Metabolic adaptation of Saccharomyces cerevisiae to high sugar

FEMS Yeast Research 3 (2003) 375^399

www.fems-microbiology.org

Genome-wide expression analyses: Metabolic adaptation of Saccharomyces cerevisiae to high sugar stress Daniel J. Erasmus, George K. van der Merwe, Hennie J.J. van Vuuren

Wine Research Centre, Faculty of Agricultural Sciences, 2205 East Mall, The University of British Columbia, Vancouver, BC, Canada V6T 1Z4 Received 12 August 2002 ; received in revised form 31 October 2002; accepted 4 November 2002 First published online 15 January 2003

Abstract The transcriptional response of laboratory strains of Saccharomyces cerevisiae to salt or sorbitol stress has been well studied. These studies have yielded valuable data on how the yeast adapts to these stress conditions. However, S. cerevisiae is a saccharophilic fungus and in its natural environment this yeast encounters high concentrations of sugars. For the production of dessert wines, the sugar concentration may be as high as 50% (w/v). The metabolic pathways in S. cerevisiae under these fermentation conditions have not been studied and the transcriptional response of this yeast to sugar stress has not been investigated. High-density DNA microarrays showed that the transcription of 589 genes in an industrial strain of S. cerevisiae were affected more than two-fold in grape juice containing 40% (w/v) sugars (equimolar amounts of glucose and fructose). High sugar stress up-regulated the glycolytic and pentose phosphate pathway genes. The PDC6 gene, previously thought to encode a minor isozyme of pyruvate decarboxylase, was highly induced under these conditions. Gene expression profiles indicate that the oxidative and non-oxidative branches of the pentose phosphate pathway were upregulated and might be used to shunt more glucose-6-phosphate and fructose-6-phosphate, respectively, from the glycolytic pathway into the pentose phosphate pathway. Structural genes involved in the formation of acetic acid from acetaldehyde, and succinic acid from glutamate, were also up-regulated. Genes involved in de novo biosynthesis of purines, pyrimidines, histidine and lysine were downregulated by sugar stress. ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Osmotic stress ; Pentose phosphate pathway ; DNA array analysis ; Wine; Acetic acid ; Succinic acid

1. Introduction The regulatory circuits of the yeast Saccharomyces cerevisiae are being analyzed extensively using DNA microarray technology, and the transcriptional response of laboratory strains of S. cerevisiae to salt or sorbitol stress is well documented [1^3]. These pioneering studies have yielded valuable and novel data on how the yeast adapts to these stress conditions. Studies on laboratory strains of S. cerevisiae have served us well and we have accumulated a vast amount of information on the genetics and physiology of this yeast. However, for most part laboratory strains are derived from an exceedingly small number of

* Corresponding author. Tel. : +1 (604) 822 0418; Fax : +1 (604) 822 5143. E-mail address : [email protected] (H.J.J. van Vuuren).

progenitors, which have been crippled by successive mutations. In addition, laboratory media and growth conditions are vastly di¡erent from those that wild-type yeast strains encounter in nature or in some commercial applications. S. cerevisiae is routinely used for the production of wine and it also encounters high concentrations of sugars in its natural environment of rotting fruits. Grape musts used for wine production usually contain 16^26% (w/v) sugars [4]. For the production of noble late-harvest or ice wines, however, sugar concentrations may be as high as 50% (w/v). The metabolic pathways in S. cerevisiae under these fermentation conditions have not been studied and the transcriptional response of this yeast to sugar stress has not been investigated. The osmoregulatory response in S. cerevisiae has been well characterized [1,5^20]. In addition to the HOG pathway, the RAS-cAMP PKA pathway is involved in regulating cell growth, carbon storage and stress response [21^

1567-1356 / 02 / $22.00 ; 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-1356(02)00203-9

FEMSYR 1540 7-5-03

Cyaan Magenta Geel Zwart

376

D.J. Erasmus et al. / FEMS Yeast Research 3 (2003) 375^399

24]. S. cerevisiae adapts to increased osmotic stress by enhanced production of intracellular glycerol as the main compatible solute to counter-balance the osmotic pressure (for reviews see [10,25^27]). The key step of glycerol synthesis is catalyzed by an NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase that converts dihydroxyacetone phosphate to glycerol-3-phosphate with the production of NADþ . Two isoforms of this enzyme are encoded by the GPD1 [28] and GPD2 [29] genes. GPD1 is strongly induced by osmotic stress [29^32]. An increase in glycerol production requires an equimolar increase of cytoplasmic NADH formation [10,33]. Under high osmotic stress, this requirement seems to be partially met by decreased reduction of acetaldehyde to ethanol on the one hand, and an increased oxidation to acetate on the other [34]. Under conditions of stress, acetate formation, therefore, plays an important role in maintaining the redox balance in yeast cells [10,33]. Modelling of unbranched glycolysis has revealed that, unless the hexose kinase or transport systems are regulated negatively, the £ux in the upper part of the pathway exceeds that in the lower part due to the so-called ‘turbodesign’ of glycolysis [35]. Two ATPs are incorporated into the upper part of glycolysis, thereby stimulating the £ux and creating an imbalance between the upper and lower parts of the pathway resulting in the accumulation of fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate [36]. Furthermore, the downregulation of biosynthetic pathways upon exposure to osmotic stress leads to a decreased demand for and subsequent accumulation of ATP [37]. The build-up of sugar phosphates and depletion of phosphate in the cell results in substrate-accelerated death or at least failure to grow [35,38]. It has been suggested that the yeast cell prevents this accumulation of fructose-1,6-bisphosphate: by controlling the in£ux of glucose by inhibition of hexose transporters [39] ; by feedback inhibition of hexokinase by trehalose-6-phosphate limiting phosphorylation of glucose [36,40^43] ; or by creating a demand for ATP by activating the glycerol, trehalose and glycogen futile cycles which act as safety valves to prevent substrate-accelerated death [38,44]. We studied the e¡ect of 40% (w/v) sugars in Riesling grape juice on the transcriptional response of a polyploid industrial wine yeast strain. Genome-wide expression analyses revealed that the transcription of 589 genes was affected by more than two-fold. In addition to the genes involved in the glycerol, trehalose and glycogen futile cycles, we found that genes involved in the glycolytic and pentose phosphate pathways were up-regulated. Furthermore, the genes involved in production of acetic acid from acetaldehyde, and succinate from glutamate, were up-regulated. Yeast produced 1.35 g l31 acetic acid in Riesling grape juice containing 40% (w/v) sugars compared to 0.3 g l31 in Riesling grape juice with 22% (w/v) sugars.

FEMSYR 1540 7-5-03

Genes involved in de novo synthesis of purines, pyrimidines, histidine and lysine were down-regulated.

2. Materials and methods 2.1. Determination of water activity (aw ) The aw of YEPD [45] medium containing 0.7 M, 1.4 M or 2 M NaCl, and Riesling grape juice containing 22% or 40% (w/v) sugars, was determined with an Aqualab Series 3 water activity meter (Decagon Devices, Pullmann, WA, USA). Measurements were done in triplicate. 2.2. Media preparation, yeast strain and growth conditions Riesling grape juice (Okanagan Valley, BC, Canada) containing 22% (w/v) sugars (equimolar amounts of glucose and fructose) was treated with 0.02 ml pectinase (Pec5L, Scott Labs) for 3 h at 40‡C. Gelatine was added to a ¢nal concentration of 0.2 g l31 and incubated at 7‡C for 12 h to precipitate particulate matter. The juice was then ¢lter-sterilized using a 0.22-Wm ¢lter (Millipore). The grape juice was diluted with sterile deionized water (1:2) to rehydrate active dry yeast (Vin13, Anchor Yeast, South Africa) at 40‡C for 30 min. Equimolar amounts of glucose and fructose were added to grape juice (22% w/v sugars) to obtain a grape juice with 40% (w/v) sugars. 1-l batches of Riesling grape juice containing 22% or 40% (w/v) sugars were inoculated with the rehydrated yeast to a ¢nal concentration of 6U106 cells ml31 . Growth of each culture was monitored over a period of 470 h by measuring the optical density (A600nm ). Cultures used for RNA extraction were grown in 22% (w/v) sugar grape juice to mid-exponential phase (A600nm = 2.0) at 20‡C. The culture was divided into two 500-ml batches in 1-l £asks. To one batch, 500 ml of the same 22% (w/v) sugar juice was added (control). To the second batch, 500 ml grape juice containing 60% (w/v) sugars was added to yield a ¢nal concentration of approximately 40% (w/v) sugars. Both £asks were further incubated stationary at 20‡C for 2 h. Yeast cells were rapidly harvested, washed and stored at 380‡C until RNA extraction [45]. All experiments were done in duplicate with independently grown cells. 2.3. RNA extraction and sample preparation Total RNA was extracted using the hot-phenol method [45]. Methods for poly(A)þ RNA puri¢cation, ampli¢cation and labelling, and cRNA fragmentation have been described previously [46]. The only modi¢cation to these procedures was the use of 15 Wg cRNA, instead of 10 Wg, in the cRNA fragmentation reaction.



2.4. Hybridization, £uidics and scanning procedures Four oligonucleotide yeast genome arrays (YGS98, Affymetrix, Santa Clara, CA, USA) were used as targets for hybridization. Procedures for hybridization, washing, staining and scanning have been described previously [46]. The following modi¢cations to these procedures applied: hybridizations were performed at 45‡C and the arrays were read at 3 Wm using the Agilent G2500A GeneArray Scanner (Agilent Technologies, Palo Alto, CA, USA). The EukGE-WS2v3 £uidics protocol of the A¡ymetrix MASv5.0 software (A¡ymetrix, Santa Clara, CA, USA) was used to perform staining and washing procedures. The arrays were subsequently read with a confocal GeneArray Scanner. 2.5. Data analyses Data were analyzed using MASv5.0. All tunable parameters were set to default values (A¡ymetrix Statistical Algorithm Reference Guide, A¡ymetrix, Santa Clara, CA, USA). The changes in gene expression levels were determined with the Wilcoxon sign-rank test. Genes responding the same in both experiments and with change P-values of 6 0.003 (genes with an increased call) or s 0.997 (genes with a decreased call) were considered to be statistically signi¢cant. Average Signal Log (base 2) Ratio (SLR) values were used to calculate the fold change. Data were further analyzed and genes grouped into cellular roles using YPD1 [47,48]. 2.6. Quanti¢cation of acetic acid Wine samples (1 ml) were taken after 470 h of fermentation to determine the acetic acid concentration. Samples were ¢lter-sterilized (0.22 Wm) and stored at 330‡C until analyzed. A Waters 6000A HPLC with a Waters LambdaMax 281 UV detector connected to a Hewlett-Packard Integrator/printer HP3396 series II was used. The UV detector was set at V = 210 nm. A 10-Wl sample was injected via a Rheodyne type 70 injector valve onto a Supelcogel C610H analytical cation exchange column (Supelco, cat #: 59320-U) and a Supelguard C610H column (Supelco, cat #: 59319). Analyses were done at ambient temperature. A degassed, 0.22-Wm-¢ltered 10-mM H3 PO4 mobile phase was used. L-maleic acid (0.002 g l31 ) was used as internal standard. A £ow rate of 0.3 ml min31 was used to quantify acetic acid in the wine containing a high residual sugar concentration. The wine samples were diluted 1:14 with 10 mM H3 PO4 . Due to the low amount of acetic acid in wine obtained from 22% (w/v) Riesling grape juice, acetic acid was quanti¢ed by using the external standard method and an increased £ow rate of 0.5 ml min31 to reduce band broad-

FEMSYR 1540 7-5-03

377

ening. The samples were diluted 2.5-fold with 10 mM H3 PO4 . Other parameters were the same as previously described.

