1760 (2006) 1281ndash1291httpwwwelseviercomlocatebba

Biochimica et Biophysica Acta

Deviation of carbohydrate metabolism by the SIT4 phosphatasein Saccharomyces cerevisiae

Willy Jablonka a1 Simoacuten Guzmaacuten b1 Jorge Ramiacuterez b Moacutenica Montero-Lomeliacute a

a Instituto de Bioquiacutemica Meacutedica Programa de Biologia Molecular e Biotecnologia Centro de Ciecircncias da Sauacutede Bloco D subsolo sala 11Universidade Federal do Rio de Janeiro CP 68041 Rio de Janeiro RJ 21941-590 Brazil

b Departamento de Geneacutetica Molecular Instituto de Fisiologia Celular Universidad Nacional Autoacutenoma de Meacutexico

Received 27 July 2005 received in revised form 21 February 2006 accepted 22 February 2006Available online 20 March 2006

Abstract

A prominent phenotype of the yeast sit4 mutant which lacks the SerndashThr phosphatase Sit4 is hyper-accumulation of glycogen and the failureto grow on respiratory substrates We investigated whether these two phenotypes are linked by studying the metabolic response to SIT4 deletionAlthough the sit4 mutant failed to grow on respiratory substrates in the exponential growth phase respiration was de-repressed active respirationwas confirmed by measuring oxygen consumption and NADH generation However the fermentation rate and the internal glucose 6-phosphateand pyruvate levels were reduced while glycogen content was high Respiro-fermentative and respiratory substrates such as galactose glyceroland ethanol were directed toward glycogen synthesis which indicates that sit4 mutant deviates metabolism to glycogenesis by activating aglycogen futile cycle and depleting cells of Krebs cycle intermediates An important feature of the sit4 mutant was the lack of growth underanaerobic conditions suggesting that respiration is necessary to meet the energy requirements of the cell Addition of aspartic acid which canrestore Krebs cycle intermediates partially restored growth on ethanol Our findings suggest that inhibition of Sit4 activity may be essential forredirecting carbohydrate flux to gluconeogenesis and glycogen storagecopy 2006 Elsevier BV All rights reserved

Keywords SerndashThr phosphatase SIT4 Carbohydrate metabolism Glycogen

1 Introduction

The SerndashThr protein phosphatase Sit4 is a type1type 2A-related protein phosphatase [1] Sit4 is involved in severalcellular processes that promote growth in response to nutrientsignalling including cell cycling bud emergence [12] cell wallintegrity actin cytoskeleton organization and ribosomal genetranscription [3] It has also been shown to be functionallylinked to the ubiquitin-proteasome system [4] The involvementof Sit4 in nitrogen starvation has been extensively studied withthe aid of rapamycin a drug that inhibits the activity of TORkinases Sit4 in association with Tap42 and four different Sapproteins (Sit4-associated proteins) mediates TOR kinase

Corresponding author Tel +55 21 2230 6899 fax +55 21 2270 8647E-mail address monterobioqmedufrjbr (M Montero-Lomeliacute)

1 These authors contributed equally to this work

0304-4165$ - see front matter copy 2006 Elsevier BV All rights reserveddoi101016jbbagen200602014

signaling in response to nitrogen starvation or rapamycintreatment [4ndash7]

Sit4 is also involved in carbohydrate sensing although itsinvolvement is not clearly understood A puzzling phenotype ofsit4 mutants is glycogen accumulation and lack of growth onrespiratory substrates [1] Accumulation of glycogen has beenpostulated to occur by inhibition of glycogen phosphorylaseactivity directly or indirectly mediated by Sit4 [8] while failureto grow on ethanol has not been elucidated Suppressors for thisphenotype have not been found and it has been reported that sit4mutant is unable to respire [9] Evidence of involvement incarbohydrate sensing has also come from studies on theinteraction between Sit4 and Tap42 In glucose-grown cellsentry into stationary phase or rapamycin treatment leads todissociation of the complex while glucose refeeding leads to itsassociation [10] Interestingly the complex Sit4Tap42 was notfound in cells grown on respiratory substrates such as galactoseor glycerol plus ethanol [10]

1282 W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

In a systematic identification of genes that affect glycogenstorage 60 of the strains identified as glycogen hypoaccu-mulators had mutations on genes known to be required forrespiration or were in mutants that failed to use non-fermentablecarbon sources [11] However the sit4 mutant accumulatesglycogen and yet fails to use non-fermentable carbon sources Inthis report we show that the lack of growth of sit4 mutant onrespiratory substrates is related to redirection of metabolismtoward glycogen synthesis which leads to a change in geneexpression to a nutrient-deprivation profile

2 Materials and methods

21 Strains

Saccharomyces cerevisiae strains used were FY833(MATa his3Δ200 ura3-52 leu2Δ1 lys2Δ202 trp1Δ63 GAL2+) referred in the text as wild type(donated by M Ghislain) JK9-3da (MATa leu2-3112 ura3-52 rme1 trp1 his4GAL+ HMLa) [12] and TS64-1a (MATa leu2-3112 ura3-52 rme1 trp1 his4GAL+ HMLa sit4∷kanMX4) [12]

22 Gene disruption

SIT4 was disrupted in FY833 background by replacing the original SIT4open reading frame with a PCR-generated HIS3 MX6 cassette as described [13]This strain is referred in the text as sit4 mutant

23 RNA isolation and cDNA labeling

Samples for RNA isolation were taken from the aerobic batch cultivation ofstrain FY833 and FY833-Δsit4 grown to mid-logarithmic phase in YPgal (2bactopeptone 1 yeast extract 2 galactose) Total RNA was extracted asdescribed [14] Ten micrograms of total RNA were used for cDNA synthesisincorporating dUTP-Cy3 or dUTP-Cy5 employing the CyScribe First-StrandcDNA labeling kit (Amersham) Incorporation of fluorophore was analyzed byusing the absorbance at 555 nm for Cy3 and 655 nm for Cy5

24 Transcription analysis

Equal quantities of labeled cDNA were hybridized using hybridizationsolution HybIT2 (TeleChem International INC) to the collection of the Yeast50-mer-oligo library from MWGBiotech Oligo Sets (httpwwwmwgbiotechcom) which contains 6250 gene-specific oligonucleotide probes representingthe complete Saccharomyces cerevisiae genome Genome-wide transcriptionanalysis was performed in triplicate on wild type strain FY833 or FY833-Δsit4grown to mid-log phase on YP-galactose 2 A detailed description oftranscription analysis methodology and complete results are available insupplementary material (httpwwwbioqmedufrjbrlablevsupplementary_sit4html)

