 76, 115) in yeast, but the C-terminal domain ( C T D ) of R N A polymerase II is also an essential component of the poly-adenylation machinery (47, 87). Regulation of m R N A decay rates is important for maintaining or quickly altering transcript levels inside a cell. For example, transcripts with the shortest half-life w i l l attain a new steady state the most rapidly after changes in transcriptional activity. The features of an m R N A molecule that influence stability, the 5' cap and 3' p o l y - A tail also work together to stimulate translation (158). Several eukaryotic (translation) initiation factors (elF) interact with the 5' end of capped m R N A . eIF4F is a protein complex consisting of the cap-binding protein eIF4E, a scaffolding protein eIF4G and an ATP-dependent R N A helicase eIF4A that is responsible for unwinding R N A secondary structure (reviewed in 86). B y binding to the 5' cap, eIF4E serves to recruit eIF4G and eIF4A into the vicinity of the m R N A molecule. In addition to binding other members of the eIF4F complex, eIF4G also binds the p o l y - A binding protein P a b l p (157). The binding of eIF4G to P a b l p along with eIF4E leads to a circularized m R N A molecule (176). Although the functional significance of a circularized m R N A molecule has not been fully characterized, several possibilities exist (150). First, linking the 5' cap structure with the p o l y - A tail prior to translation would ensure only full-length transcripts are translated. Second, since both cap removal and deadenylation precede m R N A degradation it is possible that a circularized transcript is more stable (perhaps for repeated translation). This idea is supported by the work of Schwartz and Parker (144) but there is some evidence that deadenylation can be  18  uncoupled from decapping, without affecting m R N A stability (92). Finally, circularizing o f the m R N A molecule could serve to enhance re-initiation o f translation, as the ribosome would not need to dissociate and reassociate with the transcript. Unfortunately very little is known about how the ribosome proceeds after translation termination.  1.7.1 M e c h a n i s m o f m R N A degradation - DCP1 encodes the major decapping enzyme in S. cerevisiae. Mutations in DCP1 were first identified in a genetic screen for mutants with altered m R N A decapping in vivo (52). Biochemical analysis o f D c p l p revealed that the cleavage products o f the enzyme are 7-methylGDP and a 5'-monophospate m R N A molecule (68, 153). Removal o f the 5' cap facilitates rapid degradation by X r n l p which preferentially degrades targets with a 5'-monophospate end (154). DCP2 is also involved i n the decapping process, and encodes a putative nucleoside diphosphatase that interacts with D c p l p and is required for Dcplp-mediated decapping (32). Because the cap structure is both the site o f decapping and the assembly site for the eIF4F translation initiation complex, the relationship between m R N A turnover and translation has been scrutinized recently. In fact, some evidence indicates that m R N A decapping and translation machinery may compete for access to the 5' cap (reviewed by Schwartz and Parker (150)). For example, when components o f the eIF4F complex (eIF4E, eIF4G, eIF4A) have mutations that limit translation initiation, then the rate o f decapping o f PGK1 and MFA2 transcripts is concomitantly increased (144). Translation can also be impaired by the introduction o f a secondary structure in the 5 ' U T R (untranslated region) o f a transcript, and this also leads to an enhanced rate o f decapping (94). Furthermore, both eIF4G and P a b l p bind to D c p l p , either independently, orwhen these proteins are in the eIF4F complex, but this interaction is negatively affected by eIF4E (173).  19  1.7.2 Signal transduction and m R N A turnover - There have been significant advances in the understanding the mechanisms of signal transduction pathways that stimulate gene expression in yeast or other organisms. Less clear however, are the signal transduction pathways leading to altered m R N A turnover rates. M a n y unstable mammalian genes, such as those encoding cytokines, lymphokines and transcription factors possess adenylate- and uridylate-rich (AU-rich) elements ( A R E ) (19) in the 3' untranslated region ( U T R ) of their transcript that are involved in regulating the rate of m R N A turnover (22, 147). These A R E s usually contain several repeats of the pentameric sequence A U U U A (19, 147) which, when present in a functional A R E have a destabilizing effect on the m R N A molecule (2, 22, 155). A R E s can be bound by sequence specific proteins which act as both potent stimulators of decapping (36) and deadenylation (181). There are several A R E - b i n d i n g proteins that have been characterized thus far, including tristetraprolin (TTP), K homology-type splicing regulatory protein ( K S R P ) , A U F 1 and H u R (for a review see 9). While yeast have served as an excellent model for studying mechanisms of m R N A decay, much o f the work regarding signalling pathways that alter m R N A stability has been done in mammalian systems. For example, in mouse macrophages T T P promotes tumor-necrosis factor-alpha ( T N F a ) m R N A instability through a direct interaction with its A R E (reviewed by 9). This interaction can be overcome by phosphorylation of T T P by the mitogen-activated protein kinase p38 (the mammalian homolog of yeast HOG1)  as the hyperphosphorylated form  of T T P binds A R E sequences with lower affinity than the hypophosphorylated form (9). Taken together this evidence suggests that p38-mediated accumulation of T N F a m R N A is due to increased R N A stability instead of increased transcription, as was originally suggested. The p38 M A P kinase pathway has also been shown to promote interleukin-6 (IL-6) and IL-8 m R N A stabilization in FfeLa (human cervix) cells in response to cytokine (interleukin-1) exposure in an ARE-dependent manner (180). 20  ARE-mediated m R N A decay in yeast has recently been established. HYP2/TIF51A is an ARE-containing transcript in S. cerevisiae that is destabilized i n the absence o f glucose, or when the H O G pathway is inhibited (171). Because yeast is readily amenable to genetic manipulation, the discovery o f regulable m R N A stability in S. cerevisiae should greatly accelerate the delineation o f these signalling pathways.  1.8 Scope and nature of this w o r k The purpose o f this project was to examine the transcriptional regulation o f yeast hexose transporter genes, with a particular emphasis on the response o f HXT genes to conditions that may be encountered during wine fermentations. Our primary focus was on HXT transcription in the presence o f 0.2 % to 40 % (w/v) glucose or fructose, as well as the effect o f anaerobiosis, changing p H , osmotic pressure and glucose starvation on HXT mRNA levels. M a n y o f our results were derived from assaying beta-galactosidase activity i n HXT promoter-lacZ transformed yeast cells that were grown in media containing increasing sugar concentrations, altered p H or non-fermentable carbon. When it became clear that m R N A turnover was influencing HXT1 transcript accumulation during osmotic stress, we used either a yeast strain bearing a thermally unstable R N A polymerase II mutant, or thiolutin, a potent R N A polymerase inhibitor to investigate the decay of HXT1 m R N A . In the absence o f ongoing transcription, only changes in m R N A turnover rate can affect cellular m R N A levels i n different conditions. A t various time points after transcription arrest we assayed m R N A levels b y quantitative P C R . This project was motivated by two factors. First nearly half o f the HXT gene family members have a poorly characterized or uncharacterized function. Our work may assist in identifying roles for these transporters. Second, glucose uptake represents the first rate-limiting step o f wine fermentations, and defects in hexose transport are potentially involved in the occurrence o f stuck or sluggish fermentations. A s a result a better understanding of HXT gene 21  regulation could assist in diagnosing strains, or possibly fermentation conditions that could lead to stuck or sluggish fermentations.  22  C H A P T E R II 2.0 M A T E R I A L S A N D M E T H O D S  2.1 Strains, plasmids and media The yeast strains used in this study are listed in Table 1. Yeast cells were grown on standard media: Y P D (1 % (w/v) yeast extract (Difco), 2 % (w/v) Bacto peptone (Difco), 2 % (w/v) dextrose) or Y N B (0.67 % (w/v) yeast nitrogen base without amino acids or ammonium sulphate (Difco) supplemented with the appropriate amino acids, 0.5 % (w/v) ammonium sulphate and containing raffmose/galactose (1.5:0.5 % w/v), or glucose or fructose (0.2 % up to 40 % (w/v) as indicated). For approximating cell numbers a standard curve was generated. Cells at various densities were counted using a haemocytometer and the cell density was plotted against the absorbance o f the culture at 600 nm. For both T C Y 1 and B Y 4 7 4 2 there are approximately 3 x 10 cells/mL for every 1.0 Aeoonm- HXT promoters (0.6 - 1.8 kb D N A 7  fragments generated by restriction enzyme digests) were fused to lacZ in the vector Y E p 3 5 7 R (110, 111). The plasmids are shown in Table 2 (kindly provided by M. Johnston).  2.2 Yeast transformations and p-galactosidase assays HXT-lacZ plasmids (Table 2) were transformed into S. cerevisiae T C Y 1 cells using the high efficiency lithium acetate method (38). Successful transformants were identified by growth on agarose plates lacking uracil. Individual colonies were streaked onto Y N B plates lacking uracil in 8 c m patches and incubated for two days at 30 °C. Cells were scraped from the patches 2  and re-suspended in sterile d H 2 0 for inoculation into 50 m L of Y N B media containing glucose or fructose at various concentrations (see above). The initial inoculum was Aeoonm 0.05, and the cells were grown with shaking at 30 °C until reaching an A6oo nm of 0.6-0.8. The P-galactosidase assays were performed as described previously (6), except that 2 m L or 10 m L of cell culture 23  was analyzed instead o f 25 m L (as indicated). Each sample was analyzed in duplicate within the experiment, and the entire experiment was performed at least twice. (3-galactosidase activity values are reported in M i l l e r units (88), which are calculated as follows: M i l l e r units =  (A4 onm 2  In our experiments:  x 1000)/(A onm x t x volume) 60  t (reaction time in minutes)  =20  volume (of cell lysate in m L ) = 0.05  2.3 R N A isolation Total R N A was isolated using the hot phenol method (6). For Real-time P C R analysis, an additional step was required during the isolation to remove contamination by genomic D N A . This was performed using Qiagen RNeasy kits (catalogue # 74104), with the RNase free DNase kit (catalogue # 79254) using the manufacturer's recommended protocols (available at http://www.qiagen.com').  2.4 Real-time P C R c D N A synthesis from 2.0 pg of total R N A was performed using the Omniscript R T K i t (Qiagen) according to the manufacturer's suggested protocol. The reverse transcription reaction was primed using random hexamer oligonucleotides at a final concentration of 2.5 p M . The final c D N A product was dissolved in DEPC-treated H 0 to a final volume of 500 p L . 2  Real-time P C R was performed using the S Y B R Green P C R Master M i x (Applied Biosystems) according to the manufacturer's standard protocol, except the final reaction volume was reduced to 20 p L . Gene-specific oligonucleotide primers were used at a final concentration of 0.5 p M (primer sequences are listed in Table 3). The P C R s were performed in an A B I Prism® 7000 Sequence Detector (Applied Biosystems) with the following reaction conditions: 50 °C for two min, 95 °C for 10 min, followed by 40 cycles o f 95 °C for 15 seconds and 60 °C for one min. 24  A l l samples were assayed i n triplicate for each gene, and differences in c D N A synthesis efficiency were corrected for b y normalizing all expression values to constitutively expressed 18S r R N A .  2.5 Determination of water activity (a ) w  Water activity measurements were carried out in triplicate, using an Aqualab Series 3 water activity meter (Decagon Devices, Pullman, W A , U S A ) . The variability between assays was less than 2.0 %. The values reported represent the mean o f the three assays.  