3. Results Microarray data revealed that sugar-induced osmotic stress greatly a¡ects the yeasts transcriptome ; of the 4592 genes analyzed, the expression of 589 genes changed more than two-fold when yeast cells were grown in grape juice containing 40% (w/v) sugars (equimolar amounts of glucose and fructose). Of these 589 genes, 346 genes were up-regulated and 243 were down-regulated (Tables 1 and 2). Genes were grouped into ‘cellular-role’ categories according to the YPD1 database [47]. The expression of a large number (228) of genes with unknown functions was regulated more than two-fold. Other major responses included genes involved with cell stress and small-molecule transport as well as carbohydrate, nucleotide, and amino acid metabolism (Fig. 1). 3.1. Regulation of carbohydrate metabolic genes by high sugar concentrations (fold change indicated in brackets) Growth of the yeast in 40% (w/v) sugars down-regulated transcription of two of the hexose transport genes, HXT3 (32.9) and HXT4 (37.0). However, the HXT1 (+1.8) and HXT5 (+6.1) genes encoding hexose transporters, and the STL1 (+87.0) and YBR241C (+4.0) genes encoding sugar transport-like proteins, were up-regulated in response to high sugar concentrations. Several genes involved in glycolysis, GLK1 (+2.1), GLK1 homolog YDR516C (+2.4), TDH1 (+1.7), GPM2 (+2.3), ENO1 (+1.3), PYK2 (+1.7) and PDC6 (+26) were up-regulated by sugar stress. HXK2 (31.8), PDC5 (32.5) and ADH3 (31.9) were down-regulated. PGI1, PFK2, FBA1, TPI1, PGK1, ENO2, ADH1 and ADH2 showed no change in expression levels when yeast cells were grown in grape juice containing 40% (w/v) sugars. ZWF1 (+1.7), SOL1 (+2.5), SOL4 (+2.5), GND2 (+4.3), TKL2 (+12.1), TAL1 (+1.5) and the TAL1 homolog YGR043C (+11) involved in the pentose phosphate pathway were up-regulated (Fig. 2). However, the RPE1 (31.6) and RKI1 (32.5) genes were down-regulated. These two genes encode for D-ribulose-5-phosphate 3-epimerase and D-ribose-5-phosphate ketol-isomerase, which link the oxidative and non-oxidative branches of the pentose phosphate pathway. The down-regulation of these two genes by sugar stress might decrease the £ow of carbon from the oxidative to the non-oxidative part of the pentose phosphate pathway. Our data showed that genes involved in the biosynthetic and dissimilatory pathways for glycerol {GPD1 (+2.1), GPP2 (+11.3), GCY1 (+5.7) and DAK1 (+3.5)}, trehalose,


378


Table 1 Genes in S. cerevisiae that were up-regulated more than two-fold when cells were grown in grape juice containing 40% (w/v) sugars after growing to mid-exponential phase in grape juice with 22% (w/v) sugars Gene name

ORF

1. Aging NCA3 YJL116C PDE1 YGL248W TPK1 YJL164C 2. Amino acid metabolism ALD2 YMR170C ARG3 YJL088W ARO10 YDR380W ARO9 YHR137W BAP2 YBR068C CHA1 YCL064C GAD1 YMR250W GAT1 YFL021W GDH3 YAL062W MET13 YGL125W PUT2 YHR037W PUT4 YOR348C TKL2 YBR117C UGA1 YGR019W UGA2 YBR006W VPS36 YLR417W 3. Carbohydrate metabolism ALD4 YOR374W ALD6 YPL061W AMS1 YGL156W ATH1 YPR026W CAT8 YMR280C CYB2 YML054C DOG2 YHR043C GCY1 YOR120W GLK1 YCL040W GLO1 YML004C GLO2 YDR272W GLO4 YOR040W GND2 YGR256W GPD1 YDL022W GPH1 YPR160W GRE3 YHR104W GSC2 YGR032W GSY1 YFR015C GPP2 YER062C HXT5 YHR096C KHA1 YJL094C MLS1 YNL117W MSS11 YMR164C NTH1 YDR001C PDC6 YGR087C PGM2 YMR105C PIG1 YLR273C PYC1 YGL062W SFA1 YDL168W SOL1 YNR034W SOL4 YGR248W TKL2 YBR117C TPS1 YBR126C TPS2 YDR074W TSL1 YML100W YBR056W YBR056W YDR516C YDR516C XYL2 YLR070C 4. Cell cycle control PCL1 YNL289W

Description

SLRa

Protein of unknown function 3P,5P-Cyclic-nucleotide phosphodiesterase Putative catalytic subunit of cAMP-dependent PKA

2.4 1.0 1.6

5.3 2.0 2.9

Aldehyde dehydrogenase Ornithine carbamoyltransferase Protein of unknown function Aromatic amino acid aminotransferase II Branched-chain amino acid permease Threonine dehydratase Glutamate decarboxylase Transcription factor Glutamate dehydrogenase (NADPþ ) Putative methylenetetrahydrofolate reductase 1-Pyrroline-5-carboxylate dehydrogenase Proline-speci¢c permease Transketolase 4-Aminobutyrate aminotransferase Succinate-semialdehyde dehydrogenase (NAD(P)þ ) Protein involved in vacuolar sorting

1.4 1.1 2.7 3.3 3.6 1.3 2.5 1.2 1.5 1.3 1.1 3.1 3.6 2.7 1.7 1.6

2.6 2.1 6.3 9.5 11.7 2.5 5.5 2.3 2.7 2.5 2.1 8.6 12.1 6.5 3.2 2.9

Aldehyde dehydrogenase (NAD(P)þ ) Acetaldehyde dehydrogenase (NADPþ ) K-Mannosidase K,K-Trehalase Transcription factor L-Lactate dehydrogenase (cytochrome) 2-Deoxyglucose-6-phosphatase Aldo-keto reductase(putative glycerol dehydrogenase) Glucokinase Lactoylglutathione lyase Hydroxyacylglutathione hydrolase Hydroxyacylglutathione hydrolase Phosphogluconate dehydrogenase Glycerol-3-phosphate dehydrogenase (NADþ ) Glycine amidinotransferase Induced by osmotic stress 1,3-Beta-glucan synthase Glycogen synthase D L-glycerol-3-phosphatase Hexose transporter Putative Kþ /Hþ antiporter Malate synthase Multicopy suppressor of STA10 311 K,K-Trehalase Pyruvate decarboxylase Phosphoglucomutase Regulatory subunit, interacts with Gsy2p Pyruvate carboxylase Acylglycerone-phosphate reductase Possible 6-phosphogluconolactonase Possible 6-phosphogluconolactonase Transketolase K,K-Trehalose-phosphate synthase Trehalose phosphatase K,K-Trehalose-phosphate synthase Protein of unknown function Protein of unknown function Xylitol dehydrogenase

4.0 1.9 2.3 1.2 2.9 2.2 1.0 2.5 1.1 1.1 1.5 1.1 2.1 1.1 1.3 2.2 1.5 1.2 3.5 2.6 1.2 1.5 1.0 1.5 4.7 1.6 1.2 1.5 1.0 1.4 1.3 3.6 1.3 2.5 2.4 1.2 1.3 1.3

15.5 3.7 4.8 2.2 7.2 4.6 2.0 5.7 2.1 2.1 2.7 2.1 4.3 2.1 2.5 4.6 2.8 2.2 11.3 6.1 2.3 2.8 2.0 2.8 26.0 2.9 2.2 2.7 2.0 2.5 2.5 12.1 2.5 5.5 5.3 2.3 2.4 2.4

Cyclin-dependent protein kinase

1.2

2.3

FEMSYR 1540 7-5-03


Fold changeb


379

Table 1 (Continued). Gene name

ORF

PCL5 YHR071W TFS1 YLR178C YAK1 YJL141C 5. Cell stress AAD4 YDL243C ATH1 YPR026W CRS5 YOR031W CTA1 YDR256C CTT1 YGR088W CUP2 YGL166W DAK1 YML070W DDR48 YMR173W GCY1 YOR120W GLO1 YML004C GPD1 YDL022W GPH1 YPR160W GPX2 YBR244W GRE2 YOL151W GTS1 YGL181W GTT1 YIR038C HAL5 YJL165C GPP2 YER062C HOR7 YMR251W-A HSP104 YLL026W HSP12 YFL014W HSP26 YBR072W HSP30 YCR021C HSP42 YDR171W HSP78 YDR258C HSP82 YPL240C KHA1 YJL094C NTH1 YDR001C PAI3 YMR174C PNC1 YGL037C PPZ2 YDR436W PTP2 YOR208W SFA1 YDL168W SHC1 YER096W SIP18 YMR175W SLT2 YHR030C SSA3 YBL075C SSA4 YER103W TPS1 YBR126C TPS2 YDR074W TRX3 YCR083W TSL1 YML100W TTR1 YDR513W UBI4 YLL039C XBP1 YIL101C YAK1 YJL141C YBL064C YBL064C YDR453C YDR453C YGR086C YGR086C YNL077W YNL077W YNL194C YNL194C PIN3 YPR154W VRP1 YLR337C YSC84 YHR016C 6. Cell wall maintenance CHS1 YNL192W CWP1 YKL096W DDR48 YMR173W ECM29 YHL030W ECM4 YKR076W

Description

SLRa

Cyclin-dependent protein kinase Putative lipid-binding protein Serine-threonine protein kinase

1.4 2.0 1.2

2.5 4.0 2.2

Putative aryl-alcohol dehydrogenase K,K-Trehalase Metallothionein-like protein Catalase Catalase Transcriptional activator Glycerone kinase Protein of unknown function Aldo-keto reductase(putative glycerol dehydrogenase) Lactoylglutathione lyase Glycerol-3-phosphate dehydrogenase (NADþ ) Glycine amidinotransferase Glutathione peroxidase Induced by osmotic stress Putative zinc-¢nger transcription factor Glutathione transferase Protein kinase homolog D L-glycerol-3-phosphatase Hyperosmolarity-responsive gene Heat shock protein Heat shock protein Heat shock protein Heat shock protein Chaperone Chaperone Heat shock protein Putative Kþ /Hþ antiporter K,K-Trehalase Endopeptidase inhibitor Nicotinamidase Protein serine/threonine phosphatase Protein tyrosine phosphatase Acylglycerone-phosphate reductase Sporulation-speci¢c protein similar to Skt5p Salt-induced protein MAP kinase Heat shock protein Chaperone K,K-Trehalose-phosphate synthase Trehalose phosphatase Thioredoxin K,K-Trehalose-phosphate synthase Glutaredoxin Protein degradation tagging Transcriptional repressor Serine-threonine protein kinase Thioredoxin peroxidase Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function [PSI+] induction Actin-binding Protein of unknown function

1.4 1.2 1.0 1.5 3.3 1.5 1.8 1.7 2.5 1.1 1.1 1.3 1.8 3.8 1.1 1.3 1.1 3.5 1.1 1.5 3.6 2.3 1.5 2.4 2.1 2.2 1.2 1.5 2.4 1.7 1.5 1.2 1.0 1.5 2.2 1.1 1.8 3.6 1.3 2.5 1.1 2.4 1.2 2.2 1.8 1.2 1.6 1.9 1.4 1.2 4.9 1.6 1.3 1.0

2.5 2.2 2.0 2.7 9.8 2.8 3.5 3.1 5.7 2.1 2.1 2.5 3.5 13.9 2.1 2.5 2.1 11.3 2.1 2.8 11.7 4.8 2.8 5.3 4.3 4.4 2.3 2.8 5.3 3.2 2.8 2.2 2.0 2.8 4.4 2.1 3.5 11.7 2.5 5.5 2.1 5.3 2.3 4.4 3.4 2.2 2.9 3.6 2.5 2.2 29.9 3.0 2.4 2

Chitin synthase Cell wall mannoprotein Protein of unknown function Protein of unknown function Protein of unknown function

1.3 1.2 1.7 1.1 1.9

2.5 2.3 3.1 2.1 3.7

FEMSYR 1540 7-5-03


Fold changeb

380



ORF

GSC2 YGR032W HAL5 YJL165C KTR2 YKR061W PIR3 YKL163W SHC1 YER096W SLT2 YHR030C SPI1 YER150W STE11 YLR362W STF2 YGR008C 7. Chromatin/chromosome structure HAT1 YPL001W HPA2 YPR193C RAD50 YNL250W RAD52 YML032C SPO13 YHR014W 8. Cytokinesis CTS1 YLR286C VRP1 YLR337C 9. Di¡erentiation APG7 YHR171W MSS11 YMR164C SGA1 YIL099W SHC1 YER096W SPO1 YNL012W SPO13 YHR014W STE11 YLR362W 10. DNA repair DDR48 YMR173W DNL4 YOR005C MAG1 YER142C MGT1 YDL200C MMS21 YEL019C PHR1 YOR386W RAD2 YGR258C RAD50 YNL250W RAD52 YML032C RAD59 YDL059C THI4 YGR144W 11. Energy generation COX5B YIL111W CYB2 YML054C CYC7 YEL039C STF1 YDL130W-A YEL020C YEL020C YLR164W YLR164W YLR327C YLR327C YMR118C YMR118C 12. Lipid, fatty-acid and sterol metabolism CHO1 YER026C CHO2 YGR157W ECI1 YLR284C FOX2 YKR009C MCT1 YOR221C OPI3 YJR073C OYE3 YPL171C PDC6 YGR087C PDR1 YGL013C POT1 YIL160C YDC1 YPL087W YEL020C YEL020C 13. Mating response AFR1 YDR085C CMK2 YOL016C PTP2 YOR208W

Description

SLRa

1,3-Beta-glucan synthase Protein kinase homolog Mannosyltransferase Cell wall structural protein Sporulation-speci¢c protein similar to Skt5p MAP kinase Has similarity to Sed1p MAP kinase kinase kinase ATPase-stabilizing factor

1.5 1.1 1.4 1.0 1.5 1.1 3.1 1.2 2.5

2.8 2.1 2.5 2.0 2.8 2.1 8.6 2.3 5.5

Histone acetyltransferase Histone acetyltransferase Involved in DNA repair Involved in DNA repair Meiosis-speci¢c protein

1.1 2.2 1.0 1.3 1.3

2.1 4.6 2.0 2.5 2.5

Chitinase Actin binding

1.0 1.3

2.0 2.4

Ubiquitin-like conjugating enzyme Multicopy suppressor of STA10 312 Glucan 1,4-K-glucosidase Sporulation-speci¢c protein similar to Skt5p Phospholipase Meiosis-speci¢c protein MAP kinase kinase kinase