25 Real-Time PCR

To confirm changes in gene expression levels quantitative real-time PCRanalysis of selected transcripts was carried out using cDNAs derived from strainFY833 and FY833-Δsit4 grown to mid-logarithmic phase (ABS600 nm = 10) orstationary phase (ABS600 nm = 10) where stated Total RNA was extracted andcontaminant genomic DNAwas removed by treatment with DnaseI and reversetranscribed using Taq-Man master mix (Applied Biosystems Foster City CA)following the protocol furnished by the manufacturers The cDNA samples wereused as templates for amplification of desired genes by PCR in the presence ofSybr-Green fluorescent probe using Sybr-green PCR master mix geneexpression assay (Applied Biosystems) The conditions for PCR were 92 degCfor 2 min followed by 40 cycles at 92 degC for 15 s 55deg C for 30 s and 72 degC for90 s followed by elongation at 72 degC for 5 min and final dissociation at 95 degC

for 15 s The amount of fluorescence was detected using a 7500 real-time PCRmachine (Applied Biosystems) The number of PCR cycles (Ct) required toreach a fluorescence intensity above threshold was calculated using theSequence detection software version 13 (Applied Biosystems) by the standardcurve method Relative expression levels for the studied genes were calculatedas in [15] Briefly the mean Ct for three replicates of each gene was subtractedfrom the mean Ct for three replicates of the reference gene ACT1 in each sampleto obtain ΔCt [(ΔCt = Ct (gene)minusCt(actin)] Relative copy number (RCN) inthe sit4 mutant related to wild type was calculated using the following formulaRCN = 2minusΔΔCt whereΔΔCt =ΔCt (sit4 mutant) minusΔCt (wild type) The primersdesigned for each gene were CYC1 fc (5prime-ggtgctacacttttcaagac-3prime) and CYC1 r(5prime-tttttccttcttcaacc-3prime) GPH1 fc (5prime-cttctccacaccaaatcc-3prime) and GPH1 r (5prime-gaccaccattacctaaacc-3prime) HXT3 fc (5prime-ccatccattcattcaacaag-3prime) and HXT3 r (5prime-caggcaaagacaatcatacag-3prime) HXT7 fc (5prime-ctgctattgcagagcaaac-3prime) and HXT7 r(5prime-taccaccaacacccaaac-3prime) ACT1 fc (5prime-tacgtttccatccaagccgttt-3prime) and ACT1 r(5prime-aacatacgcgcacaaaagcaga-3prime) PGM2 fc (5prime-aacaagcatcatccggagaac-3prime) andPGM2 r (5prime-cgggatccaagccattagtaaatcattgct-3prime)

26 Overexpression of PGM2

The yeast strain FY833-Δsit4 was transformed with plasmid pPGM2 inwhich the gene PGM2 is subcloned under the control of the PMA1 promoter[15]

27 Analysis of yeast metabolites

Extraction of metabolites was carried out as described previously [17]Briefly yeast were vacuum-filtered and resuspended in 80 ethanol Afterbeing dried the precipitate was resuspended in 1 mM EDTA (pH 75) andcentrifuged at 20000timesg for 20 min at 4 degC The supernatant was used formeasuring glucose 6-phosphate glucose 1-phosphate pyruvate and ATP asdescribed [18] For glycogen and trehalose content cells were resuspended in80 ethanol and the precipitate formed was resuspended in 025 M Na2CO3boiled for 1 h and then acidified to pH 47 as described [19] Glycogen washydrolyzed to glucose with amyloglucosidase (Sigma) and trehalose washydrolyzed with trehalase (Sigma) Glucose was assayed using a glucoseoxidase kit (Dole-Brazil)

28 Glycerol incorporation into glycogen

Yeast cells were grown in YPGal medium to stationary phase washed twicewith distilled water and incubated in 05 galactose for 25 h 25 mg (wetweight) of yeast were incubated in a buffer containing 10 mMMES-TEA pH 60and 10 mM [U-14C]-glycerol (5 μCimmol) for 40 min with shaking at 30 degCIncorporation was stopped by filtering the cells on a 045 μm nitrocellulosefilter Filtered cells were resuspended in 80 ethanol and the precipitate formedwas resuspended in 025 M Na2CO3 boiled for 1 h and then transferred toscintillation solution The percentage of incorporation into glycogen wascalculated as the fraction of radioactivity precipitated with ethanol compared tothe total [U-14C]-glycerol uptake Radioactivity was measured in a scintillationcounter (Beckman)

29 Glycogen synthase glycogen phosphorylase andphosphoglucomutase assays

An aliquot of yeast cells was collected by centrifugation and lysed with glassbeads (425ndash600 μm from Sigma) in a buffer containing 50 mM TrisndashHCl1 mM EDTA 025 mM PMSF 100 mM dithiothreitol and 1 unitml of each ofthe following protease inhibitors aprotinin leupeptin and pepstatin Lysed cellswere centrifuged at 3000timesg for 10 min at 4 degC and the supernatant was used forenzyme assays Glycogen synthase activity was determined by incorporation of025 mM UDP-[U-14C]-glucose into glycogen as described [20] Assays wereperformed in the presence and absence of 6 mM glucose 6-phosphate Glycogenphosphorylase activity was determined in a coupled assay [21] Briefly extractswere incubated in a medium containing 50 mM Na2PO4 1 mM MgCl2 02(wv) glycogen 50 nM glucose 16 diphosphate 1 mM DTT 06 mM NAD+1 unitml phosphoglucomutase and 1 unitml glucose 6-phosphate

Fig 1 Respiration in the sit4 mutant is de-repressed in exponential-phasecultures Yeast strains FY833 (WT) and FY833-Δsit4 (sit4mutant) were grown toexponential phase (OD600nm = 10) in YP (yeast extract 1 peptone 2)either with 2 glucose (YPD) or with 2 galactose (YPGal) followed by fastingfor 25 h in 10 mM MES-TEA pH 60 (A) Oxygen consumption rates weremeasured in a buffer containing 10 mM MES-TEA and 2 glucose using anoxygen electrode Average plusmn SD of four independent experiments is shownIn B and C the effect of FCCP an uncoupler of oxidative phosphorylationon the rate of oxygen consumption was measured in wild-type strain (B) orthe sit4 mutant (C) grown on YPD followed by fasting as described aboveWhere indicated by arrows 2 glucose (thin line) or 2 glucose plus 5 μMFCCP (dark line) were added A representative experiment is shown Theaverage rate of respiration (natoms O2min 25 mg cells plusmn SD) (n = 3) in thewild-type strain increased from 300plusmn0019 to 465plusmn013 in the presence of5 μM FCCP while in the sit4 mutant the rate increased from 1105plusmn52 to1761plusmn72 in the presence of 5 μM FCCP

1283W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

dehydrogenase pH 70 Phosphoglucomutase was assayed in the same mediumbut glycogen and phosphoglucomutase were omitted and 1 mM glucose 1-phosphate was added