2.6 Osmotic shock assay T C Y 1 cells transformed with HXTl-lacZ were grown i n 300 m L Y N B (galactose 2 % w/v) to mid-log phase and then centrifuged at 9000 R C F for 10 minutes at room temperature. The supernatant was discarded, and the cells were resuspended i n 6 m L sterile dH^O. Flasks containing 50 m L o f Y N B media plus the indicated carbon or salt were inoculated with 1 m L (approximately 1.6 x 10 cells) o f the transformants, and then incubated at 30 °C. After four 7  hours 10 m L o f each culture was harvested in duplicate and analyzed for P-galactosidase expression. The experiment was performed twice using independent cultures.  2.7 RNA stability assay A I L volume o f Y P D was inoculated with Y 2 6 0 cells at an initial cell density of ^ 6 0 0 nm = 0.2 and grown at 25 °C until an A^o  n m  1.0 was reached (~11 hours). The cells were harvested  at 9000 R C F for 5 min at room temperature. The supernatant was discarded, and the cell pellet was resuspended in 10 m L sterile d F ^ O , prewarmed to 37 °C (the non-permissive temperature). Five 50 m L volumes o f Y P D , containing either 2 % glucose (w/v) or 40 % glucose (w/v) as the carbon source, were warmed to 37 °C and then inoculated with approximately 2 x 10 cells. A l l 9  25  flasks were immediately placed in a water bath at 37°C with shaking. A l s o , one volume o f the inoculum (approximately 2 x 10 cells) was centrifuged, washed once with ice-cold D E P C 9  treated dH^O and then quickly frozen by placing the tube in ethanol at -80 °C. This marked the zero-minute time point. For each time point (15, 30 and 60 minutes) the entire 50 m L culture was harvested by centrifugation, washed and then snap-frozen i n the same manner. The cell pellets were stored at -80 °C until the R N A extractions were performed. Alternatively, thiolutin (20 pg/mL, kindly provided by Pfizer, Groton, C T ) was used to induce transcription arrest in the S. cerevisiae strain B Y 4 7 4 2 (171). The protocol was similar to that for Y 2 6 0 cells, except that at the start o f the experiment the cells were inoculated in 400 m L of media (including thiolutin and either 2 % or 40 % (w/v) glucose), and at the indicated times (0, 10, 20, 30 and 45 minutes) 40 m L was harvested b y centrifugation and frozen at -80 °C. For quantifying m R N A as a percentage o f total R N A , polyadenylated molecules were purified from 1 mg o f total R N A using the Qiagen Oligotex m R N A M i d i kit (catalogue # 70042). Total R N A and m R N A were quantified b y measuring the absorbance at 260 nm.  2.8 Statistical analyses A two-way analysis o f variance ( A N O V A ) was used to evaluate the effect o f carbon source and sugar concentration on HXT promoter activities, and for comparing m R N A levels after transcriptional arrest in low- and high-sugar media. Differences in promoter activity were analyzed using a Fisher's least significant difference ( L S D ) test (p = 0.05). Statistics were calculated using Minitab software (release 14, Minitab Inc., U S A ) .  26  Table 1. Yeast strains used in this study. Strain  Genotype  Reference/Source  TCY1  MAT a ura3A lys2A  (170)  BY4742 BY4742 hoglA  Y260  MATahis3Al  ura3A lys2A leu2A  (15)  MATa his3Al ura3A lys2A leu2A  Invitrogen Life Technologies,  hogl::kanMX  Carlsbad, C A , U S A  MATa ura3A52 rpblAl  (101)  27  Table 2. Plasmids used in this study. A l l plasmids are derived from Y E p 3 5 7 R .  Plasmid  Description  Reference  pBM2636  HXTl-lacZ  (110, 111)  pBM2717  HXT2-lacZ  (110, 111)  pBM2819  HXT3-lacZ  (110, 111)  pBM2800  HXT4-lacZ  (110, 111)  pBM3555  HXT5-lacZ  (110, 111)  pBM3537  HXTlO-lacZ  (110, 111)  pBM3539  HXTll-lacZ  (110, 111)  pBM3538  HXT12-lacZ  (110, 111)  pBM3466  HXT13-lacZ  (110,111)  pBM3573  HXT14-lacZ  (110, 111)  pBM3472  HXT15-lacZ  (110, 111)  pBM3574  HXT16-lacZ  (110, 111)  pBM3476  HXT17-lacZ  (110, 111)  28  Table 3. Primers used in this study. Gene Forward Primer  Reverse Primer  GPD1  5 '-CC AG A AGTTTTCGCTCCAATAGTA-3'  5'-AGCAACC A A ATTGTCGGGTAG A-3'  HTB1  5' -G A A ACC AGCCGCT A A AAAG ACTTC-3'  5' -GGTA A G ATC A ATCTAAC AGCGGT-3'  HXT1  5 '-CCCGATCTAATATCTCCTCAGAAATCC-3'  5 '-CCACCGAAAGCAACCATAACAC-3'  IPP1  5 '-ACAGCAAGGGTATTGATTTGACCA-3'  5 '-AAGCTGGTGGGATGGCATCA-3'  18S rRNA  5 '-GGTGAAATTCTTGGATTTATTGAAGAC-3'  5 '-TTGATTTCTCGTAAGGTGCCGAGT-3'  Name  29  C H A P T E R III 3.0 R E S U L T S  3.1 Effect of glucose or fructose on /EYT promoter activity at concentrations ranging between 0.2 % - 40 % (w/v) To advance our understanding o f the regulation o f yeast hexose transporter gene expression, we tested whether increased sugar concentrations (up to 40 % w/v), or specific carbon sources (fructose vs glucose) affected HXT expression. A s an initial screen, we prepared X-gal plates containing either glucose or fructose from 0.2 % to 40 % (w/v) glucose or fructose as the carbon source. W e incubated T C Y 1 cells transformed with HXT1-5- and HXT10-17-lacZ on these plates for several days but observed no significant difference between glucose and fructose grown cells. In addition, the only HXT promoters that exhibited activity under these conditions were HXT1-5 and HXT13 (data not shown). T o examine the promoter activity o f these genes more closely, we assayed liquid-grown cultures o f T C Y 1 transformed with HXT 1-5, HXT13-lacZ for P-galactosidase (p-gal) activity. HXT4 activity was found to be highest at 0.2 % (w/v) glucose and fructose (Figure 1), while HXT3 was expressed across all sugar concentrations (Figure 2), in agreement with previous observations (111). Surprisingly both HXT3 and HXT4 had a spike of promoter activity in cells grown in 40 % glucose or fructose (w/v). HXT5 promoter activity remained relatively unchanged in both glucose and fructose-grown cultures until the sugar concentration exceeded 20 % (w/v). However, increasing the sugar concentration from 20 % to 40 % induced the HXT5 promoter approximately 5-fold (Figure 3). HXT2 promoter activity was increased in cells exposed to sugar concentrations at or below 2 % (w/v). Interestingly, induction of HXT2 was 2-fold higher with 2 % fructose compared to 2 % glucose (Figure 4). HXTl-lacZ  expression increased nearly linearly with increasing sugar concentrations  up to 30 % (w/v) (Figure 5). There was no statistical difference in HXT1 promoter activity in 30  cells grown in 30 % versus 40 % glucose or fructose (w/v). HXT13-lacZ was moderately induced in response to l o w glucose and fructose (< 2 % w/v) (Figure 6). Similar to HXT2, there appeared to be a differential induction of HXT13 between glucose and fructose at very low sugar concentrations (0.2 % w/v). Hexose transporters i n S. cerevisiae are somewhat redundant, considering that under most conditions there are at least two different HXT genes expressed at all times. Therefore, as a reflection o f the relative contribution o f each HXT gene to the overall complement o f transporters at a given sugar concentration, we expressed the promoter activity o f each individual HXT gene as a percentage o f the total promoter activity (in M i l l e r units) o f all o f the HXT-lacZ contructs at each sugar level. Because the induction o f the HXT genes at each sugar concentration is approximately equal between glucose and fructose-grown cultures, only results for glucose are shown. A s seen i n figure 7, at 2 % glucose (w/v), the major hexose transporters are HXT1-3, and to a lesser extent HXT5. In contrast, at 40 % glucose (w/v), HXT1 promoter activity comprises approximately 84 % o f the total activity for all o f the transporters. A t 2 % (w/v) sugar levels, HXT2-lacZ had higher activity in response to fructose as the carbon source versus glucose (Figure 4). A s a percentage o f total promoter activity in 2 % (w/v) sugars, HXT2 represents 18 % and 10 % o f the total in fructose and glucose respectively.  3.1.1 Effect of pH on HXT17 promoter activity.  During growth on X - g a l plates containing  raffinose and galactose as the carbon source (1.5:0.5 % w/v) there appeared to be an increase in HXT17 promoter activity when compared to cells grown on 2 % glucose (w/v) (Figure 8A). Initial attempts to quantify this induction using P-gal assays i n liquid-grown cultures were unsuccessful. However, one key difference between plate and liquid p-gal assays is that the X gal plates are buffered to have a neutral p H , whereas liquid Y N B media has a p H o f 4.5 - 4.7. After adjusting the p H o f the Y N B media with K O H , we observed that HXT17 promoter activity 31  in cultures grown in pH 7.7 versus 4.7 (using raffmose and galactose as the carbon source) is approximately 12-fold higher (Figure 8B).  3.1.2 HXT17 is expressed upon relief from anaerobiosis. In order to determine if oxygen availability affects the expression of HXT genes, we performed a preliminary screen by incubating HXT 10-17-lacZ transformed TCY1 cells on X-gal plates either aerobically or anaerobically with glucose (2 % w/v) as the carbon source. After at least 80 hours of anaerobiosis, there was no significant difference in the promoter activity of any of the transporters (data not shown). However, after a further 24 hours of aerobic incubation, we noticed the HXT17-transformants that were originally incubated anaerobically showed a marked increase in P-gal activity (as indicated by the blue appearance of the colonies shown in figure 9). We have confirmed this observation using S. cerevisiae strain BY4742 (data not shown). These results define novel conditions in which HXT17 is expressed.  3.1.3 HXT5, HXT13 and HXT15 are expressed during growth on ethanol, or glycerol and ethanol together. After prolonged incubation of X-gal plates containing low amounts of glucose or fructose we observed slight induction of HXT5-, HXT 13- and HXT15-lacZ (data not shown). We hypothesized that these genes may be induced in response to glucose starvation, and thus tested the expression level of these genes in cells grown on the non-fermentable carbon source ethanol alone (2 % v/v), or in combination with glycerol (2 % v/v each). As seen in figure 10, HXT5, HXT13 and to a lesser extent HXT15 are all expressed on both carbon sources. We also tested glycerol alone, but our yeast strain BY4742 was unable to grow under this condition (not shown). Because HXT 15 was expressed much lower than the other two transporters, the physiological significance of this observation is uncertain.  32  3.2 High amounts of glucose or fructose reduces water activity in Y N B media. Water activity represents the chemical potential of water in solution. Pure water has a water activity of 1.000, and the addition of solute(s) decreases this value. For yeast and other microorganisms, high osmolality or low water activity of the surrounding media causes water to flow out of a cell and into the environment, thereby illiciting a cellular stress response. A s a reflection of the capability of high sugar concentrations to inflict osmotic stress upon yeast cells, we measured the water activity of various Y N B media containing glucose or fructose concentrations up to 40 % (w/v). Glucose at 2 %, 20 % and 40 % concentrations had water activities of 0.981, 0.974 and 0.939 respectively. Fructose at 2 %, 20 % and 40 % concentrations had water activities of 0.983, 0.977, and 0.942 respectively.  3.3 High glucose (40 % w/v) and 1.4 M NaCI increase HXT1 promoter activity to the same extent Our observation that 40 % glucose appeared to increase the HXT1 promoter activity was not surprising, given that others had reported HXT1 to be up-regulated by osmotic shock using NaCI (0.7 - 1.0 M ) or sorbitol (0.95 - 1.5 M ) as the osmolyte (46, 128). The water activity of media containing 1.4 M N a C I is approximately 0.952 (33). This is similar to the water activity of 40 % glucose (w/v) which we found to be approximately 0.939. To compare the effects of NaCI and 40 % glucose (w/v) on HXT1 promoter activity we designed an experiment to test the effect of a short-term osmotic shock on HXTl-lacZ induction. Short-term stress exposure reflects the immediate response, rather than long term adaptation to a stressor and therefore is distinct from experiments like those shown in figures 1-6. A s seen in figure 11A, 1.4 M NaCI (8 % w/v) in the presence of glucose activated the HXT1 promoter to the same extent as 40 % glucose. Using 2 % galactose (w/v) as the carbon source however, 1.4 M NaCI failed to activate the HXT1 promoter, similar to 2 % galactose alone (Figure 1 I B ) . Unexpectedly, the level of 33  promoter activation was ~14-fold higher in 2 % glucose (w/v) versus 40 % glucose (Figure 11 A ) . This indicated that osmotic shock actually down-regulates the HXT1 promoter.  