1.0 1.0 1.5 1.5 1.1 1.3 1.2

2.0 2.0 2.8 2.8 2.1 2.5 2.3

Protein of unknown function DNA ligase (ATP) DNA-3-methyladenine glycosidase II 6-O-methylguanine-DNA methylase Protein involved in DNA repair Deoxyribodipyrimidine photolyase Structure-speci¢c single-stranded DNA endonuclease Involved in DNA repair Involved in DNA repair Involved in mitotic recombination Protein of unknown function

1.7 1.4 2.2 1.1 4.2 1.2 1.1 1.0 1.3 1.6 1.9

3.1 2.6 4.6 2.1 18.4 2.2 2.1 2.0 2.5 3.0 3.7

Cytochrome-c oxidase L-lactate dehydrogenase (cytochrome) Iso-2-cytochrome c ATPase-stabilizing factor Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function

1.6 2.2 2.0 1.3 1.5 1.7 2.7 1.9

2.9 4.6 3.9 2.5 2.8 3.2 6.5 3.6

CDP-diacylglycerol-serine O-phosphatidyltransferase Methylene-fatty-acyl-phospholipid synthase Dodecenoyl-CoA delta-isomerase 3-Hydroxyacyl-CoA dehydrogenase Malonyl-CoA :ACP transferase Phosphatidylethanolamine N-methyltransferase NADPH dehydrogenase Pyruvate decarboxylase Transcription factor Acetyl-CoA C-acyltransferase Alkaline dihydroceramidase Protein of unknown function

1.0 1.7 1.3 2.4 1.3 1.8 2.7 4.7 1.3 1.7 1.3 1.5

2.0 3.2 2.4 5.3 2.5 3.5 6.5 26.0 2.5 3.1 2.4 2.8

Receptor-signaling protein Calcium/calmodulin-dependent protein kinase Protein tyrosine phosphatase

1.2 2.1 1.2

2.3 4.1 2.2

FEMSYR 1540 7-5-03


Fold changeb


381


ORF

STE11 YLR362W 14. Meiosis APG1 YGL180W AUT7 YBL078C RAD50 YNL250W RAD52 YML032C RAD59 YDL059C SAE3 YHR079C-A SMA1 YPL027W SPO1 YNL012W SPO13 YHR014W SPO71 YDR104C TPS1 YBR126C XBP1 YIL101C 15. Membrane fusion PEP12 YOR036W YHR138C YHR138C 16. Mitosis RAD52 YML032C 17. Nuclear-cytoplasmic transport GSP2 YOR185C NPL4 YBR170C 18. Nucleotide metabolism DAL1 YIR027C FUI1 YBL042C THI11 YJR156C URA10 YMR271C YNK1 YKL067W 19. Other metabolism BTN2 YGR142W CWP1 YKL096W HSP30 YCR021C XBP1 YIL101C AAD4 YDL243C ALD3 YMR169C ARG3 YJL088W ARO9 YHR137W COQ4 YDR204W CTA1 YDR256C DAL1 YIR027C DAL3 YIR032C FRE4 YNR060W GAT1 YFL021W GCY1 YOR120W PNC1 YGL037C THI4 YGR144W UGA1 YGR019W YAL061W YAL061W YFL057C YFL057C YMR226C YMR226C YNL274C YNL274C 20. Pol II transcription CAF17 YJR122W CAT8 YMR280C CUP2 YGL166W GAT1 YFL021W GTS1 YGL181W PDR1 YGL013C YER064C YER064C YGL131C YGL131C 21. Protein complex assembly UMP1 YBR173C 22. Protein degradation APG1 YGL180W

Description

SLRa

MAP kinase kinase kinase

1.2

2.3

Protein serine/threonine kinase Microtubule-binding Involved in DNA repair Involved in DNA repair Involved in mitotic recombination Protein of unknown function Protein of unknown function Phospholipase Meiosis-speci¢c protein Protein involved in spore wall formation K,K-Trehalose-phosphate synthase Transcriptional repressor

2.0 1.3 1.0 1.3 1.6 2.1 1.3 1.1 1.3 1.0 1.3 1.8

3.9 2.5 2.0 2.5 3.0 4.1 2.5 2.1 2.5 2.0 2.5 3.4

t-SNARE Homologous to PBI2

1.7 2.0

3.2 4.0

Involved in DNA repair

1.3

2.5

GTP-binding protein Structural protein

2.7 1.5

6.3 2.8

Allantoinase Uridine permease Involved in thiamine utilization pathway Orotate phosphoribosyltransferase Nucleoside-diphosphate kinase

1.2 1.3 1.6 1.4 1.0

2.2 2.4 3.0 2.5 2.0

Protein of unknown function Cell wall mannoprotein Heat shock protein Transcriptional repressor Putative aryl-alcohol dehydrogenase Aldehyde dehydrogenase Ornithine carbamoyltransferase Aromatic amino acid aminotransferase II Involved in ubiquinone biosynthesis Catalase Allantoinase Ureidoglycolate hydrolase Protein of unknown function Transcription factor Aldo-keto reductase(putative glycerol dehydrogenase) Nicotinamidase Protein of unknown function 4-Aminobutyrate aminotransferase Putative polyol dehydrogenase Protein of unknown function Protein of unknown function Protein of unknown function

2.0 1.2 1.5 1.8 1.4 2.6 1.1 3.3 1.1 1.5 1.2 1.5 2.3 1.2 2.5 1.7 1.9 2.7 2.1 1.4 1.1 1.0

4.0 2.3 2.8 3.4 2.5 5.9 2.1 9.5 2.0 2.7 2.2 2.8 5.0 2.3 5.7 3.2 3.7 6.5 4.3 2.6 2.1 2.0

CCR4 transcriptional complex component Transcription factor Transcription factor Transcription factor Putative zinc-¢nger transcription factor Transcription factor Protein of unknown function Protein of unknown function

1.1 2.9 1.5 1.2 1.1 1.3 1.2 1.3

2.1 7.2 2.8 2.3 2.1 2.5 2.3 2.4

Involved in ubiquitin-mediated proteolysis

1.2

2.2

Protein serine/threonine kinase

2.0

3.9

FEMSYR 1540 7-5-03


Fold changeb

382



ORF

APG5 YPL149W APG7 YHR171W AUT7 YBL078C LAP4 YKL103C MDJ1 YFL016C PAI3 YMR174C RPT5 YOR117W SNX4 YJL036W TFS1 YLR178C UBC5 YDR059C UBC8 YEL012W UBI4 YLL039C UBP5 YER144C UBP6 YFR010W UFD1 YGR048W UMP1 YBR173C YDR330W YDR330W YLR387C YLR387C 23. Protein folding CPR6 YLR216C ERO1 YML130C HSP104 YLL026W HSP12 YFL014W HSP26 YBR072W HSP30 YCR021C HSP42 YDR171W HSP78 YDR258C HSP82 YPL240C MDJ1 YFL016C SSA1 YAL005C SSA3 YBL075C SSA4 YER103W SSE2 YBR169C YNL077W YNL077W 24. Protein modi¢cation HAT1 YPL001W HPA2 YPR193C KHA1 YJL094C KTR2 YKR061W PGM2 YMR105C UBC8 YEL012W UBP15 YMR304W UBP5 YER144C UBP6 YFR010W 25. Protein synthesis MRP8 YKL142W YGR201C YGR201C 26. Protein translocation APG7 YHR171W HSP78 YDR258C PEX18 YHR160C SSA1 YAL005C SSA3 YBL075C SSA4 YER103W 27. Recombination MMS21 YEL019C RAD50 YNL250W RAD52 YML032C RAD59 YDL059C 28. RNA processing/modi¢cation NCA3 YJL116C NGR1 YBR212W REX3 YLR107W SOL1 YNR034W

Description

SLRa

Involved in autophagy Ubiquitin-like conjugating enzyme Microtubule-binding Vacuolar aminopeptidase I Heat shock protein Proteinase inhibitor Adenosinetriphosphatase Protein of unknown function Putative lipid-binding protein Ubiquitin-conjugating enzyme Ubiquitin-conjugating enzyme Protein degradation tagging Ubiquitin-speci¢c protease Ubiquitin-speci¢c protease Molecular function unknown Involved in ubiquitin-mediated proteolysis Protein of unknown function Protein of unknown function

1.7 1.0 1.3 1.3 1.4 2.4 1.0 1.2 2.0 1.4 1.2 2.2 1.1 1.2 1.3 1.2 1.5 1.3

3.1 2.0 2.5 2.5 2.5 5.3 2.0 2.3 4.0 2.5 2.2 4.4 2.1 2.2 2.4 2.2 2.8 2.5

Peptidyl-prolyl isomerase Involved in protein disul¢de bond formation in the ER Heat shock protein Heat shock protein Heat shock protein Heat shock protein Chaperone Chaperone Heat shock protein Heat shock protein Adenosine triphosphatase Heat shock protein Chaperone Heat shock protein Protein of unknown function

1.4 1.2 1.5 3.6 2.3 1.5 2.4 2.1 2.2 1.4 1.1 1.8 3.6 2.0 1.2

2.5 2.2 2.8 11.7 4.8 2.8 5.3 4.3 4.4 2.5 2.1 3.5 11.7 3.9 2.2

Histone acetyltransferase Histone acetyltransferase Putative Kþ /Hþ antiporter Mannosyl transferase Phosphoglucomutase Ubiquitin-conjugating enzyme Ubiquitin-speci¢c protease Ubiquitin-speci¢c protease Ubiquitin-speci¢c protease

1.1 2.2 1.2 1.4 1.6 1.2 1.1 1.1 1.2

2.1 4.6 2.3 2.5 2.9 2.2 2.1 2.1 2.2

Structural protein of ribosomes Protein of unknown function

1.9 1.5

3.6 2.7

Ubiquitin-like conjugating enzyme Chaperone Protein binding Adenosine triphosphatase Heat shock protein Chaperone

1.0 2.1 1.8 1.1 1.8 3.6

2.0 4.3 3.4 2.1 3.5 11.7

Protein involved in DNA repair Involved in DNA repair Involved in DNA repair Involved in mitotic recombination

4.2 1.0 1.3 1.6

18.4 2.0 2.5 3.0

Protein of unknown function Negative growth regulatory protein 3P-5P Exonuclease Possible 6-phosphogluconolactonase

2.4 1.9 1.0 1.4

5.3 3.7 2.0 2.5

FEMSYR 1540 7-5-03


Fold changeb


383


ORF

SYF2 YGR129W YGR250C YGR250C YTH1 YPR107C ISF1 YMR081C SYF2 YGR129W 29. Signal transduction BAG7 YOR134W CMK1 YFR014C PDE1 YGL248W PPZ2 YDR436W PTP2 YOR208W SLT2 YHR030C STE11 YLR362W TPK1 YJL164C YGR043C YGR043C 30. Small molecule transport BAP2 YBR068C COX5B YIL111W ENA2 YDR039C ENA5 YDR038C FRE4 YNR060W FUI1 YBL042C HXT5 YHR096C KHA1 YJL094C ODC1 YPL134C PDR1 YGL013C PDR10 YOR328W PTK2 YJR059W PUT4 YOR348C RAV2 YDR202C STL1 YDR536W VPS36 YLR417W YBR241C YBR241C YER119C YER119C YIL166C YIL166C YKL146W YKL146W YLR004C YLR004C 31. Genes of unknown function ADY2 YCR010C CSR2 YPR030W DGA1 YOR245C FMS1 YMR020W FYV10 YIL097W GPM2 YDL021W GRE1 YPL223C KKQ8 YKL168C MGA1 YGR249W MPM1 YJL066C MSC1 YML128C NGL3 YML118W OM45 YIL136W PHM7 YOL084W PHM8 YER037W PRM10 YJL108C PST2 YDR032C RIO1 YOR119C RTA1 YGR213C SDS24 YBR214W SLZ1 YNL196C SPG1 YGR236C SPS100 YHR139C SRL3 YKR091W SSH4 YKL124W TOS3 YGL179C

Description

SLRa

Involved in pre-mRNA splicing Protein of unknown function Polyadenylation factor subunit Protein of unknown function Involved in pre-mRNA splicing

1.2 1.6 1.1 1.5 1.2

2.3 2.9 2.1 2.8 2.3

GTPase activating protein Calmodulin-dependent protein kinase I 3P,5P-Cyclic-nucleotide phosphodiesterase Protein serine/threonine phosphatase Protein tyrosine phosphatase MAP kinase MAP kinase kinase kinase Protein serine/threonine kinase Transaldolase

5.5 1.1 1.0 1.5 1.2 1.1 1.2 1.6 3.5

43.7 2.1 2.0 2.8 2.2 2.1 2.3 2.9 11.0

Branched chain amino acid permease Cytochrome-c oxidase Putative Naþ pump Naþ ATPase Protein of unknown function Uridine permease Hexose transporter Putative K+/H+antiporter Oxodicarboxylate carrier Transcription factor member of ATP-binding casette (ABC) family Putative serine/threonine protein kinase Proline-speci¢c permease Regulator of (Hþ )-ATPase in Vacuolar membrane Sugar transporter-like protein Protein involved in vacuolar sorting Putative hexose transporter Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function