210 Respiration and fermentation rates

Yeast cells (25 mg wet weight) grown in YPD or YPGal medium werewashed twice with distilled water and incubated in a water-jacketed airtightchamber in 3 ml containing 10 mM MES-Triethanolamine (MES-TEA) pH 60and 2 glucose or 019 ethanol Oxygen consumption was recorded using anoxygen electrode Fermentation was assayed by incubating 25 mg yeast cells(wet weight) in 10 mM MES-TEA pH 60 2 glucose or galactose Formationof ethanol was assayed as described [18] One unit is equivalent to 1 nmolethanol10 min mg dry weight

211 NADH measurement

Yeast cells (25 mg wet weight) grown in YPD or YPGal medium werewashed twice with distilled water and incubated in a medium containing 10 mMMES-TEA pH 60 NADH was measured in whole cells as described [22] usingan excitation wavelength of 340 nm and an emission wavelength of 461 nm in arecording spectrofluorimeter

3 Results

31 Respiratory capacity of sit4 mutant

Metabolism of yeast grown in glucose to the logarithmicphase is preferentially fermentative whereas respiration isrepressed As cells reach stationary phase and glucose isexhausted respiration is de-repressed and cells start consumingethanol which was generated from fermentation [23] On theother hand galactose is a non-repressive sugar and cells exhibita fermento-respiratory metabolism [23]

We tested the respiratory capacity of the sit4mutant grown tomid-logarithmic phase in both carbon sources The rate ofoxygen consumption in the sit4 mutant grown in glucose wasde-repressed (Fig 1A) whereas the rate of oxygen consumptionin galactose-grown cells was not different from the wild-typestrain as it is already de-repressed in this carbon source Theseresults were not expected as the sit4 mutant is unable to growon ethanol or glycerol [1] Oxygen consumption in both thewild-type strain and the sit4 mutant was inhibited by KCN

If sit4 mutant was respiratory capable an explanation forfailure to grow on ethanol might be that mitochondria areuncoupled To test this idea we added FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) a potent uncoupler ofoxidative phosphorylation which collapses the mitochondrialmembrane potential by disrupting the mitochondrial H+

gradient FCCP led to an activation of respiration (Fig 1Band C) showing that mitochondria were previously coupledCoupling was even tighter in the sit4 mutant than in the wild-type strain since activation of respiration was greater whenFCCP was added (Fig 1C) These results show that failure ofsit4 mutant to grow on ethanol is neither due to respiratoryincompetence nor to uncoupling of the mitochondrial electro-chemical gradient The results are not in accordance with areport that states that in the sit4 mutant the respiration rate isalmost null although sit4 mutant is able to reduce tetrazoliumsalts [9] In order to confirm respiration in the mutant we have

used another strain TS64-1a with different genetic back-ground but with the same respiratory capacity as in our mutant(data not shown)

To confirm the results shown we have also measuredrespiration by another method monitoring NADH cycling inwhole cells NADH is generated mainly in the oxidationreactions of glycolysis and of the Krebs cycle while it is

1284 W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

consumed mainly by the electron transfer chain and bygluconeogenesis It can be monitored in whole cells by mea-suring the fluorescence emitted by NADH [22] The experimentin Fig 2A shows that NADH was generated upon addition ofglucose to both the wild-type strain and the sit4 mutantAddition of glucose led to a rapid peak of generation of NADHfollowed by a slower rate Adding the mitochondrial uncouplerpentachlorophenol (PCIP) reduced NADH levels as it increasedthe consumption of NADH by the electron transfer chain whilethe addition of NaCN an inhibitor of respiration led toaccumulation of NADH formed by fermentation NADH canalso be generated by respiratory metabolism using ethanol assubstrate during oxidation of ethanol to acetaldehyde byalcohol dehydrogenase and during the Krebs cycle In Fig 2BNADH was generated by yeast when ethanol was used assubstrate Addition of PCIP resulted in net oxidation of NADHas respiration was enhanced while inhibition of the electron

Fig 2 The sit4 mutant generates NADH on addition of glucose (A) or ethanol(B) Wild-type (thin lines) and sit4 mutant (dark lines) were grown on YP plus2 galactose and subjected to fasting for 25 h Afterwards NADH levels weremeasured in intact yeast resuspended in 10 mM MES-TEA pH 60 in aspectrofluorimeter at an excitation wavelength of 340 nm and emissionwavelength of 461 nm NADH generation was measured after addition ofglucose (A) or ethanol (B) Yeast (Y) 2 glucose or ethanol 20 μM PCIP (amitochondrial uncoupler) and 500 μM NaCN were added sequentially whereindicated by arrows and the intensity of fluorescence was recorded Arepresentative experiment is shown

transfer chain by NaCN led to reduction of NAD+ The patternmeasured for the sit4 mutant was not different from wild-typecells although NADH was generated to a greater extent in thesit4 mutant when ethanol was used These results clearly showthat lack of growth on ethanol is not due to a defect in the Krebscycle nor in the electron transport chain

32 Gluconeogenesis in sit4 mutant

Other mutants that fail to grow on respiratory substrates arethose defective in gluconeogenic genes such as fructose 16-bisphosphatase phosphoenolpyruvate carboxykinase and iso-citrate lyase [2425] To test whether the sit4 mutant is capableof driving gluconeogenesis we measured incorporation of [U-14C] glycerol into glycogen During the first 40 min ofincubation at 30 degC incorporation in the sit4 mutant (196of total glycerol uptake) was the same as that of the wild-typeyeast cells (182 of total glycerol uptake) (data not shown)

33 Glycogen synthase and glycogen phosphorylaseactivities

To find another explanation for lack of growth on ethanol weinvestigated whether lack of growth on ethanol and theaccumulation of glycogen are related phenotypes The controlof glycogen metabolism is regulated by glucose availabilitygrowth phase and environmental stresses such as heat shock ornitrogen starvation [26] A rise in glycogen synthesis at thediauxic phase of growth in glucose correlates with induction oftranscription of all genes involved in glycogen metabolism [27]including glycogen synthase (GSY2) and glycogen phosphory-lase (GPH1) The activity of these two enzymes is also regulatedat the protein level where glycogen synthase is inactivated byphosphorylation and activated by glucose 6-phosphate andglycogen phosphorylase is inactivated by dephosphorylation[27ndash30]

In sit4mutant glycogen metabolism is not properly regulatedand glycogen synthesis is de-repressed at the exponential phaseof growth in glucose [2] The role of Sit4 phosphatase in theactivity of glycogen synthase and phosphorylase has beenstudied during different periods of growth in glucose Glycogensynthase activity was reported as the ratio between glycogensynthesis in the absence and presence of glucose 6-phosphatean allosteric activator of the phosphorylated inactive form ofglycogen synthase Results showed that glycogen synthase wasless active in sit4mutant than in wild-type cells while glycogenphosphorylase activity was much higher in the exponentialphase [8] These results are intriguing as it would be expectedto find an increase in the active form of glycogen synthase andnot in glycogen phosphorylase activity In order to explain a risein glycogen content it was suggested that glycogen phosphor-ylase activity is inhibited [8]