3.4 The effect of high glucose concentrations on HXT1 m R N A stability A s mentioned, previous studies have shown by Northern blot and/or D N A microarray that HXT1 m R N A levels are increased in response to NaCI or sorbitol (46, 128). However our results indicate that the HXT1 promoter activity is actually down-regulated by osmotic shock. To reconcile these observations, we hypothesized that for HXT1 m R N A to accumulate under osmotic stress despite the promoter being down-regulated, the turnover o f the molecule must also be decreased. To address this possibility, the yeast strain Y 2 6 0 , bearing the temperature sensitive rpbl-1 allele was shifted to the non-permissive temperature in rich media containing either 2 % glucose or 40 % glucose (w/v) as the carbon source. Total R N A was harvested at various time points following cessation of transcription, and the level of HXT1 m R N A was assayed by Real-time P C R . A s expected, in 2 % glucose (w/v) the level of HXT1 C  mRNA  declined immediately upon shifting to the non-permissive temperature, and had diminished approximately five-fold after one hour at 37 °C (Figure 12A). However, i n the presence of 40 % (w/v) glucose we observed an initial increase in HXT1 levels in the first 30 minutes, followed by a decline that reached a steady state level o f approximately 50 % of that at the time of the temperature shift. The initial increase in the m R N A level was difficult to explain. Although we pre-warmed the yeast cells to 37 °C prior to introducing the osmotic stress it is possible that transcription was able to proceed briefly before inactivation of the rpbl-1 allele. The HXT1 m R N A level was reduced to below the starting level after 30 minutes, indicating that transcriptional arrest did ultimately occur. A s confirmation of the functionality of the rpbl-1  NB - Although Real-time P C R actually assays c D N A levels, we assume a 1:1 conversion from m R N A and therefore refer back to the m R N A levels throughout this thesis. 0  34  allele, the cells that were shifted to 37 °C failed to double, and did not appear to arrest at any particular stage o f the cell cycle (not shown), both o f which are characteristic o f the Y 2 6 0 strain (101). Furthermore, i f transcription were occurring at the non-permissive temperature we would expect to see an increase i n GPD1 m R N A levels. GPD1 is up-regulated within 10 minutes o f exposure to osmotic stress (127, 129) and GPD1 expression increases greater 30-fold in response to salt-induced osmotic stress (128). However, in our experiments we did not observe a significant increase in GPD1 m R N A above initial levels after 15 minutes. However, the level o f GPD1 m R N A was significantly different in cells exposed to 40 % glucose versus 2 % glucose after as little as 15 minutes (p = 0.001, Figure 12B) indicating that this transcript may also be stabilized by osmotic stress. Furthermore HTB1, which is a histone encoding transcript with a half-life o f 5-14 minutes (45) also appeared to be stabilized during exposure to osmotic stress (Figure 12C, p = 0.048). Finally, IPP1 which encodes the inorganic pyrophosphatase enzyme and is not transcriptionally regulated by osmotic stress (127) decayed at a similar rate in both 2 % and 40 % (w/v) glucose-treated cells (Figure 12D, p = 0.292). • A s an independent method to confirm our results obtained with the temperature sensitive mutant, we tested the stability o f HXT] m R N A after yeast cells were exposed to 2 % or 40 % (w/v) glucose in the presence o f thiolutin, a potent inhibitor o f R N A polymerase I, II and III in vitro and in vivo (55, 161). The specific cellular target o f thiolutin is unknown, but it is likely distinct from R p b l p , given that it interferes with R N A polymerase I and III. A s shown in figure 13A, the relative stability ofHXTl  m R N A was dependent on the extracellular sugar  concentration (p = 0.0002) and despite large variability, the levels of HXT1 m R N A were statistically different as early as 20 minutes after transcriptional arrest. W e also tested i f the stabilization ofHXTl  during osmotic stress required the H O G  pathway. A s seen in figure 13A and B,. decay profiles for HXT1 m R N A was similar between wild-type cells and the hoglA strain. 35  3.5 Osmotic stress enhances retention of polyadenylated molecules after termination of transcription in yeast cells Due to the observation that seemingly unrelated m R N A molecules  {HXTI  and HTB1) are  both stabilized by osmotic stress, we performed a preliminary experiment to address whether other transcripts may be stabilized by osmotic stress. As a reflection of m R N A levels, we purified poly-adenylated molecules from total R N A derived from Y260 cells after 30 minutes at the non-permissive temperature in either low (2 % w/v) or high (40 % w/v) glucose. As seen in figure 14, there is a 10-fold reduction in cellular m R N A (as a percentage of total RNA) from cells grown at the permissive temperature to cells in 2 % (w/v) glucose for 30 minutes at the non-permissive temperature. Remarkably, the decrease in m R N A is only 2-fold for osmotically stressed cells at the non-permissive temperature. Therefore, relatively speaking, there is a fivefold greater retention of poly-adenylated transcripts in osmotically-challenged cells compared to cells left in 2 % (w/v) glucose (p = 0.034). As a confirmation of this result we performed the same experiment in S. cerevisiae strain BY4742 using thiolutin as the transcription inhibitor. In BY4742 cells, more polyadenylated molecules were retained in the osmotically stress cells than unstressed cells, but the relative differences were slightly less than seen in the Y260 strain (data not shown).  36  12 o •  MM  >  250 200 150 100  III  50 co  2  d d 1  o 0.2  10  20  30  40  Hexose concentration (% w/v)  Figure 1. HXT4 promoter activity decreases in response to increasing extracellular sugar concentrations. Strain T C Y 1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for P - g a l activity. Results shown represent the mean o f at least two experiments. Error bars represent the range o f values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 36.3, p = 0.05).  37  0.2  10  20  30  40  Hexose concentration (% w/v)  Figure 2. HXT3 is constitutively expressed during growth in the presence o f glucose or fructose. Strain T C Y 1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for P - g a l activity. Results shown represent the mean o f at least two experiments. Error bars represent the range o f values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 272.3, p = 0.05).  38  £  1000  0.2  2  10  20  30  40  Hexose concentration (% w/v)  Figure 3. i£Y75 promoter activity is up-regulated in response to high concentrations of extracellular glucose or fructose. Strain T C Y 1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for (3-gal activity. Results shown represent the mean of at least two experiments. Error bars represent the range o f values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 52.1, p = 0.05).  39  j/f 500 •  HM  c  * • MM  >  *  (0  CO.  400 300  c,d,e  200 100  d,e -<-  6,e  T  ILL  0 0.2  2  10  20  d,e  ,e ' d  y  d  e  JUL 30  40  Hexose concentration (%w/v)  Figure 4. HXT2 promoter activity is up-regulated at low concentrations o f extracellular glucose or fructose. Strain T C Y 1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for P-gal activity. Results shown represent the mean o f at least two experiments. Error bars represent the range o f values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 48.6, p = 0.05).  40  In  5000 -  c 3  4000 -  •  vity  3000  o  <  2000 -  \  "ca  O) •  n ll c,d  1000 d d  o 0.2  2  10  20  30  40  Hexose concentration (% w/v)  Figure 5. HXT1 promoter activity is increased in response to increasing extracellular sugar concentrations. Strain TCY1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for P - g a l activity. Results shown represent the mean o f at least two experiments. Error bars represent the range o f values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 872.4, p = 0.05).  41  200  "E 3 150 •  b,c  HH  ity(  s  > <+-» U CO  100 c,d c,d  50  T  d  d  d  d  CO  •  0 0.2  2  10  20  30  40  Hexose concentration (%w/v)  Figure 6. HXT 13 is expressed at low concentrations of extracellular glucose or fructose. Strain T C Y 1 was grown in Y N B media containing the indicated carbon source. A t mid-log phase, cells were harvested and assayed for P-gal activity. Results shown represent the mean o f at least two experiments. Error bars represent the range of values from two experiments or the standard deviation from three experiments. Means with the same letter are not significantly different (Fisher's L S D = 40.5, p = 0.05).  42  low glucose  high glucose  Figure 7. The complement of expressed hexose transporters is distinct at low (2 % w/v) and high (40 % w/v) concentrations o f extracellular glucose. The contribution of individual HXT genes to to the overall complement o f hexose transporters expressed at low and high concentrations o f glucose are displayed. The promoter activity o f each transporter is expressed as a percentage of the aggregate promoter activity for all of the transporters expressed in the indicated glucose level. Promoter activity levels are based on results presented in figures 1 - 6 .  43  Raffinose/Galactose ( 1 . 5 : 0 . 5 % w/v)  Glucose (2 % w / v )  Figure 8. HXT I7 is expressed more highly at p H 7.7 versus 4.7 when grown in the presence o f raffinose and galactose. A ) HXT17 is expressed i n cells grown with raffinose and galactose (1.5 :0.5 % w/v) but not glucose (2 % w/v) as the carbon source. T C Y 1 cells were transformed with an HXT17-lacZ construct and grown i n duplicate on Y N B plates containing the indicated carbon source. HXT17 expression is identified b y the appearance o f blue colonies. B ) HXT17 is expressed i n cells grown with raffinose and galactose (1.5:0.5 % w/v) as the carbon source at p H 7.7 versus 4.7. Strain T C Y 1 was grown i n Y N B media containing raffinose and galactose i n which the p H was adjusted to 4.7 or 7.7. A t mid-log phase, cells were harvested and assayed for (3-gal activity. Results shown are the mean o f three experiments (error bars represent one standard deviation). 44  A  B  Figure 9. HXT17 is expressed in cells that are exposed to an aerobic environment after 80 hours of anaerobic growth. TCY1 cells were transformed with an HXT17-lacZ construct and grown in duplicate on Y N B plates containing 2 % glucose (w/v) as the carbon source. A) Cells were grown in an anaerobic chamber for 80 hours, and then transferred to an aerobic environment for a further 24 hours. B) Cells were grown aerobically for the full duration of the experiment. HXT17 expression is identified by the appearance of blue colonies.  45  10000  EtOH Gly/EtOH  HXT5  HXT13  HXT15  Figure 10. HXT5, HXT13 and HXT15 are expressed in cells grown in Y N B media containing ethanol (2 % v/v), or ethanol and glycerol (2 % v/v each). B Y 4 7 4 2 cells were grown i n Y N B media containing the indicated carbon source (ethanol - E t O H , glycerol - G l y ) . A t mid-log phase, cells were harvested and assayed for P - g a l activity. Results shown are the mean o f two experiments (error bars represent the range o f values from two experiments).  46  _  (0  5000  Glc 2% + NaCl 8%  Glc 40%  Glc 2%  Osmolyte (w/v)  B «r 400  1  300  >200 >  o ro 100 ro OJ  Gal 2%  Gal 2% + NaCl 8%  Glc 2% + NaCl 8%  Osmolyte (w/v)  Figure 11. Osmotic shock-induced HXT] expression requires glucose but decreases HXT] promoter activity relative to non-stressed cells. A ) Osmotic shock down-regulates the HXT] promoter. T C Y 1 cells were transformed with an HXTl-lacZ construct and grown on Y N B plates containing 2 % galactose (w/v) as the carbon source. A t mid-log phase cells were harvested and inoculated into Y N B media containing either 2 % glucose (with or without 8 % N a C l ) or 40 % glucose (w/v). After four hours cells were harvested and analyzed for P-gal activity. B ) Glucose is required for HXT] promoter activation in response to osmotic shock. T C Y 1 cells were prepared like A , but inoculated into Y N B media containing glucose (2 % w/v) or galactose (2 % w/v) with or without the presence o f 8 % N a C l (w/v). The results shown represent the mean o f two experiments (error bars represent the range o f values from two experiments).  47  B  20  10  30  40  50  60  10  Time at non-permissive temperature (min)  20  30  40  50  60  Time at non-permissive temperature (min)  2 % Glc  40 % G l c  5 0 J  -5  -10 -15 -20 10  20  30  40  50  Time at non-permissive temperature (min)  60  0  20  40  60  Time at non-permissive temperature (min)  Figure 12. Several distinct transcripts are stabilized by osmotic stress. Y 2 6 0 cells were grown to mid-log phase and then shifted to the non-permissive temperature to inactivate the rpbl-1 allele. A t the indicated times after the temperature shift, the cells were harvested and m R N A levels were quantified by Real-time P C R . A ) HXT1 B ) GPD1 C ) HTB1 D ) IPP1 (error bars represent range o f values from two experiments). A t any single time point, data points marked with an asterisk are significantly different.  