3.6 1.6 1.2 1.2 2.3 1.3 2.6 1.2 1.4 1.3 1.2 1.4 3.1 1.1 6.5 1.6 2.0 1.3 1.3 1.0 1.1

11.7 2.9 2.3 2.2 4.8 2.4 6.1 2.3 2.5 2.5 2.3 2.6 8.6 2.1 87.4 2.9 4.0 2.4 2.4 2.0 2.1

Protein of unknown function Protein of unknown function Diacyl glycerol acyltransferase Protein of unknown function Protein of unknown function Phosphoglycerate mutase Osmotic stress induced Protein kinase Heat shock transcription factor homolog Mitochondrial membrane protein Protein of unknown function Protein of unknown function 45 kDa mitochondrial outer membrane protein Protein of unknown function Protein of unknown function Pheromone-regulated membrane protein Protein of unknown function Protein of unknown function Involved in 7-aminocholesterol resistance Protein of unknown function Sporulation-speci¢c protein Protein of unknown function Involved in spore wall formation, Protein of unknown function Confers le£unomide resistance when overexpressed Putative serine/threonine protein kinase

1.2 1.6 1.2 1.5 1.3 1.3 3.1 1.1 1.4 1.1 2.7 1.1 1.9 2.5 2.7 2.4 1.1 1.1 1.4 1.4 4.0 2.3 2.8 1.3 1.9 1.2

2.3 2.9 2.2 2.8 2.4 2.4 8.3 2.1 2.6 2.1 6.3 2.1 3.7 5.5 6.3 5.3 2.1 2.1 2.5 2.6 15.5 4.9 6.7 2.4 3.6 2.2

FEMSYR 1540 7-5-03


Fold changeb

384



ORF

Description

SLRa

Fold changeb

TOS5 UGX2 WHI4 YAR027W YBL048W YBL049W YBL065W YBR005W YBR047W YBR053C YBR062C YBR085c-a YBR116C YBR137W YBR230C YBR280C YCL042W YCL044C YCR082W YCR105W YDL113C YDL124W YDL204W YDL218W YDL222C YDL223C YDR034W-B YDR036C YDR070C YDR247W YDR306C YDR366C YDR391C YDR425W YDR476C YDR540C YER028C YER034W YER067W YER079W YER121W YET1 YFL030W YFL044C YFR003C YFR017C YGL045W YGL121C YGL144C YGL157W YGL185C YGR052W YGR066C YGR110W YGR131W YGR146C YGR161C YGR237C YGR243W YGR268C YHL021C YHR033W YHR087W YHR122W

YKR011C YDL169C YDL224C YAR027W YBL048W YBL049W YBL065W YBR005W YBR047W YBR053C YBR062C YBR085c-a YBR116C YBR137W YBR230C YBR280C YCL042W YCL044C YCR082W YCR105W YDL113C YDL124W YDL204W YDL218W YDL222C YDL223C YDR034W-B YDR036C YDR070C YDR247W YDR306C YDR366C YDR391C YDR425W YDR476C YDR540C YER028C YER034W YER067W YER079W YER121W YKL065C YFL030W YFL044C YFR003C YFR017C YGL045W YGL121C YGL144C YGL157W YGL185C YGR052W YGR066C YGR110W YGR131W YGR146C YGR161C YGR237C YGR243W YGR268C YHL021C YHR033W YHR087W YHR122W

Protein of unknown function Protein of unknown function Putative RNA binding protein Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Yeast BAP31 homolog Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function

2.0 2.2 2.3 2.3 1.6 2.0 1.8 1.2 1.7 1.3 1.0 2.4 2.8 1.2 2.1 1.2 1.7 1.3 1.1 1.0 1.1 3.3 1.9 1.8 2.6 2.7 1.9 1.1 3.0 1.3 1.1 2.4 1.1 1.3 1.2 1.9 2.6 1.4 1.2 2.0 2.0 1.3 1.8 1.1 1.6 2.0 1.1 2.6 1.3 1.3 1.0 1.7 1.6 1.8 1.7 1.3 1.6 1.2 3.4 1.9 3.5 2.7 4.7 1.0

3.9 4.4 4.9 4.9 3.0 4.0 3.5 2.3 3.1 2.5 2.0 5.1 6.7 2.2 4.3 2.3 3.2 2.5 2.1 2.0 2.1 9.5 3.7 3.5 6.1 6.3 3.7 2.1 7.7 2.5 2.1 5.1 2.1 2.4 2.3 3.7 5.9 2.6 2.2 4.0 4.0 2.4 3.4 2.1 2.9 3.9 2.1 5.9 2.4 2.4 2.0 3.2 2.9 3.5 3.1 2.4 3.0 2.3 10.6 3.7 10.9 6.3 26.0 2.0

FEMSYR 1540 7-5-03



385


ORF

Description

SLRa

Fold changeb

YHR140W YHR159W YHR198C YHR199C YHR209W YIL055C YIL057C YIL077C YIL108W YIL113W YIR014W YJL107C YJL144W YJL161W YJL185C YJL213W YJR008W YJR096W YJR119C YKL034W YKL071W YKL086W YKL107W YKL123W YKL151C YKL161C YKR049C YLR042C YLR054C YLR080W YLR132C YLR149C YLR194C YLR202C YLR225C YLR247C YLR251W YLR252W YLR257W YLR270W YLR271W YLR323C YLR346C YLR350W YLR408C YLR414C YLR454W YML083C YMR034C YMR040W YMR090W YMR103C YMR107W YMR114C YMR173W-A YMR181C YMR191W YMR196W YMR210W YMR315W YMR318C YMR322C YNL092W YNL094W

YHR140W YHR159W YHR198C YHR199C YHR209W YIL055C YIL057C YIL077C YIL108W YIL113W YIR014W YJL107C YJL144W YJL161W YJL185C YJL213W YJR008W YJR096W YJR119C YKL034W YKL071W YKL086W YKL107W YKL123W YKL151C YKL161C YKR049C YLR042C YLR054C YLR080W YLR132C YLR149C YLR194C YLR202C YLR225C YLR247C YLR251W YLR252W YLR257W YLR270W YLR271W YLR323C YLR346C YLR350W YLR408C YLR414C YLR454W YML083C YMR034C YMR040W YMR090W YMR103C YMR107W YMR114C YMR173W-A YMR181C YMR191W YMR196W YMR210W YMR315W YMR318C YMR322C YNL092W YNL094W

Protein Protein Protein Protein Protein Protein Protein Protein Protein protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein

1.6 1.2 1.0 1.3 2.1 1.8 2.1 1.0 1.6 3.5 1.4 2.7 2.3 2.2 2.1 2.6 1.5 1.9 1.3 1.2 1.7 3.2 4.1 1.1 1.9 1.1 1.6 3.6 1.4 1.1 1.3 1.8 1.5 1.1 1.1 1.3 2.5 2.4 1.1 1.7 1.7 1.2 1.2 1.0 1.1 1.7 1.1 1.3 1.1 1.9 2.2 1.1 2.3 1.1 1.1 1.1 1.5 1.8 1.2 2.3 1.0 1.9 3.5 1.3

3.0 2.2 2.0 2.5 4.1 3.5 4.3 2.0 3.0 10.9 2.5 6.5 4.8 4.6 4.3 5.9 2.8 3.6 2.5 2.3 3.1 8.9 16.6 2.1 3.7 2.1 3.0 11.7 2.6 2.1 2.5 3.5 2.7 2.1 2.1 2.4 5.7 5.3 2.1 3.1 3.1 2.2 2.3 2.0 2.1 3.1 2.1 2.4 2.1 3.6 4.6 2.1 4.9 2.1 2.1 2.1 2.8 3.4 2.2 4.8 2.0 3.6 11.3 2.4

of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function tyrosine phosphatase of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function of unknown function

FEMSYR 1540 7-5-03


386



ORF

YNL115C YNL115C YNL134C YNL134C YNL195C YNL195C YNL200C YNL200C YNR014W YNR014W YNR034w-a YNR034w-a YOL032W YOL032W YOL048C YOL048C YOL083W YOL083W YOL131W YOL131W YOL150C YOL150C YOL161C YOL161C YOR019W YOR019W YOR049C YOR049C YOR052C YOR052C YOR054C YOR054C YOR062C YOR062C YOR137C YOR137C YOR173W YOR173W YOR220W YOR220W YOR289W YOR289W YOR338W YOR338W YOR385W YOR385W YPL004C YPL004C YPL047W YPL047W YPL052W YPL052W YPL070W YPL070W YPL113C YPL113C YPL168W YPL168W YPL222W YPL222W YPL247C YPL247C YPR093C YPR093C YPR127W YPR127W ZTA1 YBR046C 32. Vesicular transport APG1 YGL180W APG5 YPL149W AST2 YER101C AUT7 YBL078C DDI1 YER143W GYP7 YDL234C PEP12 YOR036W VPS36 YLR417W YKL091C YKL091C

Description

SLRa

Fold changeb

Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Zeta-crystalline homolog

1.2 1.4 1.6 1.4 2.6 1.5 2.0 1.0 1.9 5.4 1.6 1.4 1.4 1.6 1.2 1.3 1.3 1.5 2.8 2.6 1.6 2.7 1.5 1.1 1.1 1.3 1.2 3.0 1.0 2.1 1.1 1.1 1.6 1.9

2.2 2.5 2.9 2.5 5.9 2.8 3.9 2.0 3.6 42.2 3.0 2.6 2.5 2.9 2.3 2.5 2.5 2.8 6.7 6.1 2.9 6.5 2.7 2.1 2.1 2.5 2.3 8.0 2.0 4.1 2.1 2.1 3.0 3.6

Protein serine/threonine kinase 2.0 Involved in autophagy 1.7 Protein involved in targeting of plasma membrane [Hþ ] ATPase 1.0 Microtubule-binding 1.3 T- and V-snare complex binding protein 1.7 GTPase-activating protein 1.4 t-SNARE 1.7 Protein involved in vacuolar sorting 1.6 Protein of unknown function 1.2

3.9 3.1 2.0 2.5 3.1 2.6 3.2 2.9 2.3

Genes were grouped into cellular-role categories according to YPD1. SLR, average of two sets of data. b Fold change calculated from average SLR. a

{TPS1 (+2.5), TPS3 (+1.7), TSL1 (+5.3), PGM2 (+2.9), TPS2 (+5.5), NTH1 (+2.8) and ATH1 (+2.2)} and glycogen {GSY1 (2.2), GSY2 (+1.8), GLC3 (+1.9) and GPH1 (+2.5)} are up-regulated by sugar stress. These genes have previously been shown to respond to salt stress [1^3]. The up-regulation of the FBP1 (+1.7) gene, responsible for the conversion of fructose-1,6-bisphosphate to fructose-6phosphate during gluconeogenesis, is intriguing and unexpected (Fig. 2). It has previously been shown that the FBP1 gene is repressed by 2% (w/v) glucose [49]. In addition, the FBP26 gene encoding fructose-2,6-bisphosphatase was also up-regulated (+1.7).

FEMSYR 1540 7-5-03

3.2. Genes responsible for the formation of acetic acid from acetaldehyde, and succinate from glutamate, are up-regulated Four isogenes, ALD2 (+2.6), ALD3 (+5.9), ALD4 (+15.5) and ALD6 (+3.7), encoding NAD(P)þ -dependent aldehyde dehydrogenases, were up-regulated (Fig. 2). It has previously been shown that Ald4p and Ald6p convert acetaldehyde to acetate [50]. Yeast grown in the 40% (w/v) sugar juice produced 1.35 g l31 acetic acid compared to 0.3 g l31 at the lower sugar concentration (22% w/v). Genes involved in the conversion of glutamate to succinate via 4-aminobutanoate and succinate-semialdehyde



387

Table 2 Genes in S. cerevisiae that were down-regulated more than two-fold when cells were grown in grape juice containing 40% (w/v) sugars after growing to mid-exponential phase in grape juice with 22% (w/v) sugars Gene name

ORF

1. Aging SIM1 YIL123W 2. Amino acid metabolism ACO1 YLR304C AGP1 YCL025C ARO1 YDR127W ASP1 YDR321W BAP3 YDR046C GCV1 YDR019C GCV2 YMR189W GLT1 YDL171C HIS1 YER055C HIS3 YOR202W HIS4 YCL030C HIS7 YBR248C HOM3 YER052C ILV6 YCL009C LYS1 YIR034C LYS12 YIL094C LYS2 YBR115C LYS4 YDR234W LYS9 YNR050C MET6 YER091C MIS1 YBR084W SAM1 YLR180W SAM4 YPL273W SER2 YGR208W SER3 YER081W SHM2 YLR058C BNA4 YBL098W YDR111C YDR111C YOR108W YOR108W 3. Carbohydrate metabolism ACO1 YLR304C ADH3 YMR083W CAT5 YOR125C HXT3 YDR345C HXT4 YHR092C MAE1 YKL029C MNN1 YER001W PDC5 YLR134W PYC2 YBR218C RKI1 YOR095C YJR024C YJR024C 4. Cell adhesion AGA1 YNR044W 5. Cell cycle control CBF2 YGR140W CDC21 YOR074C CLB2 YPR119W CLB6 YGR109C EGT2 YNL327W FAR1 YJL157C FKH1 YIL131C GNA1 YFL017C HSL7 YBR133C MCD1 YDL003W PCL9 YDL179W PDS1 YDR113C SDA1 YGR245C SIM1 YIL123W YBR242W YBR242W