We investigated whether in galactose a non-repressivesugar glycogen accumulation and glycogen synthase as well asphosphorylase activities were regulated as previously shown Inexponentially growing cells using galactose as substrateglycogen accumulation was enhanced in the sit4 mutant (Fig

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3A) Accumulation was specific for glycogen as trehaloseanother reserve carbohydrate was not accumulated (data notshown) On the other hand the glycogen synthase activity ratio(plusmnGlu 6-P) was low in exponential phase in both strains thewild-type and sit4 mutant showing that the enzyme was in thephosphorylated inactive form (Fig 3B) In contrast to reportedresults [8] glycogen synthase activity ratio (plusmnglucose-6phosphate) in the sit4 mutant grown in glucose was lowerthan wild type Both results obtained in galactose grown cells(Fig 3B) or in glucose grown cells to exponential phase [8]indicate that glycogen synthase is in the inactive phosphory-lated form in sit4 mutant which does not explain hyper-accumulation of glycogen In stationary phase (Fig 3B)glycogen synthase activity (plusmnglucose-6 phosphate) is increased

Fig 3 SIT4 is necessary for the proper regulation of glycogen content and glycogen pthe sit4 mutant grown to exponential phase (open bars) or stationary phase (grey bashown (B) Glycogen synthase activities were measured in cell lysates prepared fromin YPGal medium Glycogen synthase activities were measured at pH 78 in a buffer c025 mM [14C]-UDPglucose (265 mCimmol) 033 glycogen 015 mgml proteiphosphate The ratio (plusmnglucose 6-phosphate) is represented (gray bars) The averagetranscript (D) were measured in cell lysates or cDNA respectively prepared from wibars) phase Glycogen phosphorylase activity was measured in a buffer containing01 mMNAD+ 1 unit phosphoglucomutase 1 unit glucose 6-phosphate dehydrogenais shown The amount of relative GPH1 transcript (D) in sit4mutant was compared toand methods The average plusmn SD of five independent experiments is shown

in wild type strain (03 fold) and in sit4 mutant (31 fold)These results indicate that glycogen synthase is properlyregulated in sit4 mutant and probably not responsible forhyper-accumulation of glycogen

In accordance with Posas et al [8] glycogen phosphorylaseactivity was enhanced in exponentially growing sit4 mutantcompared to the wild-type strain grown in galactose (Fig 3C)which also does not explain glycogen accumulation Toelucidate whether glycogen phosphorylase activation was dueto up-regulation of transcription we analyzed the amount ofGPH1 (glycogen phosphorylase) transcript using the RealTime-PCR technique Cells were grown in galactose to earlylogarithmic phase or stationary phase (Fig 3D) Results showthat in exponential phase of growth the amount of GPH1

hosphorylase activity (A) Glycogen levels were measured in wild-type strain andrs) in YPGal medium The averageplusmnSD of three independent experiments iswild type and the sit4 mutant grown to exponential (exp) or stationary (st) phaseontaining 50 mM TrisndashHCl 20 mM EDTA 25 mMNaF 15 mMUDP-glucosen and the absence (open bars) or presence (hatched bars) of 6 mM glucose 6-of two experiments is shown Glycogen phosphorylase activity (C) and GPH1ld type and the sit4 mutant grown to exponential (open bars) or stationary (grey50 mM Na2PO4 1 mM MgCl2 50 nM glucose 16 diphosphate 1 mM DTTse and 015 mgml protein The average plusmn SD of three independent experimentswild type and analyzed by quantitative Real-time PCR as described in Materials

Fig 4 The sit4 mutant suppresses cellular glycogen consumption only inrespiratory substrates and enhances glycogen synthesis (A) Glycogenconsumption Yeast cells were grown to stationary phase (OD600nm=15) inYPGal medium and further incubated for 25 h in low galactose medium (05galactose) to ensure glycogen synthesis Total intracellular glycogen wasmeasured at this time in controls (no further addition) and after incubation in 2of glucose (Glu) 2 galactose (Gal) or 2 ethanol (EtOH) for 25 h at 30 degC (B)Glycogen synthesis Yeast cells were grown to stationary phase (OD600sub nm=15) in YPGal medium and further incubated for 25 h in 2glucose medium to ensure glycogen depletion Total intracellular glycogen wasmeasured at this time (control) and after further incubation in 2 glucose 2galactose or 2 ethanol for 25 h at 30 degC Average plusmn SD of three independentexperiments is shown

1286 W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

transcript is 16 fold higher in the sit4 mutant compared to wildtype These results correlate with activation of glycogenphosphorylase activity in the sit4 mutant Entry into stationaryphase enhances transcription of GPH1 4 fold in the wild typestrain but not in the sit4mutant (Fig 3D) suggesting that properregulation of transcription of GPH1 is lost in sit4 mutant Theseresults are intriguing as they show that hyper-accumulation ofglycogen in exponential phase is not due to activation ofglycogen synthase or to inactivation of glycogen phosphorylaseactivity

34 Turnover of glycogen metabolism in sit4 mutant

We tested whether the activities of glycogen synthase andglycogen phosphorylase measured in vitro were reflected in theturnover of glycogen metabolism First we tested if the sit4mutant can degrade glycogen Cells previously grown tostationary phase in YPGal medium were incubated in completemedium containing only 05 galactose to assure a high contentof glycogen both in the wild-type strain and the sit4 mutantGlycogen degradation was monitored after supplementingfasted cells with glucose galactose or ethanol and degradationwas compared with that in controls in which no supplement wasgiven (Fig 4A) After 25 h of incubation glycogen waspartially consumed in the wild type following addition ofglucose galactose or ethanol In contrast in the sit4 mutant itwas consumed only on glucose addition but not on addition ofgalactose or ethanol These results show that glycogendegradation is impaired in the sit4 mutant in non-repressivecarbon sources

Glycogen synthesis is difficult to measure in the sit4 mutantsince it already contains high glycogen levels in the exponentialphase of growth In order to deplete intracellular glycogen wetook advantage of the fact that in glucose medium glycogenlevels are lower than in galactose or ethanol as shown in Fig4A Cells were grown to stationary phase in YPGal medium andthen shifted to YPD medium for 25 h to lower the levels ofglycogen After this maneuver glycogen levels were low inboth strains shown in control bars (Fig 4B) Cells were thenshifted to a medium containing 2 glucose galactose or etha-nol All three carbon sources induced an increase in glycogenaccumulation in both strains but in the sit4 accumulation wassubstantially enhanced when cells were shifted to galactose or toethanol In these experiments it is evident that galactose andethanol metabolism were directed toward glycogen accumula-tion in the sit4 mutant

35 The fermentation rate is diminished in sit4 mutant andmetabolism is deviated to glycogen synthesis

Galactose is metabolized in yeast by the Leloir pathway[31] Glucose 1-P formed may be used by the cell to formglucose 6-phosphate or UDP-glucose driving the metabolicflux towards glycolysis or glycogen synthesis respectively Totest for deviation of metabolism to glycogen synthesis wemeasured the fermentation rate and key internal metabolitesThe fermentation rate in the sit4 strain was diminished 7 fold