48  A 5 i  -30  -I  0  ,  10  I 1  ,  ,  ,  20  30  40  50  Time after thiolutin was added (min)  -15  H  0  10  20  30  40  50  T i m e after thiolutin w a s a d d e d (min)  Figure 13. HXT1 m R N A is stabilized by osmotic stress after transcriptional arrest induced by thiolutin. B Y 4 7 4 2 (A) or hoglA (B) cells were grown to mid-log phase and then treated with thiolutin (20 mg/L) to block R N A transcription. A t the indicated times after thiolutin addition the cells were harvested and HXT1 m R N A levels were quantified by Real-time P C R . The mean values from two experiments are displayed (error bars represent range o f values from two experiments). A t any single time point, data points marked with an asterisk are significantly different. 49  <  2-0-  oc  re o o  1.5 -  Figure 14. Global m R N A decay is reduced in osmotically stressed yeast cells. Poly-adenylated m R N A molecules were purified from total R N A derived from Y 2 6 0 cells in mid-log phase at the permissive temperature, or after 30 minutes at the non-permissive temperature with or without glucose-induced osmotic stress. m R N A levels are expressed as a percentage o f total cellular R N A . The results represent the mean of two experiments (error bars represent the range o f values from two experiments).  50  CHAPTER IV 4.0 D I S C U S S I O N S. cerevisiae has been used for centuries by humans for the production o f alcoholic beverages. Once thought to occur as a spontaneous act of Nature, Louis Pasteur first introduced the world to the science of wine-making when he discovered i n the late 1850's that ethanol production is carried out by living organisms. Remarkably, i n the nearly 150 years since Pasteur's discovery little has changed in terms of how wine is fermented, although we now understand significantly more about the biology and biochemistry o f this process. The juice from crushed grapes contains an ideal balance o f sugars, acids and other nutrients to allow it to be fermented by either indigenous yeast or by a commercially available wine yeast strain, without any further human intervention. However, wine yeasts (mostly Saccharomyces species) are not the only organisms that can readily proliferate in grape must. M a n y other microorganisms, including other (non-Saccharomyces) yeast and bacteria can easily contaminate and spoil a wine. Today wine and spirit production is a multi-billion dollar industry that spans six continents. A s a result, there is significant financial incentive to ensure that wine fermentations proceed smoothly to provide high quality wine. One issue that has chronically plagued the world wine industry is that of stuck and sluggish fermentations whereby wines take considerably longer to ferment to dryness, or never reach dryness at all. Wines that are intended to be dry (for example premium red wines) that finish with residual sugar are often deemed to be of low quality. These wines retrieve lower prices (or are not marketable altogether) and can significantly impact the revenue o f affected wineries. A s a result it is important that the wine industry have a detailed understanding at the molecular level how yeast adapts to and thrives in the challenging environment o f wine fermentations. During a typical fermentation, yeast cells are exposed to many stresses including high ethanol, osmotic stress (caused by sugar concentrations that can reach 40 % w/v), a depletion of nutrients, low p H and large temperature  51  changes. Understanding how S. cerevisiae responds to these conditions is critical for developing diagnostic tools to identify yeast strains that are prone to stuck or sluggish fermentations. The primary role o f S. cerevisiae during wine fermentations is to convert the available hexoses (6-carbon sugars) in grape must into ethanol, carbon dioxide and flavour compounds. Because hexoses cannot freely diffuse across the yeast cell membrane, a group of proteins termed hexose transporters (encoded by HXT genes) are required to facilitate the process. Because sugar uptake is the first rate-limiting step during a wine fermentation, it is conceivable that defects in hexose transporter regulation could result i n stuck or sluggish wine fermentations. However, of the twenty known HXT or HXT-likc genes i n S. cerevisiae, only nine have an assigned function. W e therefore set out to characterize the transcriptional regulation of HXT genes, with a particular emphasis on conditions that may be encountered during a wine fermentation.  4.1 Effect of glucose or fructose on HXT promoter activity at concentrations of 0.2 % - 40 % (w/v) For the HXT genes that have been characterized thus far the primary determinant o f expression level is the extracellular glucose concentration. Each transporter protein has a unique affinity for glucose (and fructose), with higher affinity transporters being expressed at the lowest concentrations o f glucose and vice versa. Despite the fact that S. cerevisiae can readily metabolize glucose at extracellular concentrations o f less than 0.1 % to greater than 40 % (w/v), the transcriptional regulation o f yeast HXT genes has only been tested up to 8 % glucose (w/v). We hypothesized that the HXT genes with previously unknown function may be transcribed only at high (> 10 % w/v) sugar concentrations. Furthermore, although glucose and fructose are present in equimolar amounts i n grape must, previous studies have focused primarily on glucosemediated regulation of HXT genes. Therefore, using HXT promoter-lacZ fusion constructs, we  52  tested the promoter activity of HXT1-5 and HXT10-17 in glucose and fructose concentrations ranging from 0.2 % to 40 % (w/v). In agreement with previous observations (111), we showed that HXT2 and HXT4 are expressed when extracellular sugar concentrations are low (< 2 % w/v Figures 1,4). Furthermore, HXT3 is expressed constitutively, and HXT1 is induced in the presence of > 1 % w/v glucose or fructose. With the exception of HXT2, no differences in promoter activation were observed between glucose-grown and fructose-grown cells. It has been reported that hxt2A strains of S. cerevisiae are prone to sluggish fermentations (cited in 10). Because stuck fermentations typically arrest with 1.5 - 2.0 % (w/v) fructose and only 0.1 - 0.5 % glucose it was interesting that the transcriptional activation of HXT2 at these levels was approximately two-fold higher in fructose-grown cells than glucose-grown cells. However, two observations indicate that mis-regulation of HXT2 alone is unlikely to cause a stuck fermentation. First, as we have shown, Hxt2p represents only 10-20 % of the hexose transporter complement expressed around 2 % (w/v) sugars, based on promoter activity (Figure 7). However, often only one functional transporter is required to support growth on glucose (178). Second, by deleting individual HXT genes in an industrial strain of S. cerevisiae it was shown that HXT2 is involved in growth initiation during wine fermentations when the extracellular sugar concentration was 20 % (w/v), but the fermentation kinetics proceed normally afterwards (78). This observation implies that Hxt2p is not required at the end of fermentations when glucose and fructose concentrations are becoming depleted, and therefore mis-regulation of HXT2 is likely not associated with the residual fructose remaining in stuck fermentations. Taken together, our results suggest that HXT I, HXT3 and HXT5 appear to be the major transporters at high sugar concentrations, making them most relevant during early stages of wine fermentations. Conversely HXT2, HXT4 and HXT13 are expressed at low concentrations of extracellular glucose or fructose, suggesting these transporters could be important at the end of wine fermentations when only trace amounts of sugars remain. These observations do not 53  entirely agree with the report o f Luyten et al. which describes the role o f individual transporters during the course o f a wine fermentation, and further emphasizes the complexity o f gene regulation for the HXT gene family. It could be that combinations o f factors, including nitrogen availability, and/or ethanol concentration affects the in vivo performance o f these transporter proteins. Alternatively, there may be protein-protein interactions among different transporters in vivo that are necessary for proper function and these interactions may affect the activity o f these proteins when they are studied in isolation (30, 110).  4.1.1  Effect of p H on HXT17 promoter activity  S. cerevisiae is able to proliferate i n a wide range o f external pHs. Growth is optimal at p H 4-5, and the typical p H during wine fermentations ranges from 3-4. Growth in more alkaline conditions illicits a stress response in S. cerevisiae, and the transcriptional response to alkaline p H is at least partially mediated by R i m l O l p (70). We observed an induction of HXT17 promoter activity in response to a shift from p H 4.7 to 7.7 i n a low-glucose environment (Figure 8B). The transcriptional response o f S. cerevisiae to alkaline conditions has not been widely studied, although D N A microarray data have revealed that as many as 500 genes have altered expression i n response to increasing extracellular p H (21, 70, 146). In the study o f Serrano et al, HXT4 was induced within 5 minutes o f shifting from p H 6.4 to p H 7.6 in rich media containing 2 % (w/v) glucose (146). Additionally the expression levels of HXT8, HXT9, HXT11 and HXT12 were repressed at least three-fold by the same stress. The significance o f p H dependent HXT expression is unclear, however it is possible that individual transporters have altered affinities for glucose depending on the local proton concentration. The predicted isoelectric point for the alkaline-induced genes HXT4 and HXT 17 is 6.37 and 6.95 respectively, indicating a net-negative charge at p H 7.6 or 7.7. Conversely, the isoelectric points for alkalinerepressed HXT9, HXT 11 and HXT 12 are 8.17, 8.54, and 8.48 respectively indicating these 54  transporters would have a net-positive charge. However HXT8, which was reported as alkalinerepressed has a p i o f 5.32 (the lowest o f all HXT genes) which, fails to support the pattern. Still, considering that it is only the extracellular portions o f these transporter proteins that are exposed to changing p H , then an examination of the net-charge o f these domains may prove interesting. Based on recent reports, there appears to be a link between alkaline p H tolerance and iron and "copper uptake. M o r e specifically, several mutants that displayed a severe sensitivity to alkali were shown to be defective in gene products that are required for efficient copper and iron uptake (145). Interestingly, the expression o f high affinity copper uptake genes is regulated by M a c l p (58), and a constitutively active allele of this protein MACT'  pl  was shown to increase the  expression of HXT17 more than two-fold (40). However, it should be pointed out that when the same group used a copper-chelator to mimic copper starvation (when MAC1 -dependent genes are transcriptionally active) HXT17 was only marginally induced (1.1 - 1.4 fold increased). Whether copper (or iron) uptake is involved in the transcriptional activation of HXT17 remains to be seen. A n interesting hypothesis would be that HXT17 serves as a symporter for particular minerals (presumably copper and iron) along with glucose. Given that HXT9 and HXT 11 were identified in a screen for multi-drug resistance (102), and HXT1 and HXT3 are suppressors o f a potassium transport defect (62) it is conceivable that many o f the HXT genes have additional (non-hexose) substrates.  4.1.2 HXT17 is expressed upon relief from anaerobiosis Because we were unable to reproduce anaerobic conditions i n liquid-grown cultures o f S. cerevisiae we were not able to precisely quantify the induction of HXT 17 seen upon removing cells from an anaerobic environment. Because the glucose consumption rate is five-fold greater in anaerobically grown yeast cells than aerobically-grown yeast cells (30) it is possible that the local glucose concentration surrounding the anaerobically grown cells was sufficiently low to 55  allow derepression of HXT17 (in a p H and glucose dependent manner, discussed in section 4.1.1). In support o f this hypothesis, i f aerobically-grown cells are incubated for an additional 48-96 hours, a slight induction of HXT17 can be seen on glucose-containing plates (data not shown). B y that time the local concentration o f glucose should be sufficiently low to allow the p H dependent derepression of HXT17.  