Description

SLRa

Fold changeb

Involved in control of DNA replication

31.6

32.9

Aconitate hydratase Amino acid permease 3-Dehydroquinate dehydratase Asparaginase Branched-chain amino acid permease Aminomethyltransferase Glycine dehydrogenase (decarboxylating) Glutamate synthase ATP phosphoribosyltransferase Imidazoleglycerol-phosphate dehydratase Histidinol dehydrogenase Imidazoleglycerol-phosphate synthase Aspartate kinase Acetolactate synthase Saccharopine dehydrogenase (NADþ , L-lysine forming) Homo-isocitrate dehydrogenase Aminoadipate-semialdehyde dehydrogenase Homoaconitate hydratase Saccharopine dehydrogenase (NADPþ , L-glutamate forming) 5-Methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase Formate-tetrahydrofolate ligase Methionine adenosyltransferase AdoMet-homocysteine methyltransferase Phosphoserine phosphatase Phosphoglycerate dehydrogenase Glycine hydroxymethyltransferase Kynurenine 3-mono oxygenase Protein of unknown function Protein of unknown function

31.2 31.2 31.5 31.7 31.4 32.7 31.9 31.4 31.5 31.4 31.4 31.4 31.0 31.1 31.8 31.4 31.6 31.5 31.4 31.5 31.1 31.3 31.3 31.3 31.7 32.5 31.9 31.1 31.9

32.2 32.2 32.7 33.1 32.5 36.5 33.7 32.6 32.7 32.5 32.6 32.5 32.0 32.1 33.5 32.6 33.0 32.8 32.5 32.8 32.1 32.5 32.4 32.5 33.1 35.5 33.7 32.1 33.6

Aconitate hydratase Acylglycerone-phosphate reductase Regulator of gluconeogenic enzymes Hexose transporter Hexose transporter Malate dehydrogenase K-1,3-Mannosyltransferase Pyruvate decarboxylase Pyruvate carboxylase Ribose-5-phosphate ketol-isomerase Protein of unknown function

31.2 31.0 31.3 31.6 32.8 31.7 31.2 31.3 31.1 31.3 31.0

32.2 32.0 32.5 32.9 37.0 33.2 32.2 32.5 32.1 32.5 32.0

Cell adhesion receptor

31.1

32.1

Centromere-binding factor Thymidylate synthase G2/M-speci¢c cyclin Cyclin Protein of unknown function Cyclin-dependent protein kinase inhibitor Protein of unknown function Glucosamine-phosphate N-acetyltransferase Protein kinase inhibitor Protein of unknown function Cyclin-dependent protein kinase Control of anaphase Protein of unknown function Involved in control of DNA replication Protein of unknown function

31.0 31.2 31.0 31.7 32.1 33.2 31.5 31.3 31.0 32.0 32.0 31.1 31.4 31.6 31.1

32.0 32.3 32.0 33.2 34.3 38.9 32.7 32.4 32.0 33.9 33.9 32.1 32.6 32.9 32.1

FEMSYR 1540 7-5-03


388



ORF

6. Cell polarity FKH1 YIL131C IST2 YBR086C SPA2 YLL021W 7. Cell stress CST13 YBR158W HTB2 YBL002W IST2 YBR086C MTO1 YGL236C SUN4 YNL066W ZRC1 YMR243C 8. Cell structure SDA1 YGR245C 9. Cell wall maintenance ECM13 YBL043W ECM22 YLR228C FEN1 YCR034W FLO9 YAL063C GNA1 YFL017C MUC1 YIR019C PLB2 YMR006C PMT4 YJR143C SCW11 YGL028C TIR4 YOR009W YFL051C YFL051C YMR215W YMR215W 10. Chromatin/chromosome structure CBF2 YGR140W ESC4 YHR154W FKH1 YIL131C HHO1 YPL127C HTB2 YBL002W MCD1 YDL003W POL2 YNL262W STU2 YLR045C YHL050C YHL050C YHM2 YMR241W 11. Di¡erentiation ASH1 YKL185W EGT2 YNL327W HMS2 YJR147W MEP2 YNL142W MUC1 YIR019C SPA2 YLL021W 12. DNA repair MSH6 YDR097C PDS1 YDR113C PMS1 YNL082W POL2 YNL262W RNH35 YNL072W 13. DNA synthesis CDC45 YLR103C CDC47 YBR202W POL1 YNL102W POL2 YNL262W RNH35 YNL072W 14. Energy generation AAC3 YBR085W ACO1 YLR304C CAT5 YOR125C CEM1 YER061C COQ2 YNR041C HAP4 YKL109W MAE1 YKL029C

Description

SLRa

Fold changeb

Protein of unknown function Similarity to Ca and Na channel proteins Cytoskeletal regulatory protein-binding

31.5 31.0 31.3

32.7 32.0 32.4

Protein of unknown function Histone H2B Similarity to Ca and Na channel proteins Protein of unknown function Protein of unknown function Di-, tri-valent inorganic cation transporter

31.6 31.1 31.0 31.0 32.0 31.2

32.9 32.1 32.0 32.0 34.0 32.3

Protein of unknown function

31.4

32.6

Protein of unknown function Protein of unknown function Putative 1,3-beta-glucan synthase subunit Putative cell wall protein involved in £occulation Glucosamine-phosphate N-acetyltransferase Glucan 1,4-K-glucosidase Lysophospholipase Dolichyl-phosphate-mannose-protein mannosyltransferase Soluble cell wall protein Protein of unknown function Protein of unknown function Protein of unknown function

31.5 31.2 31.4 32.3 31.3 32.6 32.1 31.1 31.4 31.2 31.6 33.0

32.8 32.2 32.6 34.8 32.4 36.1 34.1 32.1 32.5 32.3 32.9 38.0

Centromere-binding factor Protein of unknown function Protein of unknown function Histone H1 Histone H2B Protein of unknown function Epsilon-DNA polymerase Structural protein of cytoskeleton Protein of unknown function Protein of unknown function

31.0 31.5 31.5 31.9 31.1 32.0 31.4 31.1 31.3 31.6

32.0 32.8 32.7 33.6 32.1 33.9 32.5 32.1 32.5 32.9

Speci¢c transcriptional repressor Protein of unknown function Transcription factor Ammonium transporter Glucan 1,4-K-glucosidase Cytoskeletal regulatory protein-binding

31.1 32.1 31.6 31.2 32.6 31.3

32.1 34.3 33.0 32.3 36.1 32.4

Required for mismatch repair in mitosis and meiosis Control of anaphase Required for mismatch repair in mitosis and meiosis Epsilon-DNA polymerase Ribonuclease H

31.4 31.1 31.0 31.4 31.8

32.5 32.1 32.0 32.5 33.5

DNA replication factor Chromatin binding K-DNA polymerase Epsilon-DNA polymerase Ribonuclease H

31.2 31.0 31.4 31.4 31.8

32.3 32.0 32.6 32.5 33.5

ATP/ADP antiporter Aconitate hydratase Regulator of gluconeogenic enzymes 3-Oxoacyl-[acyl-carrier protein] synthase Para-hydroxybenzoate: polyprenyl transferase Transcriptional activator Malate dehydrogenase

32.1 31.2 31.3 31.6 31.1 31.4 31.7

34.1 32.2 32.5 32.9 32.1 32.6 33.2

FEMSYR 1540 7-5-03



389


ORF

MTO1 YGL236C YPR004C YPR004C 15. Lipid, fatty-acid and sterol metabolism CEM1 YER061C CPT1 YNL130C ECM22 YLR228C FEN1 YCR034W LAC1 YKL008C PLB2 YMR006C SUR2 YDR297W SUR4 YLR372W 16. Mating response AGA1 YNR044W FAR1 YJL157C MFA2 YNL145W PRY3 YJL078C 17. Meiosis BBP1 YPL255W ISC10 YER180C SPS1 YDR523C STU2 YLR045C TYS1 YGR185C WTM2 YOR229W 18. Mitosis ASE1 YOR058C BBP1 YPL255W CBF2 YGR140W CIN2 YPL241C MCD1 YDL003W NUD1 YOR373W NUF1 YDR356W PDS1 YDR113C SCP160 YJL080C STU2 YLR045C 19. Nuclear-cytoplasmic transport KAP122 YGL016W KAP123 YER110C TYS1 YGR185C 20. Nucleotide metabolism AAH1 YNL141W ADE1 YAR015W ADE12 YNL220W ADE13 YLR359W ADE17 YMR120C ADE2 YOR128C ADE4 YMR300C ADE5,7 YGL234W ADE6 YGR061C ADE8 YDR408C CDC21 YOR074C DUT1 YBR252W FCY2 YER056C FUN26 YAL022C FUR1 YHR128W GUA1 YMR217W GUK1 YDR454C HPT1 YDR399W IMD1 YAR073W IMD4 YML056C MIS1 YBR084W MTD1 YKR080W RNR1 YER070W URA1 YKL216W URA2 YJL130C

Description

SLRa

Fold changeb

Protein of unknown function Protein of unknown function

31.0 31.0

32.0 32.0

3-Oxoacyl-[acyl-carrier protein] synthase Diacylglycerol cholinephosphotransferase Protein of unknown function Putative 1,3-beta-glucan synthase subunit LAG1 longevity gene homolog Lysophospholipase Sphingosine hydroxylase Protein of unknown function

31.6 31.1 31.2 31.4 31.1 32.1 31.7 31.3

32.9 32.1 32.2 32.6 32.1 34.1 33.2 32.4

Cell adhesion receptor Cyclin-dependent protein kinase inhibitor a-Factor mating pheromone precursor Protein of unknown function

31.1 33.2 31.9 31.2

32.1 38.9 33.7 32.3

Structural protein of cytoskeleton Protein required for spore formation Required for spore wall formation Structural protein of cytoskeleton Tyrosine-tRNA ligase Transcriptional modulator

31.3 31.0 31.1 31.1 31.3 31.0

32.5 32.0 32.1 32.1 32.5 32.0

Microtubule-binding Structural protein of cytoskeleton Centromere-binding factor Protein of unknown function Protein of unknown function Structural protein of cytoskeleton Structural protein of cytoskeleton Control of anaphase RNA-binding protein Structural protein of cytoskeleton

31.0 31.3 31.0 31.1 32.0 31.0 31.1 31.1 31.2 31.1

32.0 32.5 32.0 32.1 33.9 32.0 32.1 32.1 32.3 32.1

Karyopherin-beta family member Karyopherin-beta family member Tyrosine-tRNA ligase

31.1 31.7 31.3

32.1 33.1 32.5

Adenine deaminase Phosphoribosylaminoimidazole-succinocarboxamide synthase Adenylosuccinate synthase Adenylosuccinate lyase IMP cyclohydrolase Phosphoribosylaminoimidazole carboxylase Amidophosphoribosyltransferase Phosphoribosylformylglycinamidine cyclo-ligase Phosphoribosylformylglycinamidine synthase Phosphoribosylglycinamide formyltransferase Thymidylate synthase dUTP pyrophosphatase Purine-cytosine permease Protein of unknown function Uracil phosphoribosyltransferase GMP synthase (glutamine hydrolyzing) Guanylate kinase Hypoxanthine phosphoribosyltransferase IMP dehydrogenase IMP dehydrogenase Formate-tetrahydrofolate ligase Methylenetetrahydrofolate dehydrogenase (NADþ ) Ribonucleoside-diphosphate reductase Dihydroorotate oxidase Aspartate carbamoyltransferase

33.3 33.0 31.3 31.7 32.8 31.9 33.1 31.7 32.5 31.7 31.2 31.4 31.4 31.3 31.8 31.1 31.1 31.8 31.4 31.4 31.1 32.9 31.6 31.1 31.1

39.5 37.7 32.4 33.1 36.7 33.7 38.3 33.2 35.7 33.2 32.3 32.6 32.6 32.5 33.4 32.1 32.1 33.4 32.5 32.5 32.1 37.2 33.0 32.1 32.1

FEMSYR 1540 7-5-03


390



ORF

URA3 YEL021W URA7 YBL039C 21. Other metabolism ATF2 YGR177C COQ2 YNR041C DPH5 YLR172C FRE2 YKL220C HEM13 YDR044W HEM3 YDL205C MEP2 YNL142W SAM4 YPL273W SSU1 YPL092W BNA4 YBL098W 22. Phosphate metabolism PHO11 YAR071W PHO4 YFR034C 23. Pol I transcription RPA135 YPR010C RPA190 YOR341W RPB8 YOR224C RPC10 YHR143W-A RRN7 YJL025W SRP40 YKR092C 24. Pol II transcription ASH1 YKL185W ECM22 YLR228C HAP4 YKL109W HTB2 YBL002W RPB8 YOR224C RPC10 YHR143W-A SPT21 YMR179W SSU72 YNL222W WTM2 YOR229W 25. Pol III transcription RPB8 YOR224C RPC10 YHR143W-A RPC11 YDR045C SRP40 YKR092C 26. Protein complex assembly NUF1 YDR356W 27. Protein folding FPR4 YLR449W ZUO1 YGR285C 28. Protein modi¢cation DPH5 YLR172C HSL7 YBR133C KTR3 YBR205W MAK3 YPR051W MNN1 YER001W PMT4 YJR143C 29. Protein synthesis GCD6 YDR211W GIS2 YNL255C MTO1 YGL236C RPS22B YLR367W TEF4 YKL081W TIF4631 YGR162W TYS1 YGR185C YDR341C YDR341C ZUO1 YGR285C 30. Recombination CLB6 YGR109C PMS1 YNL082W 31. RNA processing/modi¢cation DBP2 YNL112W