(21plusmn028 units in wild-type strain versus 031plusmn003 units inthe sit4 mutant) Furthermore the amounts of Glu 6-P andpyruvate were diminished while ATP and Glu 1-P contentswere not significantly changed (Table 1) These results areconsistent with deviation of metabolism to glycogen synthesisin the sit4 mutant

An essential enzyme for the entry of galactose into glycolysisis phosphoglucomutase (PGM) In the sit4 mutant the levels ofPGM2 transcript are lower than in wild type (Table 2)However inhibition of PGM activity would result in Glu 1-Paccumulation which was not observed in the sit4 mutant Weverified that galactose was actually not converted to glucose-6-P by measuring Glu-1-P after a lithium stress Lithium induces alarge accumulation of these metabolites when wild-type yeast is

Table 1Intracellular content of glucose 6-phosphate ATP and pyruvate (nmolmg dryweight of cells) in the wild type and the sit4 mutant grown to exponential phasein YPGal

WT sit4 mutant

Glucose 6-phosphate 07 plusmn 02 (n = 6) 03 plusmn 004 (n = 6)ATP 23 plusmn 04 (n = 6) 19 plusmn 03 (n = 6)Pyruvate 42 plusmn 07 (n = 3) 19 plusmn 01 (n = 3)Glucose 1-phosphate 67 plusmn 12 (n = 3) 64 plusmn 17 (n = 3)Glucose 1-phosphate + 6 mM LiCl 153 plusmn 37 (n = 3) 62 plusmn 18 (n = 3)

The intracellular content of glucose 1-phosphate is shown in yeast cells grown asindicated or further incubated for 3 h with 6 mM lithium (see explanation intext) Data are averages plusmn SD of the number of experiments shown inparentheses

1287W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

grown in galactose because it blocks PGM activity [16] In thesit4 mutant no accumulation of Glu-1-P was observed in thepresence of lithium (Table 1) which confirms the data showingthat galactose is not metabolized to glucose-6P in the sit4mutant Furthermore we tried to suppress glycogen accumu-lation by driving metabolism toward formation of Glu 6-P byoverexpressing PGM2 as previously shown for pyruvatecarboxylase mutants [32] However overexpression of PGM2in the sit4mutant could not drive flux toward Glu 6-P formation(data not shown)

36 Microarray analysis of sit4 mutant grown in galactose

We compared the expression profile of wild-type strainversus sit4 mutant grown in galactose to exponential phase bymicroarray analysis cDNA labeled with Cy3-UTP or Cy5-UTPwas synthesized from total RNA of sit4 mutant and wild-typecells and hybridized against a Saccharomyces cerevisiae 50-meroligo-library Genes were identified as having changed theirexpression level based on a significant difference between theirmean expression level in sit4 mutant and the mean expressionlevel in the wild-type strain With this experiment we expectedto see the global changes in transcription induced by sit4deletion and to compare them with the biochemical resultsOnly a small number of genes (20 up-regulated and 60 down-regulated genes) were affected by sit4 deletion Microarrayprimary results as well as analysis of up-regulated and down-

Table 2Confirmation of microarray analysis

Real-time PCR microarray

Gene Δsit4WT SD Δsit4WT SD

ACT1 10 108 06HXT3 131 01 28 07PGM2 minus17 01 minus28 01GPH1 16 02 minus20 02HXT7 42 04 273 10CYC1 20 02 363 06

A comparison of the expression ratio (sit4 mutantwild type) of selected genesbased on Real-time PCR and microarray analysis is shown The average plusmn SDof expression in three independent Real-time PCR experiments and three inde-pendent microarray analyses are shown The fold change for up-regulated genesis (sit4 mutantWT) while for down-regulated genes it is minus[(sit4 mutantWT)]minus1

regulated genes are available as supplementary material (httpwwwbioqmedufrjbrlablevsupplementary_sit4html) To sup-port our data on the microarray experiment we have selectedfive genes whose expression was changed and confirmed thelevel of expression by Real-time PCR (Table 2) The patternobserved in the microarray experiments correlated with thatobtained by Real-time PCR with the exception of GPH1 whoseup-regulated expression (16) was verified by the more reliabletechnique Real-time PCR Up-regulation of expression wasalso corroborated by stimulation of glycogen phosphorylaseactivity in sit4 mutant (Fig 3C)

In order to analyze the microarray experiment we firstclustered genes into broad biological categories using SGDGene Ontology Slim Term Mapper (httpdbyeastgenomeorg)The major functional classes of genes affected in the sit4mutantwere related to physiological processes metabolism andtransport while approximately 50 (up-regulated and down-regulated) were unknown genes (Fig 5) In a more detailedclassification we have clustered genes according to thebiological process (Table 3) where we show the percentage ofgenes affected that correspond to each class and as an examplewe show the gene that changed the most in each class Theidentity of all genes changed in each class is described in sup-plementary material

From these clusters we could identify two groups of genesthat might be regulated in response to nutrient deprivation onerelated to sugar transport and one to sugar metabolism (Table 4)In these groups the genes related to sugar transport (HXT2HXT3 HXT4 HXT7 ITR1 and GAL2) and two respiration-related genes (CYC1 and NCA3) were up-regulated It may bethat the low levels of Glu 6-P observed in the sit4 mutantinduces expression of sugar transporters in order to increase theuptake of sugars With respect to respiration-related genes weshowed that respiration in galactose-grown cells is not inducedby SIT4 deletion (Fig 1A) although metabolism is shifted fromrespiro-fermentative to respiratory Thus sit4 mutant must relyon respiration for growth

From the group of down-regulated genes related to carbo-hydrate metabolism in the sit4 mutant we identified genes

Fig 5 Major functional classes of genes affected by SIT4 deletion Results ofmicroarray data were grouped into broad biological process categories usingSGD GO Slim Term mapper The percentage of up-regulated (open bars) anddown-regulated (gray bars) genes in each class is shown It should be noted thata specific gene can be classified into more than one category

Table 4Sugar transport and carbohydrate metabolism genes affected by SIT4 deletionas measured by microarray

Gene name ORF-Reference

Gene description Fold change(SD n=3) a

Sugar TransportHXT3 ydr345c low-affinity glucose transport 28 (07)HXT7 ydr342c high-affinity glucose transport 27 (10)ITR1 ydr497c myo-inositol transport 26 (06)HXT2 ymr011 high-affinity glucose transport 25 (02)GAL2 ylr081w galactose (and glucose) permease 23 (08)HXT4 yhr092c moderate- to low-affinity glucose

transporter23 (01)

Carbohydrate MetabolismCYC1 yjr048w cytochrome-c isoform 1 36 (06)NCA3 yjl116c involved in regulation of synthesis

of ATP6P and Atp8p23 (02)