4.1.3 HXT5, HXT13 and HXT15 are expressed during growth on ethanol, or glycerol and ethanol together The expression of HXT5 is linked to growth rate i n S. cerevisiae and previous studies have shown that cells growing on glycerol or ethanol exhibit increased HXT5 expression relative to glucose-grown cells (31, 172). W e observed an induction of HXT5 when yeast cells were grown on non-fermentable carbon sources, and also saw increased HXT 13 and HXT 15 expression under the same conditions (Figure 10). HXT5 does not appear to be regulated by the glucose sensors Snf3p/Rgt2p (172) which is expected, given that many o f the conditions that induce HXT5 (low nitrogen, osmotic stress, heat stress) act independently o f the extracellular glucose concentration. Our observations suggest that HXT13 may be regulated in a similar manner. Although HXT 15 appeared to be induced on non-fermentable carbon sources, the low level o f expression suggests this transporter may not be physiologically relevant under these conditions. Hxt5p has a moderate affinity for glucose ( K = 10 m M ) but no such data is m  available for H x t l 3 p . In our experiments HXT 13 is also inducible by 0.2 % glucose (w/v) indicating it may be a high affinity transporter, similar to HXT2, HXT4 and HXT6/7. If this is the case, then Hxt5p and H x t l 3 p could serve as low- and high-affinity transporters (respectively) for the cell under conditions o f glucose starvation. Further analysis o f the promoters of HXT5 and HXT13 as well as a detailed biochemical study o f the physical properties o f these transporter proteins should help to address this point. For example, a study o f Hxt5p and H x t l 3 p protein 56  turnover rates would be interesting because highly stable proteins would be ideally expressed under starvation conditions when transcriptional and translational activity is likely to be minimal.  4.1.4 HXT1 m R N A is stabilized by glucose-induced osmotic stress W e and others have observed that HXT1 is up-regulated in response to osmotic stress (33, 46, 129). Our observation that HXT1 is increased more than 3-fold in cells grown in 40 % glucose versus 10 % (w/v) glucose (Figure 5) is counter-intuitive given that the growth rate of yeast is reduced by the osmotic stress of 40 % glucose (w/v). Because the glycolytic flux decreases concomitantly with the growth rate, there should already be sufficient glucose to meet cellular requirements at the level of 10 % (w/v). To better understand HXT1 promoter regulation, we compared the effect of osmotic shock caused by 40 % glucose or 2 % glucose with 8 % N a C l (w/v). Both of these media have similar water activity and therefore should inflict comparable osmotic stress upon yeast cells. The original intention was to delineate the extent of HXT1 induction that was attributable to glucose, and to osmotic stress. Using the same HXTl-lacZ construct, Ozcan and Johnston reported that HXT1 reached maximal expression in cells grown in 4 % glucose (w/v) and remained constant up to 8 % (111). W e therefore suspected that the stress-dependent component of HXT 1 expression would require at least 10 % glucose (w/v). First we observed that salt-induced osmotic stress could only activate the HXT1 promoter in the presence of glucose (Figure 1 IB). R g t l p is a transcriptional repressor that restricts HXT1 expression to glucose-containing environments (112). Since H o g l p is required fox HXT 1 m R N A accumulation during osmotic stress (129), our observation suggests that the H O G pathway is unable to overcome repression by R g t l p . Furthermore this result also indicates that either the H O G pathway does not act directly on the HXT1 promoter, or that osmotic stress signals to a second activator or repressor on the HXT1 promoter that acts in concert with R g t l p 57  (analogous to the dual repression of HXT2 and HXT4 b y R g t l p and M i g l p that restricts expression to low-glucose conditions). A very recent report has confirmed this hypothesis, as the transcriptional repressor S k o l p was shown to bind and repress the HXT1 promoter in the absence o f H o g l p kinase activity (164). A s little as 4 % glucose (w/v) or 2 % glucose plus NaCI (0.4 M ) was able to induce S k o l p derepression. This explains why other groups observed maximalHXT1 promoter induction in cells grown in 4 % glucose (w/v) (111), but still can not account for the increased accumulation of HXT1 m R N A as the glucose concentration increases to 40 % (w/v) because the promoter is fully derepressed. Surprisingly, comparing the HXT1 promoter activity during short-term osmotic shock in 2 % glucose (w/v) versus 40 % glucose revealed that HXT1 was actually down-regulated by osmotic stress (Figure 11 A ) . This is distinct from the experiment shown in figure 5 because it reveals the immediate response o f the promoter to the stress, whereas when growing cells in the presence o f 40 % glucose (w/v) (Figure 5) the long-term adaptation is measured. In order for m R N A molecules to accumulate despite lower promoter activity, the rate o f turnover must also be decreased. Indeed, when we tested the rate o f decay of HXT1 m R N A we found that the transcript persisted at higher levels after transcription shut-off during osmotic stress by 40 % (w/v) glucose compared to 2 % (w/v) glucose (Figure 13A). Previous studies have demonstrated both derepression and activation mechanisms leading to HXT1 expression (111, 164). Here we show that there is a third component to the regulation m R N A stabilization by osmotic stress. Given that HXT1 was not induced by osmotic stress with galactose as the carbon source, our data and data provided by others suggests two factors contributing to the high level oiHXTl m R N A accumulation during osmotic stress: i) glucosedependent promoter activation (110) and ii) osmotic stress-dependent m R N A stabilization. Several lines o f evidence suggest that the m R N A stabilization of HXT1 may occur as a result o f a positive signal through the H O G pathway. First, HOG1 is required for osmotic stress58  induction of HXT1 (129). Second, the mammalian homolog of HOG1,  p38 M A P K is involved in  the transcript-specific stabilization of several m R N A molecules (42, 61,81). Third, H o g l p is required for the transient inhibition of translation in response to osmotic stress (159), and translation-initiation is closely associated with m R N A turnover (150). W e tested HXT1  mRNA  stability after transcriptional arrest by thiolutin treatment in a hoglA strain, but found that the decay pattern was consistent with that seen in wild-type yeast cells. Therefore, although HOG1 is required for HXT1 m R N A accumulation during osmotic stress, the H O G pathway is not required for m R N A stabilization. A t this time we cannot predict what signal leads to m R N A stabilization under osmotic stress, but i f there is a signal transduction pathway it does not require de-novo transcription of a stabilizing factor, as HXT1 m R N A was stabilized in the absence of ongoing transcription. The paradigm for the transcriptional response to osmotic stress is increased GPD1 expression (127). In our study we found GPD1 m R N A to be stablized by sugar-induced osmotic stress to a similar extent as HXT1 (Figure 12B). A n intriguing explanation for these observations is that the osmotic stress-mediated stabilization of HXT1 and GPD1 could occur to ensure an adequate supply of carbon for the production of glycerol, a compatible solute that is rapidly accumulated in osmotically stressed yeast cells (12, 48). Interestingly, HTB1 which encodes a histone m R N A was stabilized in response to osmotic stress (Figure 12C). Because HTB1 has not been previously shown to respond to osmotic stress, we inquired whether there were other genes also stabilized in a similar fashion. A s a reflection of cellular m R N A levels we isolated poly-adenylated molecules from yeast cells that had been without active transcription for 30 minutes in the presence of either 2 % glucose (w/v) or 40 % glucose. A s a percentage of total cellular R N A , we observed a 5-fold greater retention of pblyadenylated molecules in osmotically stressed yeast cells compared to non-stressed cells after transcription shut-off (Figure 14). The implication of this observation is that a large portion of S. 59  cerevisiae m R N A molecules may have a decreased rate o f turnover during exposure to osmotic stress. A similar observation has been made in glucose-starved yeast cells (56), indicating that it may be part o f a general stress response. The same group also noticed that m R N A molecules were stabilized upon entry into stationary phase - and entry into stationary phase may be induced by stress exposure (136, 177). If this is true, then other stresses to which yeast is exposed such as ethanol, should reduce m R N A turnover. Although this has not been demonstrated for S. cerevisiae, ethanol can stabilize T N F a m R N A i n a p38-dependent manner in rat liver cells (60, 61).  4.2 Conclusions The HXT genes o f S. cerevisiae serve as an interesting model for gene expression. Except H x t l 2 p , all o f the HXT gene products are able to transport glucose, and yet each has a unique expression pattern. A t first glance, the complexity seems excessive, given that a system directly linked to extracellular glucose would be sufficient to always provide carbon when available. HXT 1-4 exhibit the expected expression patterns i n this regard. Perhaps the induction of HXT5 and HXT 13 on non-fermentable carbon sources occurs because these hexose transporters have particular protein characteristics that make them more useful during glucose starvation. For example, i f these proteins were more stable than other transporters they would be ideal for starvation conditions when transcriptional and translational activity is likely to be minimal. It has already been established that Hxt5p has a moderate affinity for glucose, and based on the expression pattern we observed for H x t l 3 p we suspect this is a high affinity transporter. Therefore, these two proteins would be present during growth on non-fermentable carbon to await the return o f glucose to the environment. Our observation that HXT17 is expressed at higher p H on trace amounts o f glucose is interesting because it represents the first HXT gene that is not solely regulated by carbon 60  availability. It is possible that this protein has particular characteristics that benefit the interaction with glucose at higher p H . Alternatively HXT17 could be part o f a copper-regulon that has yet to be fully defined. A s mentioned there is a link between copper and iron uptake with alkaline conditions, and it is possible that HXT17 is serving as a symporter for glucose and one o f these metals under these conditions. The m R N A stabilization we observed for HXT1 has broad implications. First, the fact that m R N A turnover is reduced during stress conditions complicates studies o f transcriptional response to stress stimuli. If indeed there are many transcripts within the cell that are also stabilized by osmotic stress then the next challenge would be to identify the specific genes regulated in this manner. Second, i f HXT1 m R N A is stabilized specifically, (as opposed to a global, non-specific decrease i n m R N A turnover) then the implication is that there is a subset o f genes that is part o f a core set o f transcripts required for stress survival, or restoration o f growth and proliferation once the stressor has passed. This set o f genes could be analogous to, or part o f the "common environmental response" or "environmental stress response" genes that are transcriptionally induced in response to a wide variety o f stress conditions. Identification o f these genes would be difficult because they may not be detected in conventional microarray experiments. More specifically, these m R N A molecules may not be required in higher copy numbers for stress survival, but rather only the steady state level needs to be maintained. If turnover is decreased then less energy needs to be expended transcribing these genes. Finally, our goal from the outset was to examine the transcriptional response o f hexose transporters to conditions that may be encountered during wine fermentations. Certainly the ethanol induction of HXT5 and HXT 13 is relevant to wine fermentations, particularly towards the end as glucose is exhausted. Whether these transporters are involved i n the finishing stages o f a wine fermentation remains to be seen. Furthermore, the osmotic stress-mediated stabilization o f HXT1 is relevant as the must into which yeast are inoculated at the start o f fermentations 61  presents an osmotic shock to the yeast cells. If transcriptional activity and m R N A turnover are reduced during early stages of fermentation then the yeast cell may be limited in adaptability to additional stressors that may occur thereafter.  