Description

SLRa

Fold changeb

Orotidine-5P-phosphate decarboxylase CTP synthase

31.0 31.1

32.0 32.1

Alcohol O-acetyltransferase Para-hydroxybenzoate: polyprenyl transferase Diphthine synthase Ferric reductase Coproporphyrinogen oxidase Hydroxymethylbilane synthase Ammonium transporter AdoMet-homocysteine methyltransferase Sul¢te transporter Kynurenine 3-mono oxygenase

31.5 31.1 31.6 31.5 31.0 31.2 31.2 31.3 31.7 31.9

32.8 32.1 32.9 32.8 32.0 32.2 32.3 32.4 33.1 33.7

Acid phosphatase Transcription factor

31.0 31.4

32.0 32.5

DNA-directed RNA polymerase I DNA-directed RNA polymerase I DNA-directed RNA polymerase III DNA-directed RNA polymerase III RNA polymerase I transcription factor Chaperone

31.7 31.5 31.4 31.1 32.2 31.0

33.2 32.8 32.5 32.1 34.6 32.0

Speci¢c transcriptional repressor Protein of unknown function Transcriptional activator Histone H2B DNA-directed RNA polymerase III DNA-directed RNA polymerase III Involved in transcriptional regulation of Ty1 LTRs Complex assembly protein Transcriptional modulator

31.1 31.2 31.4 31.1 31.4 31.1 31.1 31.5 31.0

32.1 32.2 32.6 32.1 32.5 32.1 32.1 32.7 32.0

DNA-directed RNA polymerase III DNA-directed RNA polymerase III DNA-directed RNA polymerase III Chaperone

31.4 31.1 31.5 31.0

32.5 32.1 32.7 32.0

Structural protein of cytoskeleton

31.1

32.1

Peptidyl-prolyl isomerase Chaperone

31.2 31.0

32.3 32.0

Diphthine synthase Protein kinase inhibitor Mannosyltransferase N-acetyltransferase K-1,3-Mannosyltransferase Dolichyl-phosphate-mannose-protein mannosyltransferase

31.6 31.0 31.2 31.0 31.2 31.1

32.9 32.0 32.2 32.0 32.2 32.1

Translation initiation factor Transcription factor Protein of unknown function Structural protein of ribosome Translation elongation factor Translation initiation factor Tyrosine-tRNA ligase Arginine-tRNA ligase Chaperone

31.0 31.1 31.0 31.4 31.2 31.3 31.3 31.2 31.0

32.0 32.1 32.0 32.6 32.3 32.4 32.5 32.2 32.0

Cyclin Required for mismatch repair in mitosis and meiosis

31.7 31.0

33.2 32.0

RNA helicase

32.2

34.4

FEMSYR 1540 7-5-03



391


ORF

GAR1 YHR089C HAS1 YMR290C HRP1 YOL123W MAK16 YAL025C NIP7 YPL211W NOP1 YDL014W NOP13 YNL175C RNH35 YNL072W RRP5 YMR229C SCP160 YJL080C TRM1 YDR120C ZUO1 YGR285C 32. RNA splicing and turnover MRS2 YOR334W RNH35 YNL072W 33. Septation SUN4 YNL066W 34. Signal transduction MEP2 YNL142W MFA2 YNL145W RPI1 YIL119C YBR242W YBR242W 35. Small molecule transport AAC3 YBR085W AGP1 YCL025C AUS1 YOR011W BAP3 YDR046C CTP1 YBR291C FCY2 YER056C FET4 YMR319C FRE2 YKL220C FUN26 YAL022C HXT3 YDR345C HXT4 YHR092C IST2 YBR086C MEP2 YNL142W MRS2 YOR334W ODC2 YOR222W OPT2 YPR194C PHO3 YBR092C PHO91 YNR013C SSU1 YPL092W YBT1 YLL048C YDR119W YDR119W YGR096W YGR096W YHM2 YMR241W YHR032W YHR032W YMC2 YBR104W ZRC1 YMR243C ZRT2 YLR130C 36. Genes of unknown function FYV14 YDL213C KEL3 YPL263C KRR1 YCL059C MKC7 YDR144C NEW1 YPL226W PPT1 YGR123C RLI1 YDR091C RRP13 YGR103W TOS2 YGR221C TOS4 YLR183C YAL065C YAL065C YAR064W YAR064W YAR068W YAR068W

Description

SLRa

Fold changeb

Small nuclear ribonucleoprotein RNA helicase Putative polyadenylated-RNA-binding protein Putative nuclear protein Protein binding Small nuclear ribonucleoprotein Protein of unknown function Ribonuclease H RNA binding RNA-binding protein tRNA (guanine-N2-)-methyltransferase Chaperone

31.1 32.1 32.3 31.5 31.4 31.4 31.4 31.8 31.3 31.2 31.2 31.0

32.1 34.1 34.8 32.8 32.6 32.5 32.6 33.5 32.4 32.3 32.3 32.0

Magnesium ion transporter Ribonuclease H

31.1 31.8

32.1 33.5

Protein of unknown function

32.0

34.0

Ammonium transporter a-Factor mating pheromone precursor Small GTPase regulatory/interacting protein Protein of unknown function

31.2 31.9 31.6 31.1

32.3 33.7 32.9 32.1

ATP/ADP antiporter Amino acid permease Protein involved in uptake of sterols Branched-chain amino acid permease Tricarboxylate carrier Purine-cytosine permease Iron transporter Ferric reductase Protein of unknown function Hexose transporter Hexose transporter Similarity to Ca and Na channel proteins Ammonium transporter Magnesium ion transporter Mitochondrial 2-oxodicarboxylate transporter Oligopeptide transporter Acid phosphatase Low-a⁄nity phosphate transporter Sul¢te transporter Similarity to mammalian bile transporter Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Mitochondrial carrier protein Di-, tri-valent inorganic cation transporter Low-a⁄nity zinc ion transporter

32.1 31.2 31.5 31.4 32.5 31.4 31.2 31.5 31.3 31.6 32.8 31.0 31.2 31.1 31.7 34.1 31.7 31.0 31.7 31.5 31.7 31.1 31.6 31.8 31.5 31.2 31.3

34.1 32.2 32.7 32.5 35.7 32.6 32.3 32.8 32.5 32.9 37.0 32.0 32.3 32.1 33.2 316.6 33.1 32.0 33.1 32.8 33.1 32.1 32.9 33.4 32.7 32.3 32.5

Protein of unknown function Kelch-repeat protein Involved in cell division and spore germination Aspartyl protease related to Yap3p Protein of unknown function Protein serine/threonine phosphatase Required for vegetative growth and sporulation Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function

31.3 31.0 32.0 31.3 31.1 31.0 31.3 31.3 32.0 31.5 32.7 31.5 31.5

32.5 32.0 34.0 32.4 32.1 32.0 32.5 32.4 33.9 32.8 36.5 32.7 32.7

FEMSYR 1540 7-5-03


392



ORF

YAR075W YAR075W YBL028C YBL028C YBL029W YBL029W YBL032W YBL032W YBL095W YBL095W YBR028C YBR028C YBR074W YBR074W YBR075W YBR075W YBR147W YBR147W YBR206W YBR206W YBR300C YBR300C YCR051W YCR051W YCR087C-A YCR087C-A YCR087W YCR087W YDL121C YDL121C YDR020C YDR020C YDR089W YDR089W YDR133C YDR133C YER156C YER156C YGL101W YGL101W YGR001C YGR001C YGR068C YGR068C YGR280C YGR280C YHL026C YHL026C YHR149C YHR149C YIL064W YIL064W YIL096C YIL096C YIL158W YIL158W YJL097W YJL097W YJL118W YJL118W YJL200C YJL200C YJL218W YJL218W YJR030C YJR030C YJR070C YJR070C YLR049C YLR049C YLR106C YLR106C YLR154C YLR154C YLR446W YLR446W YLR455W YLR455W YMR003W YMR003W YMR209C YMR209C YMR317W YMR317W YMR321C YMR321C YNL087W YNL087W YNL174W YNL174W YNL246W YNL246W YNR009W YNR009W YNR065C YNR065C YOL155C YOL155C YOR243C YOR243C YOR315W YOR315W YPL056C YPL056C YPL158C YPL158C YPL264C YPL264C YPR157W YPR157W YVH1 YIR026C 37. Vesicular transport AKR2 YOR034C EMP70 YLR083C GEA1 YJR031C LAC1 YKL008C SVL3 YPL032C

Description

SLRa

Fold changeb

Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Putative aconitate hydratase Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein of unknown function Protein tyrosine phosphatase

31.0 31.5 31.6 31.0 31.9 31.5 31.5 31.7 31.3 31.0 31.1 31.5 32.1 31.0 31.0 31.9 32.2 31.0 31.3 31.3 31.0 31.7 31.5 31.5 31.5 31.3 31.1 31.7 31.3 31.4 31.8 31.1 31.8 32.0 31.3 31.4 31.1 33.3 31.7 31.6 31.5 31.6 31.3 31.3 31.1 31.2 32.0 31.3 31.1 31.3 32.6 31.3 31.0 31.0 31.1 31.3

32.0 32.8 32.9 32.0 33.6 32.8 32.7 33.2 32.4 32.0 32.1 32.7 34.1 32.0 32.0 33.7 34.4 32.0 32.5 32.4 32.0 33.1 32.7 32.7 32.7 32.5 32.1 33.2 32.5 32.6 33.5 32.1 33.4 33.9 32.4 32.6 32.1 39.5 33.2 33.0 32.8 32.9 32.5 32.5 32.1 32.3 33.9 32.5 32.1 32.4 35.9 32.5 32.0 32.0 32.1 32.4

Involved in constitutive endocytosis of Ste3p Protein of unknown function ARF small monomeric GTPase LAG1 longevity gene homolog Protein of unknown function

31.0 31.3 31.2 31.1 31.3

32.0 32.4 32.3 32.1 32.4

Genes were grouped into cellular-role categories according to YPD1. SLR, average of two sets of data. b Fold change calculated from average SLR. a

FEMSYR 1540 7-5-03



393

Fig. 1. Classi¢cation of genes into cellular-role categories according to YPD1. Bars indicate the number of genes classi¢ed in a particular cellular role changing two-fold or more. A: cell stress ; B: small-molecule transport; C: carbohydrate metabolism ; D: nucleotide metabolism ; E: amino acid metabolism ; F: other metabolism ; G : cell wall maintenance; H: protein degradation; I: lipid and fatty-acid and sterol metabolism; J: RNA processing/modi¢cation; K: Pol II transcription; L: protein folding ; M: meiosis; N: cell cycle control ; O: vesicular transport; P: protein modi¢cation; Q: DNA repair protein translocation; R: energy generation; S: di¡erentiation ; T: chromatin/chromosome structure ; U: protein synthesis; V: signal transduction.

were all up-regulated (Fig. 3). The GAD1 gene encoding glutamate decarboxylase was induced 5.5-fold, transcription of the UGA1 gene encoding for 4-aminobutyrate amino-transferase increased 6.5-fold, and the UGA2 (UGA5) gene encoding the NADPþ -dependent succinate-semialdehyde dehydrogenase, was up-regulated 3.2-fold. Genes encoding enzymes in the main oxidative and minor reductive reactions of the tricarboxylic acid cycle used by yeast for the formation of succinic acid during fermentation [51] were not a¡ected by osmotic stress (data not shown).

of histidine, HIS1 (32.7), HIS4 (32.6), HIS7 (32.5), HIS3 (32.5) and HIS5 (31.8), were down-regulated in response to osmotic stress (Fig. 4). Genes encoding enzymes involved in ¢ve of the eight steps of lysine biosynthesis (Fig. 4) were down-regulated: LYS20 (31.7), LYS4 (32.8), LYS21 (31.5), LYS12 (32.6), LYS1 (33.5), LYS2 (33) and LYS9 (32.5). The ARO4 (31.8) and ARO1 (32.7) genes involved in the biosynthesis of aromatic amino acids were also down-regulated (Fig. 2). 3.5. Sugar stress decreases the growth rate of S. cerevisiae

3.3. Genes involved in the de novo purine and pyrimidine biosynthesis are down-regulated Genes encoding proteins that incorporate pentose phosphate pathway intermediates into nucleotides {PRS1 (31.7), PRS3 (31.9), PRS5 (31.4)} were down-regulated (Fig. 2). Genes involved in purine biosynthesis from phosphoribosyl-pyrophosphate were down-regulated in response to sugar-induced osmotic stress. The ADE4 (38.3), ADE5,7 (33.2), ADE8 (33.2), ADE6 (35.7), ADE2 (33.7), ADE1 (37.7), ADE13 (33.1), ADE17 (36.7), ADE12 (32.4), IMD4 (32.5) and GUA1 (32.1) genes encoding enzymes responsible for the de novo synthesis of GMP and AMP were down-regulated (Fig. 4). A further three genes, AAH1 (39.5), GUK1 (32.1) and RNR1 (33), involved in biosynthetic pathways downstream of GMP and AMP, were down-regulated as well. In addition, the URA2 (32.1), URA1 (32.1), URA5 (31.9), URA3 (32), FUR1 (33.4) and URA7 (32.1) genes in the pyrimidine biosynthetic pathway were also downregulated (Fig. 4). 3.4. Genes involved in de novo biosynthesis of histidine, lysine and aromatic amino acids are down-regulated Five of the seven genes involved in de novo biosynthesis

FEMSYR 1540 7-5-03

The growth rate of the yeast in grape juice with the low aw (40% w/v sugars) was considerably lower (Wmax = 0.023) than the growth rate in grape juice with only 22% (w/v) sugars (Wmax = 0.071). The ¢nal optical density (A600nm ) of yeast grown in 22% and 40% (w/v) sugars was 6.0 and 2.70, respectively. The aw of Riesling grape juice containing 22% (w/v) sugars and that of YEPD medium supplemented with 0.7 M NaCl were almost identical (0.982 and 0.981, respectively). The aw of the 40% (w/v) sugar Riesling grape juice was 0.939 and the YEPD media containing 1.4 M and 2 M NaCl had aw values of 0.952 and 0.918, respectively.