PGM2 ymr105c phosphoglucomutase minus28 (01)FBA1 ykl060c fructose-bisphosphate aldolase minus22 (01)GPH1 ypr160w glycogen phosphorylase minus20 (02)GCY1 yor120w galactose-induced protein of

aldoketo reductase familyminus20 (01)

MMD1 yil051c required for maintenance ofmitochondrial DNA

minus19 (01)

FBA2 ykl060c-r fructose-bisphosphate aldolase minus18 (01)GPD1 ydl022w glycerol-3-phosphate

dehydrogenase (NAD+)cytoplasmic

minus15 (04)

a The fold change for up-regulated genes is (sit4 mutantWT) while for downregulated genes is minus(sit4 mutantWT)]ndash1

Table 3Major biological classes of genes affected in sit4 mutant related to wild type

Biological Process Up-regulated a

(Example b)Down-regulated a

(Example b)

Unknown genes 30 (YOR280c) 25 (YER181C)Hexose transporters 30 (HXT3)Cell wall related genes 25 (CHS1) 6 (YPG1)Carbohydrate metabolism 15 (CYC1) 16 (PGM2)Membrane transporters 5 (BPA2) 5 (PMA1)Transcriptional activation 5 (OYE2) 3 (BRO1)Protein metabolism 5 (YSP3)Heat shock response 5 (SSA4)Amino acid metabolism 5 (MET18)Cellular fusion 3 (MFA2)Purine and pyrimidinemetabolism

5 (URA1)

Protein modification 6 (NCE12)DNA metabolism ty-elementtransposition

8 (TY2A)

a The percentage of genes changed in sit4 mutant is shown from a total of 20up-regulated and 60 down-regulated genesb The most affected gene in each subclass is shown as an example

1288 W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

related to glycogen turnover (PGM2 and GPH1) and genesrelated to glycolysis (FBA1 FBA2 and GPD1) However asshown above down-regulation of GPH1 gene was not con-firmed by Real-time PCR analysis or by phosphorylase activityboth measures show it was up-regulated in the sit4mutant grownin galactose to exponential phase (Table 2 and Fig 3) Genesrelated to glycolysis (FBA1GPD1GPD2) are down-regulatedwhich is consistent with the observed inhibition of fermentationWe would have expected to see changes in limiting steps ofglycolysis but neither fructose 16-bisphosphate aldolase orglycerol 3-phosphate dehydrogenase is a limiting enzyme ofglycolysis These results confirm the hypothesis that deletion ofSIT4 leads to entry into a futile cycle of glycogen synthesis anddegradation where fermentation is down-regulated leading to anutritional stress reflected by induction of expression of hexosetransporters and respiration-related genes

Transcriptional changes are quantitative and qualitativelimited in the microarray data and do not reflect the defectsinduced by SIT4 deletion [1ndash3] Probably SIT4 inactivationaffects proteins related to metabolism by de-phosphorylationTogether with regulation of carbohydrate metabolism thesechanges should now be addressed by performing a phospho-proteome analysis where direct targets of the phosphatasemight be revealed

37 sit4 mutant relies on respiration for growth

If galactose metabolism is being driven toward glycogensynthesis and glucose 6-phosphate is depleted (Table 1) thenhow is ATP homeostasis maintained Biochemical and micro-array experiments provide a clue as they indicate that bothrespiration and transcription of several respiration-related genesare induced in sit4 mutant We investigated whether respirationis actually important for viability of the sit4 mutant In Fig 6Awe show that sit4 is an aerobic obligate in galactose but not inglucose medium Inhibition of respiration by addition of 25 mM

KCN also abolished growth in galactose medium These resultssuggest that ATP homeostasis in the sit4 mutant grown ingalactose is maintained by respiration This result led us to thinkthat the reason the sit4mutant does not grow on ethanol might bethat deviation to glycogen synthesis depletes intermediatemetabolites of the Krebs and glyoxylate cycles To test thisidea the sit4 mutant was grown in galactose or ethanol inthe presence of aspartic acid so that replenishment of theseintermediates could occur via transamination of aspartate tooxaloacetate by aspartate amino transferase (Fig 6B) Thepartial recovery of growth in this medium indicates that thismaneuver suppresses lack of growth on ethanol

4 Discussion

In principle the failure of yeast to grow on respiratorysubstrates can be due to three main reasons mutants lackingmitochondria mutation in genes essential for the Krebs cycleand the electron transfer chain andor mutation in genesessential for gluconeogenesis In this report we have exploredwhether the sit4 mutant meets one of these conditions Bymeasuring oxygen consumption we found that the sit4 mutantwas respiratory competent since oxygen was consumed onaddition of fermentative as well as non-fermentative carbonsources Even during exponential growth on glucose mediumwhere fermentation is the primary fate of glucose and respirationis repressed in wild-type yeast the mutant showed higher levelsof respiration This is in agreement with a recent observation

Fig 6 The sit4 mutant is an aerobic obligate and lack of growth is suppressed by aspartic acid (A) Yeast strains FY833(WT) and FY833-Δsit4 JK9-3da (WT) andTS64-1a (JK93-Δsit4) were grown to saturation on YPD and 5 μl of a diluted culture (OD600 sim 03 003 and 0003) in each panel were plated onto YP mediumcontaining the indicated carbon sources in aerobic (top row) or anaerobic medium (second row left) or aerobic medium in the presence of 25 mM KCN (second rowright) For anaerobic growth plates were incubated in an anaerobic jar system form AnaeroGen (Oxoid) (B) FY833-Δsit4 was plated on YP plus 2 galactose or YPplus 2 ethanol with or without 2 aspartic acid in aerobic medium

1289W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

where sit4 mutant cells stain red in the presence of tetrazolium[9] This method has been used widely as a screening test forrespiration deficiency in yeast where the colorless tetrazoliumsalt diffuses into respiring yeast and accepts electrons from themitochondrial electron transport chain reducing it to a pinkcompound known as formazan which imparts a red color toactively respiring cells [33] We have also determined the rate ofrespiration and the rate of fermentation as well as glycogenaccumulation in a second sit4 mutant with another geneticbackground (TS64-1a) and results were the same as shown hereDe-repression of respiration in our experiments was not due toan uncoupling of the mitochondrial electron transport chain asaddition of FCCP or PCIP induced respiration and NADHoxidation