62  CHAPTER V 5.0 F U T U R E D I R E C T I O N S B y studying the transcriptional regulation o f the HXT gene family we have identified two unique modes o f gene regulation. HXT1 m R N A appears to be stabilized i n a hyper-osmotic environment. A l s o , HXT17 is induced in a carbon source and pH-dependent manner. In both cases the signal transduction pathway and/or regulatory components that lead to transcription have not been identified. W e therefore proposed the following studies to increase our understanding o f these modes o f gene regulation.  5.1 Identification of the f u l l complement of m R N A molecules that are stabilized by osmotic stress Based on our observations it appears that there are a large number o f transcripts in S. cerevisiae that exhibit decreased turnover rates in response to osmotic stress. T o identify these genes, D N A microarray technology could be employed together with the S. cerevisiae strain Y260. Y 2 6 0 bears a temperature sensitive allele o f an R N A polymerase subunit rpbl-1, and when shifted to the non-permissive temperature global m R N A transcription is quickly arrested. After growing Y 2 6 0 cells in standard rich media (2 % glucose w/v) the cells could be shifted to the non-permissive temperature and inoculated into low (2 % w/v) or high (40 % w/v) glucose environments. A t one or more time points after transcriptional arrest, the cells can be harvested and global m R N A levels compared between cells exposed to the two different conditions using D N A microarrays. A similar technique has been used to study global m R N A decay rates in S. cerevisiae (175). For our study we would simply need to expand this method to compare and contrast m R N A decay rates in cells exposed to two different environments.  63  5.2 Identification of the regulatory components that mediate pH-dependent HXT17 expression Our data suggests that there are two distinct regulatory components to the HXT17 promoter. The first is a glucose-dependent element that likely involves the repressor M i g l p , and the second is an as-yet uncharacterized pH-dependent element. To elucidate the genes that are required for the pH-dependent induction of HXT17 a mutagenesis screen could be carried out. Mutagenised yeast cells that have been transformed with an HXT17-lacZ construct could be grown on Y N B X - g a l plates containing either raffinose or glucose as the carbon source. Colonies expressing the lacZ gene turn blue on X - g a l plates. Therefore, colonies that remain white on raffinose plates, or that turn blue on glucose plates are defective for proper HXT17 expression. Mutated genes could then be identified by re-introducing a c D N A library containing all known open reading frames from S. cerevisiae. Presumably this screen should also confirm the identity o f the glucosedependent promoter regulators such as the classical glucose repression regulators S n f l p and M i g l p . However, the function o f these proteins in regulating HXT17 expression could also be directly examined using the specific mutant strains.  64  LITERATURE CITED 1.  2. 3.  4.  5. 6.  7. 8.  9.  10.  11. 12. 13. 14.  15.  Ahuatzi, D., P. Herrero, T. De La Cera, and F. Moreno. 2004. The glucose regulated nuclear localization of hexokinase 2 in Saccharomyces cerevisiae is M i g l dependent. J B i o l Chem:published online ahead of print. Akashi, M., G. Shaw, M. Hachiya, E. Elstner, G. Suzuki, and P. Koeffler. 1994. Number and location o f A U U U A motifs: role i n regulating transiently expressed R N A s . B l o o d 83:3182-7. Akhtar, N., A. K. Pahlman, K. Larsson, A. H. Corbett, and L. Adler. 2000. SGD1 encodes an essential nuclear protein oi Saccharomyces cerevisiae that affects expression of the GPD1 gene for glycerol 3-phosphate dehydrogenase. F E B S Lett 483:87-92. Alepuz, P. M., E. de Nadal, M. Zapater, G. Ammerer, and F. Posas. 2003. Osmostress-induced transcription by H o t l depends on a Hogl-mediated recruitment of the R N A P o l II. E M B O J 22:2433-42. Ashrafi, K., S. S. Lin, J. K. Manchester, and J. I. Gordon. 2000. Sip2p and its partner S n f l p kinase affect aging in S. cerevisiae. Genes Dev 14:1872-1885. Ausubel, F. M. 1995. Short protocols in molecular biology : a compendium of methods from Current protocols in molecular biology, 3rd ed. John W i l e y & Sons, New York, N.Y. Barnett, J. A. 1976. The utilization of sugars by yeasts. A d v Carbohydr Chem Biochem 32:125-234. Bender, A., and J. R. Pringle. 1989. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl A c a d Sci U S A 86:9976-80. Bevilacqua, A., M. C. Ceriani, S. Capaccioli, and A. Nicolin. 2003. Posttranscriptional regulation of gene expression by degradation o f messenger R N A s . J Cell Physiol 195:356-72. Bisson, L. F. 1999. Stuck and sluggish fermentations. A m J E n o l Viticult 50:107-119.  Bisson, L. F., D. M. Coons, A. L. Kruckeberg, and D. A. Lewis. 1993. Yeast sugar transporters. Crit Rev Biochem M o l B i o l 28:259-308. Blomberg, A., and L. Adler. 1992. Physiology of osmotolerance in fungi. A d v Microb •Physiol 33:145-212. Boles, E., and C. P. Hollenberg. 1997. The molecular genetics of hexose transport in yeasts. F E M S M i c r o b i o l Rev 21:85-111. Boorstein, W. R., and E. A. Craig. 1990. Regulation o f a yeast HSP70 gene by a c A M P responsive transcriptional control element. E M B O J 9:2543-53.  Brachmann, C. B., A. Davies, G. J. Cost, E. Caputo, J. Li, P. Hieter, and J. D. Boeke. 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set o f strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115-32.  16.  Brewster, J. L., T. de Valoir, N. D. Dwyer, E. Winter, and M. C. Gustin. 1993. A n  17.  osmosensing signal transduction pathway in yeast. Science 259:1760-3. Brown, A. D. 1978. Compatible solutes and extreme water stress i n eukaryotic microorganisms. A d v Microb Physiol 17:181-242.  18.  Cabrera, C. V., J. J. Lee, J. W. Ellison, R. J. Britten, and E. H. Davidson. 1984. Regulation o f cytoplasmic m R N A prevalence in sea urchin embryos. Rates of appearance and turnover for specific sequences. J M o l B i o l 174:85-111. 65  19.  Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, and A . Cerami.  20.  1986. Identification o f a common nucleotide sequence in the 3'-untranslated region o f m R N A molecules specifying inflammatory mediators. Proc Natl A c a d Sci U S A 83:1670-4. Carlson, M. 1999. Glucose repression in yeast. Curr Opin M i c r o b i o l 2:202-7.  21.  Causton, H. C , B. Ren, S. S. Koh, C. T. Harbison, E. Kanin, E. G. Jennings, T. I. Lee, H. L. True, E. S. Lander, and R. A . Young. 2001. Remodeling o f yeast genome  22.  23.  24.  25.  26. 27.  28. 29.  expression in response to environmental changes. M o l B i o l C e l l 12:323-37. Chen, C. Y., and A . B. Shyu. 1995. A U - r i c h elements: characterization and importance in m R N A degradation. Trends Biochem Sci 20:465-70.  Colombo, S., P. Ma, L. Cauwenberg, J. Winderickx, M. Crauwels, A . Teunissen, D. Nauwelaers, J. H. de Winde, M. F. Gorwa, D. Colavizza, and J. M. Thevelein. 1998. Involvement o f distinct G-proteins, Gpa2 and Ras, i n glucose- and intracellular acidification-induced c A M P signalling in the yeast Saccharomyces cerevisiae. E M B O J 17:3326-41. Coons, D. M., R. B. Boulton, and L. F. Bisson. 1995. Computer-assisted nonlinear regression analysis o f the multicomponent glucose uptake kinetics oi Saccharomyces cerevisiae. J Bacteriol 177:3251-8. Coppola, J. A . , A . S. Field, and D. S. Luse. 1983. Promoter-proximal pausing by R N A polymerase II i n vitro: transcripts shorter than 20 nucleotides are not capped. Proc Natl Acad S c i U S A 8 0 : 1 2 5 1 - 5 . Cullen, P. J., and G. F. Sprague, Jr. 2000. Glucose depletion causes haploid invasive growth in yeast. Proc Natl A c a d Sci U S A 97:13619-13624. de Nadal, E., L. Casadome, and F. Posas. 2003. Targeting the M E F 2 - l i k e transcription . factor S m p l b y the stress-activated H o g l mitogen-activated protein kinase. M o l Cell B i o l 23:229-37. De Vit, M., J. Waddle, and M. Johnston. 1997. Regulated nuclear translocation o f the M i g l glucose repressor. M o l B i o l C e l l 8:1603-1618. DeVit, M. J., and M. Johnston. 1999. The nuclear exportin M s n 5 is required for nuclear export o f the M i g l glucose repressor of Saccharomyces cerevisiae. Curr B i o l 9:1231-41.  30.  Diderich, J. A . , M. Schepper, P. van Hoek, M. A . H. Luttik, J. P. van Dijken, J. T. Pronk, P. Klaassen, H. F. M. Boelens, M. J. T. de Mattos, K. van Dam, and A . L. Kruckeberg. 1999. Glucose uptake kinetics and transcription of HXT genes in chemostat  31.  Diderich, J. A . , J. M. Schuurmans, M. C. Van Gaalen, A . L. Kruckeberg, and K.  cultures o f Saccharomyces cerevisiae. J B i o l Chem 274:15350-15359.  32.  33.  34.  Van Dam. 2001. Functional analysis o f the hexose transporter homologue HXT5 in Saccharomyces cerevisiae. Yeast 18:1515-24. Dunckley, T., and R. Parker. 1999. The Dcp2 protein is required for m R N A decapping in Saccharomyces cerevisiae and contains a functional M u t T motif. E M B O J 18:541122. Erasmus, D. J., G. K. van der Merwe, and H. J. J. van Vuuren. 2003. Genome-wide expression analyses: Metabolic adaptation o f Saccharomyces cerevisiae to high sugar stress. F E M S Yeast Res 3:375-99.  Flick, K. M., N. Spielewoy, T. I. Kalashnikova, M. Guaderrama, Q. Zhu, H. C. Chang, and C. Wittenberg. 2003. Grrl-dependent inactivation of M t h l mediates glucose-induced dissociation o f R g t l from HXT gene promoters. M o l B i o l C e l l 14:323041.  35.  Gancedo, J. M. 1998. Yeast carbon catabolite repression. M i c r o b i o l M o l B i o l Rev 62:334-61. 66  36.  Gao, M., C. J. Wilusz, S. W. Peltz, and J. Wilusz. 2001. A novel mRNA-decapping activity in H e L a cytoplasmic extracts is regulated by A U - r i c h elements. E M B O J 20:1134-43.  37. 38.  39. 40.  41.  42.  43.  44.  45.  46. 47. 48. 49.  50. 51.  52.  53.  Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B. Eisen, G. Storz, D. Botstein, and P. O. Brown. 2000. Genomic expression programs in the response of yeast cells to environmental changes. M o l B i o l C e l l 11:4241-57. Gietz, R. D., and R. H. Schiestl. 1995. Transforming yeast with D N A . Methods in Molecular and Cellular B i o l o g y 5:255-269.  Gorner, W., E. Durchschlag, M. T. Martinez-Pastor, F. Estruch, G. Ammerer, B. Hamilton, H. Ruis, and C. Schuller. 1998. Nuclear localization o f the C 2 H 2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12:586-97. Gross, C , M. Kelleher, V. R. Iyer, P. O. Brown, and D. R. Winge. 2000. Identification o f the copper regulon in Saccharomyces cerevisiae by D N A microarrays. J B i o l Chem 275:32310-6. Guhaniyogi, J., and G. Brewer. 2001. Regulation of m R N A stability in mammalian cells. Gene 265:11-23.  Han, Q., J. Leng, D. Bian, C. Mahanivong, K. A. Carpenter, Z. K. Pan, J. Han, and S. Huang. 2002. R a c l - M K K 3 - p 3 8 - M A P K A P K 2 pathway promotes urokinase plasminogen activator m R N A stability in invasive breast cancer cells. J B i o l Chem 277:48379-48385. Hardie, D. G., D. Carling, and M. Carlson. 1998. The A M P - a c t i v a t e d / S N F l protein kinase subfamily: metabolic sensors of the eukaryotic cell? A n n u Rev Biochem 67:82155. Herrero, P., C. Martinez-Campa, and F. Moreno. 1998. The hexokinase 2 protein participates in regulatory DNA-protein complexes necessary for glucose repression o f the SUC2 gene in Saccharomyces cerevisiae. F E B S Lett 434:71-6. Herrick, D., R. Parker, and A. Jacobson. 1990. Identification and comparison o f stable and unstable m R N A s in Saccharomyces cerevisiae. M o l C e l l B i o l 10:2269-84.  Hirayama, T., T. Maeda, H. Saito, and K. Shinozaki. 1995. Cloning and characterization o f seven c D N A s for hyperosmolarity-responsive ( H O R ) genes o f Saccharomyces cerevisiae. M o l Gen Genet 249:127-38. Hirose, Y., and J. L. Manley. 1998. R N A polymerase II is an essential m R N A polyadenylation factor. Nature 395:93-6. Hohmann, S. 2002. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol M o l B i o l Rev 66:300-372. Hong, S. P., F. C. Leiper, A. Woods, D. Carling, and M. Carlson. 