4. Discussion 4.1. Genes involved in glycolysis and the synthesis and dissimilation of glycerol, trehalose and glycogen are up-regulated by sugar stress Sugar-induced osmotic stress regulated the expression of six genes encoding hexose transporters and transport-like proteins. The HXT3 and HXT4 genes were down-regulated and the HXT1, HXT5, STL1 and YBR241C genes were up-regulated. The latter four genes are also up-regu-


394


Fig. 2. Regulation of genes involved in the glycolytic, glycerol, trehalose, glycogen and pentose phosphate pathways in S. cerevisiae by sugar stress. Abbreviations: NC, no change; G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate ; GAP, glyceraldehyde-3-phosphate ; G-3-P, glycerol-3-phosphate; DHA, dihydroxyacetone; T-6-P, trehalose-6-phosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate.

lated by salt or sorbitol stress [1] and these genes seem to respond to osmotic stress rather than to the type of osmolyte present. The GLK1, YDR516C, GPD1, GPP2, TDH1, GPM2, ENO1, PYK2, and PDC6 genes were all up-regulated while the expression of the PGI1, PFK2, FBA1, TPI1, PGK1, ENO2, ADH1 and ADH2 genes remained unchanged (Fig. 2). Three isogenes, PDC1, PDC5 and PDC6, encode pyruvate decarboxylase that converts pyruvate into acetaldehyde and CO2 . Deletion of the PDC1 and PDC5 genes results in the inability of the yeast to ferment low concentrations of glucose (8% w/v) [52,53]. The PDC1 gene encodes for the major pyruvate decarboxylase, and its expression as well as Pdc1p activity are induced by glucose [54,55]. Deletion of PDC5 does not substantially reduce pyruvate decarboxylase activity in actively fermenting yeast cells [55,56]. PDC6 does not contribute to pyruvate decarboxylase activity in either 2% ethanol or 8% glucose [55]. However, Hohmann [53] has reported that PDC6 is required for pyruvate decarboxylase activity in media with

FEMSYR 1540 7-5-03

ethanol and galactose but not in the fermentation of glucose. In grape juice containing 40% (w/v) sugars (equimolar amounts of glucose and fructose), PDC6 was up-regulated 26-fold compared to growth in only 22% (w/v) sugars. Expression of PDC1 was una¡ected and PDC5 was down-regulated under these conditions. Salt and sorbitol stress do not induce PDC6 expression and its response seems to be speci¢c to sugar stress. Modelling of unbranched glycolysis revealed a requirement for at least a six-fold increase in pyruvate decarboxylase activity to attain a stable steady state [36]. Our data indicate that pyruvate decarboxylase activity from the highly induced Pdc6 isozyme may provide additional pyruvate decarboxylase activity required for steady-state levels of pyruvate while yeast cells are fermenting high concentrations of sugar. The hitherto regarded minor form of these three pyruvate decarboxylases seems to contribute to pyruvate decarboxylase activity under high sugar stress conditions. It has been well documented that S. cerevisiae forms glycerol and trehalose during salt or sorbitol stress



395

Fig. 3. Schematic presentation of the glutamate catabolic pathway that converts glutamate to succinate in S. cerevisiae under conditions of sugar stress {adapted from [71]}.

[31,34,57] and that futile cycles of glycerol, trehalose and glycogen [38,44] are operational under these conditions. Our data con¢rm the up-regulation of genes in these futile cycles that act as glycolytic safety valves under conditions of high sugar stress. 4.2. The pentose phosphate pathway may act as a shunt to prevent accumulation of fructose-1,6-bisphosphate in the glycolytic pathway The phosphorylation of glucose to glucose-6-phosphate, and fructose-6-phosphate to fructose-1,6-bisphosphate,

leads to an increased £ux in the upper part of glycolysis [58,59]. The accumulation of fructose-1,6-bisphosphate [35,38,40,43] deprives the cell of its phosphate pool and may lead to cell death [38]. Furthermore, modelling of unbranched glycolysis has revealed that fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate accumulate when the £ux upstream of fructose-1,6-bisphosphate exceeds the £ux downstream of glyceraldehyde-3-phosphate [36]. This accumulation of sugar phosphates in unbranched glycolysis might lead to cell death, or at least interfere with growth. It has been suggested that the accumulation of fructose-1,6-bisphos-

Fig. 4. Down-regulation of genes involved in the biosynthesis of nucleotides, histidine and lysine in S. cerevisiae by sugar stress {adapted from YPD [47], KEGG [70], [66]}. Abbreviations: PRPP, phosphoribosyl pyrophosphate; AICAR, 5P-phosphoribosyl-5-amino-4-imidazolecarboxamide ; IMP, inosine 5P-monophosphate ; AMP, adenosine 5P-monophosphate; GMP, guanosine 5P-monophosphate ; CTP, cytidine 5P-triphosphate; K-Kg, K-ketoglutarate.

FEMSYR 1540 7-5-03


396


phate can be circumvented in the yeast cell by down-regulating the HXT genes, thereby limiting the glucose £ux in glycolysis [39]. Alternatively, feedback inhibition of HXK1 by trehalose-6-phosphate could limit the glucose-6-phosphate concentration in the cell [41,42]. Gene expression pro¢les obtained in this study indicated that ZWF1 (+1.7), SOL1 (+2.5), SOL4 (+2.5) and GND2 (+4.3) genes in the oxidative part of the pentose phosphate pathway that encode enzymes responsible for the conversion of glucose-6-phosphate to ribulose-5-phosphate, were all up-regulated by sugar stress. If proteins and metabolic regulators follow suit, more glucose-6-phosphate may be shunted from the glycolytic pathway into the pentose phosphate pathway. The RPE1 (31.6) and RKI1 (32.5) genes, however, were down-regulated which could limit the £ux of ribulose-5-phosphate from the oxidative part to the non-oxidative part of the pentose phosphate pathway, thus theoretically leading to the accumulation of ribulose-5-phosphate. Furthermore, transcription of the TKL2, YGR034C, and TAL1 genes in the non-oxidative part of the pentose phosphate pathway was signi¢cantly up-regulated under conditions of severe sugar stress (Fig. 2). These results suggest that the non-oxidative part of the pentose phosphate pathway may function as a shunt to remove fructose-6-phosphate and glyceraldehyde-3-phosphate from the glycolytic pathway. If glucose-6-phosphate and fructose-6-phosphate indeed £ow into the pentose phosphate pathway under conditions of severe osmotic stress, it could limit the build-up of fructose-1,6-bisphosphate during glycolysis. It is conceivable that fructose-6-phosphate and glyceraldehyde-3-phosphate will £ow back into the glycolytic pathway during the later stages of fermentation when sugar stress is no longer severe. 4.3. Hyper-osmotic stress down-regulates genes involved in de novo biosynthesis of purines, pyrimidines, histidine and lysine Osmotic stress signi¢cantly reduced the growth rate of S. cerevisiae in Riesling grape juice containing 40% (w/v) sugars (Wmax = 0.023 vs. 0.071 at 22% sugars). Growth arrest of yeast that occurs upon transfer to high-osmolarity medium results in a decreased demand for de novo biosynthesized metabolites [1,60]. Down-regulation of the pathway can be implemented by the sudden decrease in ATP consumption for biosynthetic purposes due to growth arrest [38]. Increased AMP levels act via ADP or ATP to repress genes involved in purine and histidine biosynthesis [61]. Our data indicate that genes in the pathways leading to de novo biosynthesis of purines, pyrimidines, histidine and lysine were down-regulated by osmotic stress (Fig. 4). Purine and histidine biosynthesis share 5aminoimidazole-4-carboxamide ribotide as an intermediate [62] and these pathways are co-regulated by Bas1p, Bas2p as well as Gcn4p [62^67].

FEMSYR 1540 7-5-03

4.4. Sugar stress up-regulates genes in pathways leading to acetic and succinic acids It has often been speculated that bacterial contaminants are responsible for the production of acetic acid during ice wine production. Our data show conclusively that sugar stress up-regulates four isogenes encoding aldehyde dehydrogenases (Fig. 2). Under conditions of severe sugar stress encountered during ice wine production, the yeast produced 1.35 g l31 of acetic acid compared to 0.3 g l31 in grape must with only 22% (w/v) sugars. Under conditions of stress, acetate formation plays an important role in maintaining the redox balance in yeast cells since they require NADþ for this reaction to proceed [10]. Succinic acid is the main dicarboxylic acid produced by S. cerevisiae during wine fermentations [68] and its production is stimulated by the presence of glutamate [69]. According to Radler [51], wine yeast produces succinic acid from glutamate via 2-oxo-glutarate and succinylCoA, or from sugars via oxalacetate, L-malate and fumarate. However, our data demonstrated that the pathway for the production of succinic acid from glutamate in grape must that is most activated by high sugar stress is via 4-aminobutanoate and succinate-semialdehyde (Fig. 3). Sugar stress increased the transcription of all genes involved in the production of succinic acid from glutamate through this pathway. 4.5. Conclusions and future perspectives S. cerevisiae has developed extensive regulatory mechanisms to cope with osmotic stress. In addition to the synthesis of glycerol as a compatible solute, glycerol, glycogen and trehalose futile cycles act as safety valves to avoid substrate-accelerated death. Our data show that when the yeast ¢nds itself under severe sugar stress, control of carbon £ux through the glycolytic and the pentose phosphate pathways might be more complex than was previously thought. By shunting more glucose-6-phosphate and fructose-6-phosphate into the oxidative and non-oxidative branches of the pentose phosphate pathway, respectively, the yeast cell may prevent accumulation of fructose-1,6bisphosphate in the glycolytic pathway and concomitant depletion of phosphate resulting in substrate-accelerated death. Kinetic data and the quanti¢cation of intermediates in these pathways are required to con¢rm this hypothesis. Laboratory conditions previously used were inappropriate to detect expression of the Pdc6 isozyme. This isozyme, previously thought to be a minor isozyme, functions under conditions of sugar stress in which the yeast cell often ¢nds itself. It is clear that the yeast, and not bacterial contaminants as was previously thought, produces additional acetic acid during the fermentation of grape musts with high sugar concentrations. It is also evident that the yeast has evolved more than one mechanism to control the redox balance in the cell: activation of trehalose synthesis



and degradation, possible up-regulation of the pentose phosphate pathway, and increased acetic acid and succinic acid production are some of the options open to yeast growing under osmotic stress. The growth of S. cerevisiae was inhibited under conditions of severe sugar stress and genes in the pathways leading to purine, pyrimidine, histidine and lysine biosynthesis were down-regulated. This makes sense since the yeast no longer requires these macromolecules when growth is inhibited. We have made good progress to unravel the molecular response of S. cerevisiae to osmotic stress. However, we will not fully understand the molecular mechanisms that this yeast has evolved to cope with stress until we have elucidated the functions of at least some of the 228 orphan genes regulated by sugar-induced osmotic stress. Genomics will yield new insights into fermentation processes and their control only if we study S. cerevisiae under fermentation conditions using high sugar concentrations that this yeast normally encounters in nature.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements We thank Terry Cooper, Hans Westerho¡ and the UBC Wine Research Centre yeast group for a critical review of the manuscript and Russ Morris of the University of British Columbia Media Group for preparing the artwork. This research was supported by NSERC/AAFC/BCWI Grant 240132 to H.J.J.V.V.