Gluconeogenesis is essential for growth on non-fermentablecarbon sources Intermediate metabolites from gluconeogenesisare used in glycogen and trehalose formation and ribose syn-thesis [23] Our experiments show that in the sit4 mutantgluconeogenesis is active since incorporation of [U-14C] gly-cerol into glycogen was the same as in the wild-type This wasalso confirmed by de novo synthesis of glycogen when galactoseor ethanol was added to glycogen-depleted yeast (Fig 4B) As

failure to grow on respiratory substrates is not due to inhibitionof respiration nor inhibition of gluconeogenesis we postulatedthat it could be related to hyperaccumulation of glycogen and theaccompanying depletion of intermediary metabolites Westudied galactose metabolism where accumulation of glycogenis more pronounced and tried to find a rate-limiting step inethanol production Galactose is metabolized by the Leloir cycle(Fig 7) Glucose 1-phosphate derived from galactose 1-phosphate and UDP-glucose has two main fates formation ofglycogen or entry into glycolysis via isomerization to glucose 6-phosphate However fermentation and glucose 6-phosphatelevels are low in the sit4mutant which indicates that glucose 1-phosphate is directed toward glycogen synthesis This wasclearly confirmed in Fig 4B where we show that glycogensynthesis was activated by galactose but not glucose in sit4mutant cells that had been previously depleted of glycogenHowever one would expect glycogen synthase to be activatedand glycogen phosphorylase to be inhibited On the contrarytotal glycogen synthase activity remains the same as in wild-typecells and phosphorylase is activated In yeast it has been welldocumented that glycogen and trehalose can enter a futile cycleunder different types of stress such as heat salt and oxidative

Fig 7 Galactose metabolism in the sit4 mutant is directed toward glycogensynthesis In sit4mutant glycogen is hyper-accumulated Galactose metabolismis re-directed to a futile cycle of glycogen synthesis and glycogen degradationGalactose-1P and glucose-1P are not hyper-accumulated and glucose-6P levelsare low Arrows indicate the flux in the direction of galactose catabolismHowever all reactions represented are reversible Abbreviations are GKgalactokinase GALT galactose-1-phosphate uridyltransferase GALE UDP-glucose 4 epimerase PGM phosphoglucomutase

1290 W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

stresses [1834] and the mechanism involves activation of eithertrehalase or glycogen phosphorylase Our results indicate thatyeast enters a glycogen futile cycle upon Sit4 inactivationHowever contrary to the response to stress where bothglycogen and trehalose futile cycles are activated deletion ofSIT4 leads to glycogen accumulation but not trehalose accu-mulation Activation of these two futile cycles may be signaledthrough different mechanisms We further studied whetheractivation of glycogen phosphorylase activity in the exponentialphase was due to an up-regulation of transcription Abundanceof GPH1 transcripts was increased 16 fold in the sit4 mutantcompared to wild-type (Fig 3D) which explains activation

Comparison of the expression profile between the wild-typestrain and the sit4 mutant showed that the latter overexpressesgenes that are normally activated by glucose deprivation such asthe glucose transporters The Saccharomyces cerevisiae genomehas 20 different glucose transporters that are regulated byglucose availability [for a review see 35] Of the seven trans-porters known to transport glucose (HXT1-7) sit4 mutant up-regulates HXT2 HXT3 HXT4 and HXT7 In addition a galac-tose transporter (GAL2) and a myo-inositol transporter (ITR1)were up-regulated (Table 4) These results confirm that the sit4strain grown in galactose senses carbohydrate deprivationAmong metabolites that could signal deprivation glucose 6-P isa possible candidate as it is diminished in the sit4 mutant

Sit4 is a downstream target of the TOR kinases [10] and twopoints in this response have not been clarified The first is whe-ther the response to rapamycin an inhibitor of the TOR pathwayis mediated by Sit4 activation or by its inhibition Severalresponses mediated by TOR kinases require Sit4 an example isthe transcription factor GLN3 where TOR kinase promotes the

association of Gln3 with the cytoplasmic Ure2 proteinpreventing transcription of genes that are normally inducedwhen nitrogen is limiting [36ndash38] Sit4 is essential fordephosphorylation of Gln3 and its localization in the nucleus[36ndash38] Our results show that deletion of SIT4 mimicsinhibition of TOR kinases by rapamycin the result is a nutrientdeprivation response and glycogen storage [3940] indicatingthat Sit4 activity controls nutrient sensing especially inrespiratory substrates A second point to be elucidated is theidentification of the specific readouts signaled by TOR kinasesmediated by Sit4 Schmelzle et al [41] have shown that twomain nutrient-responsive pathways in yeast TOR and RAScAMP may intersect and have shown that Gln3 localization andNpr1 phosphorylation are regulated by Tap42Sit4 whileribosome biogenesis HXT1 expression and stress response(MSN) and glycogen are mediated by RAScAMP Our resultsindicate that SIT4 deletion leads to glycogen accumulationwhich suggests that accumulation of glycogen by rapamycininvolves Sit4 inactivation

In summary our results suggest that inhibition of Sit4 activityenhances a glycogen futile cycle in respiratory substrates As aresponse to glycogen accumulation the level of glucose 6-phosphate drops and a nutrient-deprivation response is activatedleading to activation of respiration However cells are unable togrow in ethanol or in anaerobic medium as they lack necessarymetabolites which can be furnished by adding aspartic acid

Acknowledgements

This research was supported by Fundaccedilatildeo Carlos ChagasFilho de Amparo agrave Pesquisa do Estado do Rio de Janeiro(FAPERJ) Conselho Nacional de Desenvolvimento Cientiacuteficoe Tecnoloacutegico-Brasil (CNPq) to Moacutenica Montero-Lomeli MMontero-Lomeliacute and W Jablonka are recipients of fellowshipsfrom CNPq We thank Joseacute Luis Santillaacuten Torres and LorenaChaacutevez Gonzaacutelez from Microarray Unit Instituto de FisiologiaCelular Universidad Nacional Autoacutenoma de Meacutexico We thankSocircnia CF Silva for her technical assistance Maria CeacuteliaBertolini (Instituto de Quiacutemica Universidade Estadual de SatildeoPaulo) for glycogen synthase measurements and Dr AntonioPentildea (Instituto de Fisiologia Celular Universidad NacionalAutoacutenoma de Meacutexico) for his help in measuring glycerolincorporation and NADH We especially thank Dr Martha MSorenson for critical reading of the manuscript

References

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[2] A Sutton D Immanuel KT Arndt The SIT4 protein phosphatasefunctions in late G1 for progression into S phase Mol Cell Biol 11 (1991)2133ndash2148

[3] J Torres CJ Di Como E Herrero MA de la Torre-Ruiz Regulation ofthe cell integrity pathway by rapamycin-sensitive TOR function in buddingyeast J Biol Chem 277 (2003) 43495ndash43504

[4] T Singer S Haefner M Hoffmann M Fischer J Ilyina W Hilt Sit4phosphatase is functionally linked to the ubiquitin-proteasome systemGenetics 164 (2003) 1305ndash1321

1291W Jablonka et al Biochimica et Biophysica Acta 1760 (2006) 1281ndash1291

[5] MM Luke F Della Seta CJ Di Como H Sugimoto R Kobayashi KTArndt The SAP a new family of proteins associate and function positivelywith the SIT4 phosphatase Mol Cell Biol 16 (1996) 2744ndash2755