2003. Activation of yeast S n f l and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci U S A 100:8839-43. Honigberg, S. M., and R. H. Lee. 1998. S n f l kinase connects nutritional pathways controlling meiosis i n Saccharomyces cerevisiae. M o l C e l l B i o l 18:4548-4555. Hsu, C. L., and A. Stevens. 1993. Yeast cells lacking 5'—>3' exoribonuclease 1 contain m R N A species that are poly(A) deficient and partially lack the 5' cap structure. M o l Cell B i o l 13:4826-35. Jacobs, J. S., A. R. Anderson, and R. P. Parker. 1998. The 3' to 5' degradation of yeast m R N A s is a general mechanism for m R N A turnover that requires the S K I 2 D E V H box protein and 3' to 5' exonucleases of the exosome complex. E M B O J 17:1497-506. Jacobson, A., and S. W. Peltz. 1996. Interrelationships of the pathways of m R N A decay and translation in eukaryotic cells. Annu Rev Biochem 65:693-739.  67  54.  55.  56.  57.  58. 59. 60.  61.  62. 63. 64. 65.  66. 67.  68. 69. 70.  71.  Jiang, R., and M. Carlson. 1997. The S n f l protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sipl/Sip2/Gal83 component in the kinase complex. M o l Cell B i o l 17:2099-106. Jimenez, A . , D. J. Tipper, and J. Davies. 1973. M o d e o f action of thiolutin, an inhibitor of macromolecular synthesis in Saccharomyces cerevisiae. Antimicrob Agents Chemother 3:729-738. Jona, G., M. Choder, and O. Gileadi. 2000. Glucose starvation induces a drastic reduction in the rates o f both transcription and degradation o f m R N A in yeast. Biochim Biophys Acta 1491:37-48. Jove, R., and J. L. Manley. 1984. In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites. J B i o l Chem 259:8513-21.  Jungmann, J., H. A . Reins, J. Lee, A . Romeo, R. Hassett, D. Kosman, and S. Jentsch. 1993. M a c l , a nuclear regulatory protein related to Cu-dependent transcription factors is involved i n Cu/Fe utilization and stress resistance in yeast. E M B O J 12:5051-6. Keleher, C. A . , M. J. Redd, J. Schultz, M. Carlson, and A . D. Johnson. 1992. Ssn6T u p l is a general repressor o f transcription i n yeast. C e l l 68:709-19. Kishore, R., M. R. McMullen, E. Cocuzzi, and L. E. Nagy. 2004. Lipopolysaccharidemediated signal transduction: Stabilization of TNF-alpha m R N A contributes to increased lipopolysaccharide-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding. Comp Hepatol 3 Suppl 1:S31. Kishore, R., M. R. McMullen, and L. E. Nagy. 2001. Stabilization of tumor necrosis factor alpha m R N A by chronic ethanol: role of A + U-rich elements and p38 mitogenactivated protein kinase signaling pathway. J B i o l Chem 276:41930-7. Ko, C. H., H. Liang, and R. F. Gaber. 1993. Roles of multiple glucose transporters in Saccharomyces cerevisiae. M o l Cell B i o l 13:638-48. Kruckeberg, A . L. 1996. The hexose transporter family of Saccharomyces cerevisiae. A r c h Microbiol 166:283-92. Kruckeberg, A . L., and L. F. Bisson. 1990. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport. M o l C e l l B i o l 10:5903-13. Kuchin, S., V. K. Vyas, and M. Carlson. 2002. S n f l Protein Kinase and the Repressors N r g l and Nrg2 Regulate FLOll, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation. M o l C e l l B i o l 22:3994-4000. Kurjan, J. 1993. The pheromone response pathway in Saccharomyces cerevisiae. A n n u Rev Genet 27:147-79. Lafuente, M. J., C. Gancedo, J. C. Jauniaux, and J. M. Gancedo. 2000. M t h l receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae. M o l M i c r o b i o l 35:161-72. LaGrandeur, T. E., and R. Parker. 1998. Isolation and characterization of D c p l p , the yeast m R N A decapping enzyme. E M B O J 17:1487-96. Lakshmanan, J., A . L. Mosley, and S. Ozcan. 2003. Repression of transcription by R g t l in the absence o f glucose requires Stdl and M t h l . Curr Genet 44:19-25. Lamb, T. M., W. Xu, A . Diamond, and A . P. Mitchell. 2001. Alkaline response genes of Saccharomyces cerevisiae and their relationship to the REVI101 pathway. J B i o l Chem 276:1850-1856. Lewis, D. A . , and L. F. Bisson. 1991. The HXT1 gene product o f Saccharomyces cerevisiae is a new member o f the family o f hexose transporters. M o l C e l l B i o l 11:380413.  68  72.  73. 74.  75. 76.  77.  78. 79. 80.  81.  82.  83.  84.  Li, F. N., and M. Johnston. 1997. G r r l of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through S k p l : coupling glucose sensing to gene expression and the cell cycle. E M B O J. 16:5629-5638. Liang, H., and R. Gaber. 1996. A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6. M o l B i o l Cell 7:1953-1966. Lin, S. S., J. K. Manchester, and J. I. Gordon. 2003. Sip2, an N-myristoylated beta subunit of snfl kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at r D N A loci, and silencing. J B i o l Chem 278:13390-13397. Lingner, J., J. Kellermann, and W. Keller. 1991. Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae. Nature 354:496-8. Lingner, J., I. Radtke, E. Wahle, and W. Keller. 1991. Purification and characterization o f poly(A) polymerase from Saccharomyces cerevisiae. J B i o l Chem 266:8741-6. Lundin, M., J. Nehlin, and H. Ronne. 1994. Importance o f a flanking A T - r i c h region in target site recognition by the G C box-binding zinc finger protein M i g l . M o l Cell B i o l 14:1979-1985. Luyten, K., C. Riou, and B. Blondin. 2002. The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation. Yeast 19:713-26. Maeda, T., M. Takekawa, and H. Saito. 1995. Activation of yeast Pbs2 M A P K K by M A P K K K s or by binding of an SH3-containing osmosensor. Science 269:554-8. Maeda, T., S. M. Wurgler-Murphy, and H. Saito. 1994. A two-component system that regulates an osmosensing M A P kinase cascade i n yeast. Nature 369:242-5. Mahtani, K. R., M. Brook, J. L. Dean, G. Sully, J. Saklatvala, and A. R. Clark. 2001. Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator o f tumor necrosis factor alpha m R N A stability. M o l C e l l B i o l 21:6461-9. Maier, A., B. Volker, E. Boles, and G. F. Fuhrmann. 2002. Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single H x t l , F£xt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters. F E M S Yeast Res 2:539-50. Marks, V. D., G. K. van der Merwe, and H. J. J. van Vuuren. 2003. Transcriptional profiling of wine yeast in fermenting grape juice: Regulatory effect of diammonium phosphate. F E M S Yeast Res 3:269-287.  Marshall-Carlson, L., J. L. Celenza, B. C. Laurent, and M. Carlson. 1990. Mutational analysis o f the Snf3 glucose transporter of Saccharomyces cerevisiae. M o l Cell B i o l 10:1105-15.  85.  86.  87.  88.  Martinez-Pastor, M. T., G. Marchler, C. Schuller, A. Marchler-Bauer, H. Ruis, and F. Estruch. 1996. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). E M B O J 15:2227-35. McCarthy, J. E. G. 1998. Posttranscriptional control o f gene expression in yeast. Microbiol M o l B i o l Rev 62:1492-1553.  McCracken, S., N. Fong, K. Yankulov, S. Ballantyne, G. Pan, J. Greenblatt, S. D. Patterson, M. Wickens, and D. L. Bentley. 1997. The C-terminal domain of R N A polymerase II couples m R N A processing to transcription. Nature 385:357-61. Miller, J. H. 1972. Experiments in molecular genetics. C o l d Spring Harbor Laboratory, [Cold Spring Harbor, N.Y.].  69  89. 90.  91.  92. 93.  94. 95.  96.  97. 98. 99. 100. 101.  102. 103.  104.  105.  106. 107.  Moreno, F., and P. Herrero. 2002. The hexokinase 2-dependent glucose signal transduction pathway o f Saccharomyces cerevisiae. F E M S M i c r o b i o l Rev 26:83-90. Moriya, H., and M. Johnston. 2004. Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I. Proc Natl A c a d Sci U S A 101:1572-1577. Morris, G. J., L. Winters, G. E. Coulson, and K. J. Clarke. 1986. Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae. J Gen Microbiol 132 ( Pt 7):2023-34. Morrissey, J. P., J. A. Deardorff, C. Hebron, and A. B. Sachs. 1999. Decapping o f stabilized, polyadenylated m R N A in yeast pabl mutants. Yeast 15:687-702. Mosley, A. L., J. Lakshmanan, B. K. Aryal, and S. Ozcan. 2003. Glucose-mediated phosphorylation converts the transcription factor R g t l from a repressor to an activator. J B i o l Chem 278:10322-7. Muhlrad, D., C. J. Decker, and R. Parker. 1995. Turnover mechanisms of the stable yeast PGK1 m R N A . M o l Cell B i o l 15:2145-56. Muhlrad, D., and R. Parker. 1992. Mutations affecting stability and deadenylation o f the yeast MFA2 transcript. Genes Dev 6:2100-11.  Nakafuku, M., T. Obara, K. Kaibuchi, I. Miyajima, A. Miyajima, H. Itoh, S. Nakamura, K. Arai, K. Matsumoto, and Y. Kaziro. 1988. Isolation o f a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl A c a d Sci U S A 85:1374-8. Nath, N., R. R. McCartney, and M. C. Schmidt. 2003. Yeast P a k l kinase associates with and activates S n f l . M o l Cell B i o l 23:3909-3917. Nehlin, J., M. Carlberg, and H. Ronne. 1991. Control o f yeast GAL genes by M i g l repressor: A transcriptional cascade in the glucose response. E M B O J 10:3373-3377. Nehlin, J., and H. Ronne. 1990. Yeast M i g l repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. E M B O J 9:2891-2898. Nehlin, J. O., M. Carlberg, and H. Ronne. 1989. Yeast galactose permease is related to yeast and mammalian glucose transporters. Gene 85:313-9. Nonet, M., C. Scafe, J. Sexton, and R. Young. 1987. Eucaryotic R N A polymerase conditional mutant that rapidly ceases m R N A synthesis. M o l C e l l B i o l 7:1602-11.  Nourani, A., M. Wesolowski-Louvel, T. Delaveau, C. Jacq, and A. Delahodde. 1997. Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: Involvement o f two hexose transporters. M o l Cell B i o l 17:5453-5460. O'Rourke, S. M., and I. Herskowitz. 1998. The H o g l M A P K prevents cross talk between the H O G and pheromone response M A P K pathways in Saccharomyces cerevisiae. Genes D e v 12:2874-86. O'Rourke, S. M., and I. Herskowitz. 2002. A third osmosensing branch in Saccharomyces cerevisiae requires the Msb2 protein and functions in parallel with the S h o l branch. M o l C e l l B i o l 22:4739-49. O'Rourke, S. M., and I. Herskowitz. 2004. Unique and redundant roles for H O G M A P K pathway components as revealed by whole-genome expression analysis. M o l B i o l Cell 15:532-42. O'Rourke, S. M., I. Herskowitz, and E. K. O'Shea. 2002. Yeast go the whole H O G for the hyperosmotic response. Trends Genet 18:405-12. Ostling, J., M. Carlberg, and H. Ronne. 1996. Functional domains in the M i g l repressor. M o l C e l l B i o l 16:753-761.  70  108.  109. 110. 111.  112.  113.  114. 115. 116.  Ozcan, S., J. Dover, and M. Johnston. 1998. Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. E M B O J . 17:2566-2573. Ozcan, S., J. Dover, A. G. Rosenwald, S. Wolfl, and M. Johnston. 1996. Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction o f gene expression. Proc Natl Acad Sci U S A 93:12428-12432. Ozcan, S., and M. Johnston. 1999. Function and regulation o f yeast hexose transporters. Microbiol M o l B i o l Rev 63:554-69. Ozcan, S., and M. Johnston. 1995. Three different regulatory mechanisms enable yeast hexose transporter ( H X T ) genes to be induced b y different levels o f glucose. M o l Cell B i o l 15:1564-72. Ozcan, S., T. Leong, and M. Johnston. 1996. R g t l p o f Saccharomyces cerevisiae, a key regulator o f glucose-induced genes, is both an activator and a repressor o f transcription. M o l C e l l B i o l 16:6419-6426. Papamichos-Chronakis, M., T. Gligoris, and D. Tzamarias. 2004. The S n f l kinase controls glucose repression in yeast by modulating interactions between the M i g l repressor and the C y c 8 - T u p l co-repressor. E M B O Rep.  Papamichos-Chronakis, M., T. Petrakis, E . Ktistaki, I. Topalidou, and D. Tzamarias. 2002. Cti6, a P H D domain protein, bridges the C y c 8 - T u p l corepressor and the S A G A coactivator to overcome repression at GALL M o l Cell 9:1297-305. Patel, D., and J. S. Butler. 1992. Conditional defect in m R N A 3' end processing caused by a mutation i n the gene for poly(A) polymerase. M o l C e l l B i o l 12:3297-304. Petit, T., J. A. Diderich, A. L. Kruckeberg, C. Gancedo, and K. Van Dam. 2000. Hexokinase regulates kinetics o f glucose transport and expression o f genes encoding hexose transporters in Saccharomyces cerevisiae. J Bacteriol 182:6815-8.  117.  Posas, F., J. R. Chambers, J. A. Heyman, J. P. Hoeffler, E . de Nadal, and J. Arino.  118.  2000. The transcriptional response o f yeast to saline stress. J B i o l Chem 275:17249-55. Posas, F., and H. Saito. 1998. Activation of the yeast Ssk2 M A P kinase kinase kinase b y the S s k l two-component response regulator. E M B O J 17:1385-94.  119.  Posas, F., S. M. Wurgler-Murphy, T. Maeda, E . A. Witten, T. C. Thai, and H. Saito. 1996. Yeast H o g l M A P kinase cascade is regulated b y a multistep phosphorelay mechanism in the S l n l - Y p d l - S s k l "two-component" osmosensor. Cell 86:865-75.  120.  