[17]

[18]

[19]

References [1] Rep, M., Krantz, M., Thevelein, J.M. and Hohmann, S. (2000) The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J. Biol. Chem. 275, 8290^8300. [2] Posas, F., Chambers, J.R., Heyman, J.A., Hoe¥er, J.P., de Nadal, E. and Arino, J. (2000) The transcriptional response of yeast to saline stress. J. Biol. Chem. 275, 17249^17255. [3] Yale, J. and Bohnert, H.J. (2001) Transcript expression in Saccharomyces cerevisiae at high salinity. J. Biol. Chem. 276, 15996^16007. [4] Margalit, Y. (1997) Concepts in Wine Chemistry. The Wine Appreciation Guild Ltd., San Francisco, CA. [5] Brewster, J.L., de Valoir, T., Dwyer, N.D., Winter, E. and Gustin, M.C. (1993) An osmosensing signal transduction pathway in yeast. Science 259, 1760^1763. [6] Maeda, T., Wurgler-Murphy, S.M. and Saito, H. (1994) A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369, 242^245. [7] Schuller, C., Brewster, J.L., Alexander, M.R., Gustin, M.C. and Ruis, H. (1994) The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene. EMBO J. 13, 4382^4389. [8] Maeda, T., Takekawa, M. and Saito, H. (1995) Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3- containing osmosensor. Science 269, 554^558. [9] Martinez-Pastor, M.T., Marchler, G., Schuller, C., Marchler-Bauer, A., Ruis, H. and Estruch, F. (1996) The Saccharomyces cerevisiae

FEMSYR 1540 7-5-03

[20]

[21]

[22] [23]

[24]

[25] [26] [27]

[28]

397

zinc ¢nger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J. 15, 2227^2235. Nevoigt, E. and Stahl, U. (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 21, 231^241. Posas, F. and Saito, H. (1997) Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK : sca¡old role of Pbs2p MAPKK. Science 276, 1702^1705. Ferrigno, P., Posas, F., Koepp, D., Saito, H. and Silver, P.A. (1998) Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J. 17, 5606^5614. Gorner, W., Durchschlag, E., Martinez-Pastor, M.T., Estruch, F., Ammerer, G., Hamilton, B., Ruis, H. and Schuller, C. (1998) Nuclear localization of the C2H2 zinc ¢nger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev. 12, 586^597. O’Rourke, S.M. and Herskowitz, I. (1998) The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 12, 2874^2886. Posas, F., Witten, E.A. and Saito, H. (1998) Requirement of STE50 for osmostress-induced activation of the STE11 mitogen-activated protein kinase kinase kinase in the high-osmolarity glycerol response pathway. Mol. Cell. Biol. 18, 5788^5796. Proft, M. and Serrano, R. (1999) Repressors and upstream repressing sequences of the stress-regulated ENA1 gene in Saccharomyces cerevisiae: bZIP protein Sko1p confers HOG-dependent osmotic regulation. Mol. Cell. Biol. 19, 537^546. Reiser, V., Ruis, H. and Ammerer, G. (1999) Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell. 10, 1147^1161. Rep, M., Reiser, V., Gartner, U., Thevelein, J.M., Hohmann, S., Ammerer, G. and Ruis, H. (1999) Osmotic stress-induced gene expression in Saccharomyces cerevisiae requires Msn1p and the novel nuclear factor Hot1p. Mol. Cell. Biol. 19, 5474^5485. Reiser, V., Salah, S.M. and Ammerer, G. (2000) Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42. Nat. Cell. Biol. 2, 620^627. Rep, M., Proft, M., Remize, F., Tamas, M., Serrano, R., Thevelein, J.M. and Hohmann, S. (2001) The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage. Mol. Microbiol. 40, 1067^1083. Cameron, S., Levin, L., Zoller, M. and Wigler, M. (1988) cAMPindependent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell 53, 555^566. Broach, J.R. and Deschenes, R.J. (1990) The function of ras genes in Saccharomyces cerevisiae. Adv. Cancer Res. 54, 79^139. Gimeno, C.J., Ljungdahl, P.O., Styles, C.A. and Fink, G.R. (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to ¢lamentous growth regulation by starvation and RAS. Cell 68, 1077^1090. Smith, A., Ward, M.P. and Garrett, S. (1998) Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation. EMBO J. 17, 3556^3564. Blomberg, A. and Adler, L. (1992) Physiology of osmotolerance in fungi. Adv. Microb. Physiol. 33, 145^212. Hohmann, S. (1997) In: Yeast Stress Responses (Hohmann, S. and Mager, H., Eds.), pp. 101^146. Springer, New York. Prior, B.A. and Hohmann, S. (1997) In: Yeast Sugar Metabolism (Zimmermann, F.K. and Entian, K.D., Eds.), pp. 313^337. Technomic, Lancaster. Larsson, K., Ansell, R., Eriksson, P. and Adler, L. (1993) A gene encoding sn-glycerol 3-phosphate dehydrogenase (NADþ ) complements an osmosensitive mutant of Saccharomyces cerevisiae. Mol. Microbiol. 10, 1101^1111.


398


[29] Eriksson, P., Andre, L., Ansell, R., Blomberg, A. and Adler, L. (1995) Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. Mol. Microbiol. 17, 95^107. [30] Varela, J.C., van Beekvelt, C., Planta, R.J. and Mager, W.H. (1992) Osmostress-induced changes in yeast gene expression. Mol. Microbiol. 6, 2183^2190. [31] Albertyn, J., Hohmann, S., Thevelein, J.M. and Prior, B.A. (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol. Cell. Biol. 14, 4135^4144. [32] Hirayama, T., Maeda, T., Saito, H. and Shinozaki, K. (1995) Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae. Mol. Gen. Genet. 249, 127^138. [33] Van Dijken, J.P. and Sche¡ers, W.A. (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol. Rev. 32, 199^224. [34] Blomberg, A. and Adler, L. (1989) Roles of glycerol and glycerol-3phosphate dehydrogenase (NADþ ) in acquired osmotolerance of Saccharomyces cerevisiae. J. Bacteriol. 171, 1087^1092. [35] Teusink, B., Walsh, M.C., van Dam, K. and Westerho¡, H.V. (1998) The danger of metabolic pathways with turbo design. Trends Biochem. Sci. 23, 162^169. [36] Teusink, B., Passarge, J., Reijenga, C.A., Esgalhado, E., van der Weijden, C.C., Schepper, M., Walsh, M.C., Bakker, B.M., van Dam, K., Westerho¡, H.V. and Snoep, J.L. et al. (2000) Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes ? Testing biochemistry. Eur. J. Biochem. 267, 5313^ 5329. [37] Blomberg, A. (1995) Global changes in protein synthesis during adaptation of the yeast Saccharomyces cerevisiae to 0.7 M NaCl. J. Bacteriol. 177, 3563^3572. [38] Blomberg, A. (2000) Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol. Lett. 182, 1^8. [39] Hohmann, S., Neves, M.J., de Koning, W., Alijo, R., Ramos, J. and Thevelein, J.M. (1993) The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII. Curr. Genet. 23, 281^289. [40] Van Aelst, L., Hohmann, S., Zimmermann, F.K., Jans, A.W. and Thevelein, J.M. (1991) A yeast homologue of the bovine lens ¢bre MIP gene family complements the growth defect of a Saccharomyces cerevisiae mutant on fermentable sugars but not its defect in glucoseinduced RAS-mediated cAMP signalling. EMBO J. 10, 2095^2104. [41] Blazquez, M.A., Lagunas, R., Gancedo, C. and Gancedo, J.M. (1993) Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases. FEBS Lett. 329, 51^54. [42] Luyten, K., de Koning, W., Tesseur, I., Ruiz, M.C., Ramos, J., Cobbaert, P., Thevelein, J.M. and Hohmann, S. (1993) Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake. Eur. J. Biochem. 217, 701^713. [43] Thevelein, J.M. and Hohmann, S. (1995) Trehalose synthase: guard to the gate of glycolysis in yeast? Trends Biochem. Sci. 20, 3^10. [44] Franc]ois, J. and Parrou, J.L. (2001) Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 125^145. [45] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1995) In: Current Protocols in Molecular Biology. John Wiley and Sons Inc., New York. [46] Jelinsky, S.A. and Samson, L.D. (1999) Global response of Saccharomyces cerevisiae to an alkylating agent. Proc. Natl. Acad. Sci. USA 96, 1486^1491. [47] Costanzo, M.C., Crawford, M.E., Hirschman, J.E., Kranz, J.E., Olsen, P., Robertson, L.S., Skrzypek, M.S., Braun, B.R., Hopkins,

FEMSYR 1540 7-5-03

[48]

[49]

[50]

[51] [52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63] [64]

[65] [66]

K.L., Kondu, P., Lengieza, C., Lew-Smith, J.E., Tillberg, M. and Garrels, J.I. et al. (2001) YPD, PombePD and WormPD: model organism volumes of the BioKnowledge library, an integrated resource for protein information. Nucleic Acids Res. 29, 75^79. Hodges, P.E., McKee, A.H., Davis, B.P., Payne, W.E. and Garrels, J.I. (1999) The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data. Nucleic Acids Res. 27, 69^73. Zaragoza, O., Vincent, O. and Gancedo, J.M. (2001) Regulatory elements in the FBP1 promoter respond di¡erently to glucose-dependent signals in Saccharomyces cerevisiae. Biochem. J. 359, 193^ 201. Remize, F., Andrieu, E. and Dequin, S. (2000) Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae : role of the cytosolic Mg 2þ -and mitochondrial Kþ -acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation. Appl. Environ. Microbiol. 66, 3151^3159. Radler, F. (1994) In: Wine Microbiology and Biotechnology (Fleet, G.H., Ed.), pp. 166^179. Harwood Academic Publishers, Chur. Hohmann, S. and Cederberg, H. (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur. J. Biochem. 188, 615^621. Hohmann, S. (1991) PDC6, a weakly expressed pyruvate decarboxylase gene from yeast, is activated when fused spontaneously under the control of the PDC1 promoter. Curr. Genet. 20, 373^378. Boy-Marcotte, E., Tadi, D., Perrot, M., Boucherie, H. and Jacquet, M. (1996) High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiology 142, 459^467. Flikweert, M.T., van der Zanden, L., Janssen, W.M., Steensma, H.Y., van Dijken, J.P. and Pronk, J.T. (1996) Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12, 247^257. Hohmann, S. (1991) Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae. J. Bacteriol. 173, 7963^7969. Parrou, J.L., Teste, M.A. and Franc]ois, J. (1997) E¡ects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae : genetic evidence for a stress-induced recycling of glycogen and trehalose. Microbiology 143, 1891^1900. Heinrich, R., Montero, F., Klipp, E., Waddell, T.G. and MelendezHevia, E. (1997) Theoretical approaches to the evolutionary optimization of glycolysis : thermodynamic and kinetic constraints. Eur. J. Biochem. 243, 191^201. Melendez-Hevia, E., Waddell, T.G., Heinrich, R. and Montero, F. (1997) Theoretical approaches to the evolutionary optimization of glycolysis-chemical analysis. Eur. J. Biochem. 244, 527^543. Norbeck, J. and Blomberg, A. (1997) Metabolic and regulatory changes associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl. Evidence for osmotic induction of glycerol dissimilation via the dihydroxyacetone pathway. J. Biol. Chem. 272, 5544^5554. Rebora, K., Desmoucelles, C., Borne, F., Pinson, B. and DaignanFornier, B. (2001) Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate. Mol. Cell. Biol. 21, 7901^7912. Daignan-Fornier, B. and Fink, G.R. (1992) Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2. Proc. Natl. Acad. Sci. USA 89, 6746^6750. Arndt, K.T., Styles, C. and Fink, G.R. (1987) Multiple global regulators control HIS4 transcription in yeast. Science 237, 874^880. Mosch, H.U., Scheier, B., Lahti, R., Mantsala, P. and Braus, G.H. (1991) Transcriptional activation of yeast nucleotide biosynthetic gene ADE4 by GCN4. J. Biol. Chem. 266, 20453^20456. Stotz, A., Muller, P.P. and Linder, P. (1993) Regulation of the ADE2 gene from Saccharomyces cerevisiae. Curr. Genet. 24, 472^480. Springer, C., Kunzler, M., Balmelli, T. and Braus, G.H. (1996) Amino acid and adenine cross-pathway regulation act through the same


D.J. Erasmus et al. / FEMS Yeast Research 3 (2003) 375^399 5P-TGACTC-3P motif in the yeast HIS7 promoter. J. Biol. Chem. 271, 29637^29643. [67] Guetsova, M.L., Lecoq, K. and Daignan-Fornier, B. (1997) The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes. Genetics 147, 383^ 397. [68] Thoukis, G., Ueda, M. and Wright, D. (1965) The formation of succinic acid during alcoholic fermentation. Am. J. Enol. Vitic. 16, 1^8. [69] Heerde, E. and Radler, F. (1978) Metabolism of the anaerobic for-

FEMSYR 1540 7-5-03

399

mation of succinic acid by Saccharomyces cerevisiae. Arch. Microbiol. 117, 269^276. [70] Ogata, H., Goto, S., Sato, K., Fujibuchi, W., Bono, H. and Kanehisa, M. (1999) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 27, 29^34. [71] Coleman, S.T., Fang, T.K., Rovinsky, S.A., Turano, F.J. and MoyeRowley, W.S. (2001) Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae. J. Biol. Chem. 276, 244^250.