[6] H Wang Y Jiang The Tap42-protein phosphatase type 2A catalyticsubunit complex is required for cell cycle-dependent distribution of actin inyeast Mol Cell Biol 23 (2003) 3116ndash3125

[7] JR Rohde S Campbell SA Zurita-Martinez NS Cutler M AsheME Cardenas TOR controls transcriptional and translational programsvia Sap-Sit4 protein phosphatase signaling effectors Mol Cell Biol 24(2004) 8332ndash8341

[8] F Posas J Clotet J Arintildeo Saccharomyces cerevisiae gene SIT4 isinvolved in the control of glycogen metabolism FEBS Lett 279 (1991)341ndash345

[9] I Muntildeoz E Simon N Casals J Clotet J Arintildeo Identification ofmulticopy suppressors of cell cycle arrest at the G1-S transition inSaccharomyces cerevisiae Yeast 20 (2003) 157ndash169

[10] JC Di Como KT Arndt Nutrients via the Tor proteins stimulate theassociation of Tap42 with type 2A phosphatases Genes Dev 10 (1996)1904ndash1916

[11] WA Wilson Z Wang PJ Roach Systematic identification of the genesaffecting glycogen storage in the yeast Saccharomyces cerevisiaeimplication of the vacuole as a determinant of glycogen level Mol CellProteomics I (2002) 232ndash242

[12] E Jacinto B Guo KT Arndt T Schmelzle M Hall TIP41 interacts withTAP42 and negatively regulates the TOR signaling pathway Mol Cell8 (2001) 1017ndash1026

[13] A Wach A Brachat R Pohlmann P Philippsen New heterologousmodules for classical or PCR-based gene disruptions in Saccharomycescerevisiae Yeast 10 (1994) 1793ndash1808

[14] ME Schmitt TA Brown BL Trumpower A rapid and simple methodfor preparation of RNA from Saccharomyces cerevisiae Nucleic Acid Res18 (1990) 3091ndash3092

[15] DG Ginzinger TE Godfrey J Nigro DH Moore II S Suzuki MGPallavicini JW Gray RH Jensen Measurement of DNA copy number atmicrosatellite loci using quantitative PCR analysis Cancer Res 60 (2000)5405ndash5409

[16] CA Masuda MA Xavier KA Mattos A Galina M Montero-LomeliPhosphoglucomutase is an in vivo lithium target in yeast J Biol Chem276 (2001) 37794ndash37801

[17] B Gonzalez J Francois M Renaud A rapid and reliable method formetabolite extraction in yeast using boiling buffered ethanol Yeast 13(1997) 1347ndash1355

[18] HU Bergmeyer Methods of Enzymatic Analysis vols I II VerlagBasel 1983

[19] JL Parrou J Francois A simplified procedure for a rapid and reliableassay of both glycogen and trehalose in whole yeast cells Anal Biochem248 (1977) 186ndash188

[20] J Francois ME Villanueva HG Hers The control of glycogenmetabolism in yeast 1 Interconversion in vivo of glycogen synthaseand glycogen phosphorylase induced by glucose a nitrogen source oruncouplers Eur J Biochem 174 (1988) 551ndash559

[21] E Helmreich CF Cori The role of adenylic acid in the activation ofphosphorylase Proc Natl Acad Sci U S A 51 (1964) 131ndash138

[22] FA Hommes Oscillatory reductions of pyridine nucleotides during

anaerobic glycolysis in brewers yeast Arch Biochem Biophys 108(1964) 36ndash46

[23] R Dickinson Carbon metabolism in JR Dickinson M Schweizer(Eds) The Metabolism and Molecular Physiology of Saccharomycescerevisiae Taylor and Francis Ltd 1999 pp 23ndash55

[24] LM Steinmetz et al Systematic screen for human disease genes in yeastNat Genet 31 (2002) 400ndash404

[25] LM Steinmetz et al Systematic screen for human disease genes in yeastsupplemental information (2003) inhttpwwwyeastgenomeorg

[26] HT Ni DC LaPorte Response of a yeast glycogen synthase gene tostress Mol Microbiol 16 (1995) 1197ndash1205

[27] J Francois JL Parrou Reserve carbohydrates metabolism in theyeast Saccharomyces cerevisiae FEMS Microbiol Rev 25 (2001)125ndash145

[28] KP Huang E Cabib Separation of the glucose-6-phosphate independentand dependent forms of glycogen synthetase from yeast BiochemBiophys Res Commun 49 (1972) 1610ndash1616

[29] D Huang WA Wilson PJ Roach Glucose-6-P control of glycogensynthase phosphorylation in yeast J Biol Chem 272 (1977)22495ndash22501

[30] BA Pedersen WA Wilson PJ Roach Glycogen synthase sensitivity toglucose-6-P is important for controlling glycogen accumulation inSaccharomyces cerevisiae J Biol Chem 279 (2004) 13764ndash13768

[31] LF Leloir The enzymatic transformation of uridine diphosphate glucoseinto a galactose derivative Arch Biochem 33 (1951) 186ndash190

[32] NK Brewster DL Val ME Walker JC Wallace Regulation ofpyruvate carboxylase isozyme (PYC1 PYC2) gene expression inSaccharomyces cerevisiae during fermentative and nonfermentativegrowth Arch Biochem Biophys 311 (1994) 62ndash71

[33] M Ogur R St John S Nagai Tetrazolium overlay technique forpopulation studies of respiration deficiency in yeast Science 125 (1957)928ndash929

[34] T Hottiger P Schmutz A Wiemken Heat-induced accumulation andfutile cycling of trehalose in Saccharomyces cerevisiae J Bacteriol 169(1987) 518ndash522

[35] S Ozcan M Johnston Function and regulation of yeast hexosetransporters Microbiol Mol Biol Rev 63 (1999) 554ndash569

[36] T Beck MN Hall The TOR signalling pathway controls nuclearlocalization of nutrient-regulated transcription factors Nature 402 (1999)689ndash692

[37] ME Cardenas NS Cutler MC Lorenz CJ Di Como J Heitman TheTOR signaling cascade regulates gene expression in response to nutrientsGenes Dev 13 (1999) 3271ndash3279

[38] PG Bertram JH Choi J Carvalho W Ai C Zeng TF Chan XFZheng Tripartite regulation of Gln3p by TOR Ure2p and phosphatasesJ Biol Chem 275 (2000) 35727ndash35733

[39] J Rohde J Heitman ME Cardenas The TOR kinases link nutrientsensing to cell growth J Biol Chem 276 (2001) 9583ndash9586

[40] JL Crespo MN Hall Elucidating TOR signaling and rapamycin actionlessons from Saccharomyces cerevisiae Microbiol Mol Biol Rev 66(2002) 579ndash591

[41] T Schmelzle T Beck DE Martin MN Hall Activation of the RAScyclic AMP pathway suppresses a TOR deficiency in yeast Mol CellBiol 24 (2004) 338ndash351