Prior, C , H. Fukuhara, J. Blaisonneau, and M. Wesolowski-Louvel. 1993. Lowaffinity glucose carrier gene LGT1 o f Saccharomyces cerevisiae, a homologue o f the Kluyveromyces lactis RAG1 gene. Yeast 9:1373-7.  121. 122.  123.  124.  125.  Proft, M., A. Pascual-Ahuir, E . de Nadal, J. Arino, R. Serrano, and F. Posas. 2001. Regulation o f the S k o l transcriptional repressor by the H o g l M A P kinase in response to osmotic stress. E M B O J 20:1123-33. Proft, M., and R. Serrano. 1999. Repressors and upstream repressing sequences of the stress-regulated ENA1 gene i n Saccharomyces cerevisiae: bZIP protein S k o l p confers HOG-dependent osmotic regulation. M o l C e l l B i o l 19:537-46. Proft, M., and K. Struhl. 2002. H o g l kinase converts the S k o l - C y c 8 - T u p l repressor complex into an activator that recruits S A G A and S W I / S N F i n response to osmotic stress. M o l C e l l 9:1307-17. Randez-Gil, F., P. Herrero, P. Sanz, J. A. Prieto, and F. Moreno. 1998. Hexokinase PII has a double cytosolic-nuclear localisation in Saccharomyces cerevisiae. F E B S Lett 425:475-8. Reifenberger, E . , E . Boles, and M. Ciriacy. 1997. Kinetic characterization of individual hexose transporters o f Saccharomyces cerevisiae and their relation to the triggering mechanisms o f glucose repression. Eur J Biochem 245:324-33. 71  126.  127.  128.  129. 130. 131.  132.  133. 134.  135. 136. 137.  138.  139.  140.  Reifenberger, E., K. Freidel, and M. Ciriacy. 1995. Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact o f individual hexose transporters on glycolytic flux. M o l M i c r o b i o l 16:157-67. Rep, M., J. Albertyn, J. M. Thevelein, B. A. Prior, and S. Hohmann. 1999. Different signalling pathways contribute to the control o f GPD1 gene expression b y osmotic stress in Saccharomyces cerevisiae. Microbiology 145 ( Pt 3):715-27. Rep, M., M. Krantz, J. M. Thevelein, and S. Hohmann. 2000. The transcriptional response o f Saccharomyces cerevisiae to osmotic shock. H o t l p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J B i o l Chem 275:8290-300.  Rep, M., V. Reiser, U. Gartner, J. M. Thevelein, S. Hohmann, G. Ammerer, and H. Ruis. 1999. Osmotic stress-induced gene expression i n Saccharomyces cerevisiae requires M s n l p and the novel nuclear factor H o t l p . M o l Cell B i o l 19:5474-85. Robinson, M. J., and M. H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr Opin C e l l B i o l 9:180-6. Rodriguez, A., T. De La Cera, P. Herrero, and F. Moreno. 2001. The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem J 355:625-31.  Rolland, F., J. H. De Winde, K. Lemaire, E. Boles, J. M. Thevelein, and J. Winderickx. 2000. Glucose-induced c A M P signalling i n yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. M o l Microbiol 38:348-58. Rolland, F., J. Winderickx, and J. M. Thevelein. 2002. Glucose-sensing and signalling mechanisms i n yeast. F E M S Yeast Res 2:183-201. Rose, M., W. Albig, and K. D. Entian. 1991. Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and P H . Eur J Biochem 199:511-8. Ross, J. 1995. m R N A stability in mammalian cells. M i c r o b i o l Rev 59:423-50. Rossignol, T., L. Dulau, and B. Blondin. 2003. Genome-wide analysis of yeast gene expression during wine fermentation. Yeast 20.-S314-S314. Sanz, P., G. R. Alms, T. A. J. Haystead, and M. Carlson. 2000. Regulatory interactions between the R e g l - G l c 7 protein phosphatase and the S n f l protein kinase. M o l C e l l B i o l 20:1321-1328. Schmidt, M. C , and R. R. McCartney. 2000. 6eta-subunits o f S n f l kinase are required for kinase function and substrate definition. E M B O J . 19:4936-4943.  Schmidt, M. C , R. R. McCartney, X. Zhang, T. S. Tillman, H. Solimeo, S. Wolfl, C. Almonte, and S. C. Watkins. 1999. Stdl and M t h l proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae. M o l Cell B i o l 19:4561-71. Schmitt, A. P., and K. McEntee. 1996. Msn2p, a zinc finger D N A - b i n d i n g protein, is the transcriptional activator o f the multistress response i n Saccharomyces cerevisiae. Proc Natl A c a d Sci U S A 93:5777-82.  141.  Schuller, C , J. L. Brewster, M. R. Alexander, M. C. Gustin, and H. Ruis. 1994. The  142.  H O G pathway controls osmotic regulation of transcription via the stress response element ( S T R E ) of the Saccharomyces cerevisiae CTT1 gene. E M B O J 13:4382-9. Schuller, H. J., and K. D. Entian. 1991. Extragenic suppressors of yeast glucose derepression mutants leading to constitutive synthesis o f several glucose-repressible enzymes. J Bacteriol 173:2045-52.  72  143.  144.  145.  146.  147. 148.  149. 150. 151. 152. 153. 154. 155. 156.  157. 158.  159.  160.  161. 162.  Schulte, F., R. Wieczorke, C. P. Hollenberg, and E. Boles. 2000. The HTR1 gene is a dominant negative mutant allele of MTH1 and blocks Snf3- and Rgt2-dependent glucose signaling i n yeast. J Bacteriol 182:540-2. Schwartz, D. C , and R. Parker. 1999. Mutations in translation initiation factors lead to increased rates o f deadenylation and decapping o f m R N A s in Saccharomyces cerevisiae. M o l Cell B i o l 19:5247-56. Serrano, R., D. Bernal, E. Simon, and J. Arino. 2004. Copper and iron are the limiting factors for growth o f the yeast Saccharomyces cerevisiae in an alkaline environment. J B i o l Chem:published online ahead of print. Serrano, R., A. Ruiz, D. B e r n a l , J. R. C h a m b e r s , and J. Arino. 2002. The transcriptional response to alkaline p H in Saccharomyces cerevisiae: evidence for calcium-mediated signalling. M o l Microbiol 46:1319-33. Shaw, G., and R. Kamen. 1986. A conserved A U sequence from the 3' untranslated region o f G M - C S F m R N A mediates selective m R N A degradation. Cell 46:659-67. Shyu, A. B., J. G. Belasco, and M. E. Greenberg. 1991. T w o distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid m R N A decay. Genes Dev 5:221-31. Singer, R. H., and S. Penman. 1973. Messenger R N A in H e L a cells: kinetics of formation and decay. J M o l B i o l 78:321-34. Sonenberg, N., J. W. B. Hershey, and M. M a t h e w s . 2000. Translational control o f gene expression, 2nd ed. C o l d Spring Harbor Laboratory Press, C o l d Spring Harbor, N Y . S p r a d l i n g , A., H. Hui, and S. Penman. 1975. T w o very different components o f messenger R N A i n an insect cell line. C e l l 4:131-7. Spriggs, C. N. 2004. Remodeling of the S. cerevisiae transcriptome in response to acetic acid. M . Sc. thesis. University o f British Columbia, Vancouver. Stevens, A. 1980. A n m R N A decapping enzyme from ribosomes of Saccharomyces cerevisiae. Biochem Biophys Res Commun 96:1150-5. Stevens, A., and M. K. Maupin. 1987. A 5 ' — 3 ' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity. A r c h Biochem Biophys 252:339-47. Stoecklin, G., S. Hahn, and C. Moroni. 1994. Functional hierarchy of A U U U A motifs in mediating rapid interleukin-3 m R N A decay. J B i o l Chem 269:28591-7. Sutherland, C. M., S. A. Hawley, R. R. M c C a r t n e y , A. Leech, M. J. Stark, M. C. Schmidt, and D. G. H a r d i e . 2003. E l m l p is one o f three upstream kinases for the Saccharomyces cerevisiae SNF1 complex. Curr B i o l 13:1299-305. Tarun, S. Z., Jr., and A. B. Sachs. 1996. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. E M B O J 15:7168-77. Tarun, S. Z., Jr., S. E. Wells, J. A. Deardorff, and A. B. Sachs. 1997. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc Natl A c a d Sci U S A 94:9046-9051. Teige, M., E. S c h e i k l , V. Reiser, H. Ruis, and G. Ammerer. 2001. R c k 2 , a member o f the calmodulin-protein kinase family, links protein synthesis to high osmolality M A P kinase signaling in budding yeast. Proc Natl A c a d Sci U S A 98:5625-5630. Theodoris, G., N. M. Fong, D. M. Coons, and L. F. Bisson. 1994. High-copy suppression o f glucose transport defects by HXT4 and regulatory elements i n the promoters o f the HXT genes in Saccharomyces cerevisiae. Genetics 137:957-66. Tipper, D. J. 1973. Inhibition o f yeast ribonucleic acid polymerases by thiolutin. J Bacteriol 116:245-56. Toda, T., S. C a m e r o n , P. Sass, M. Zoller, J. D. Scott, B. McMullen, M. H u r w i t z , E. G. Krebs, and M. Wigler. 1987. Cloning and characterization ofBCYl, a locus 73  163.  164.  165. 166.  167. 168. 169.  170.  171.  172.  encoding a regulatory subunit of the cyclic AMP-dependent protein kinase i n Saccharomyces cerevisiae. M o l Cell B i o l 7:1371-7. Toda, T., S. Cameron, P. Sass, M. Zoller, and M. Wigler. 1987. Three different genes in S. cerevisiae encode the catalytic subunits o f the cAMP-dependent protein kinase. C e l l 50:277-87. Tomas-Cobos, L., L. Casadome, G. Mas, P. Sanz, and F. Posas. 2004. Expression of the HXT1 low-affinity glucose transporter requires the coordinated activities of the H O G and glucose signalling pathways. J B i o l Chem:published online ahead of print. Treitel, M., and M. Carlson. 1995. Repression by Ssn6-Tupl is directed by M i g l , a repressor/activator protein. Proc Natl A c a d Sci U S A 92:3132-3136. Treitel, M. A . , S. Kuchin, and M. Carlson. 1998. S n f l protein kinase regulates phosphorylation of the M i g l repressor in Saccharomyces cerevisiae. M o l Cell B i o l 18:6273-80. Tucker, M., and R. Parker. 2000. Mechanisms and control of m R N A decapping in Saccharomyces cerevisiae. A n n u Rev Biochem 69:571-95. Vallier, L. G., and M. Carlson. 1994. Synergistic release from glucose repression by migl and ssn mutations in Saccharomyces cerevisiae. Genetics 137:49-54. Vallier, L. G., D. Coons, L. F. Bisson, and M. Carlson. 1994. Altered regulatory responses to glucose are associated with a glucose transport defect in grrl mutants of Saccharomyces cerevisiae. Genetics 136:1279-85. van der Merwe, G. K., H. J. J. van Vuuren, and T. G. Cooper. 2001. Cis-acting sites contributing to expression o f divergently transcribed DAL1 and DAL4 genes in S. cerevisiae: a word of caution when correlating cis-acting sequences with genome-wide expression analyses. Curr Genet 39:156-65. Vasudevan, S., and S. W. Peltz. 2001. Regulated ARE-mediated m R N A decay in Saccharomyces cerevisiae. M o l Cell 7:1191-200.  Verwaal, R., J. W. Paalman, A . Hogenkamp, A . J. Verkleij, C. T. Verrips, and J. Boonstra. 2002. HXT5 expression is determined by growth rates in Saccharomyces cerevisiae. Yeast 19:1029-38.  173.  Vilela, C , C. Velasco, M. Ptushkina, and J. E . McCarthy. 2000. The eukaryotic  174.  m R N A decapping protein D c p l interacts physically and functionally with the eIF4F translation initiation complex. E M B O J 19:4372-82. Vyas, V. K., S. Kuchin, C. D. Berkey, and M. Carlson. 2003. S n f l kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth. M o l C e l l B i o l 23:1341-8.  175. 176. 177.  178.  179.  180.  Wang, Y., C. L. Liu, J. D. Storey, R. J. Tibshirani, D. Herschlag, and P. O. Brown. 2002. Precision and functional specificity in m R N A decay. Proc Natl A c a d Sci U S A 99:5860-5. Wells, S. E . , P. E . Hillner, R. D. Vale, and A . B. Sachs. 1998. Circularization of m R N A by eukaryotic translation initiation factors. M o l Cell 2:135-40. Werner-Washburne, M., E . Braun, G. Johnston, and R. Singer. 1993. Stationary phase in the yeast Saccharomyces cerevisiae. M i c r o b i o l Rev 57:383-401.  Wieczorke, R., S. Krampe, T. Weierstall, K. Freidel, C. P. Hollenberg, and E . Boles. 1999. Concurrent knock-out of at least 20 transporter genes is required to block uptake o f hexoses in Saccharomyces cerevisiae. F E B S Lett 464:123-8. Wilusz, C. J., M. Wormington, and S. W. Peltz. 2001. The cap-to-tail guide to m R N A turnover. Nat Rev M o l C e l l B i o l 2:237-46.  Winzen, R., M. Kracht, B. Ritter, A . Wilhelm, C. Y. Chen, A . B. Shyu, M. Muller, M. Gaestel, K. Resch, and H. Holtmann. 1999. The p38 M A P kinase pathway signals 74  181.  182. 183.  184.  for cytokine-induced m R N A stabilization via M A P kinase-activated protein kinase 2 and an A U - r i c h region-targeted mechanism. E M B O J 18:4969-80. X u , N . , C . Y . Chen, and A . B . Shyu. 1997. Modulation o f the fate of cytoplasmic m R N A by A U - r i c h elements: key sequence features controlling m R N A deadenylation and decay. M o l Cell B i o l 17:4611 -21. Yale, J . , and H . J . Bohnert. 2001. Transcript expression in Saccharomyces cerevisiae at high salinity. J B i o l Chem 276:15996-6007. Yang, X . , R. Jiang, and M . Carlson. 1994. A family o f proteins containing a conserved domain that mediates interaction with the yeast S n f l protein kinase complex. E M B O J 13:5878-5886. Ye, L . , J . A . Berden, K . van Dam, and A . L . Kruckeberg. 2001. Expression and activity o f the Hxt7 high-affinity hexose transporter oi Saccharomyces cerevisiae. Yeast 18:1257-67.  75  

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