Glucose-sensing and -Signalling Mechanisms in Yeast

MiniReview

Glucose-sensing and -signalling mechanisms in yeast

Filip Rolland 1, Joris Winderickx, Johan M. Thevelein �

Laboratorium voor Moleculaire Celbiologie, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31,B-3001 Leuven-Heverlee, Flanders, Belgium

Received 20 September 2001; received in revised form 14 January 2002; accepted 28 January 2002

First published online 12 March 2002

Abstract

Glucose has dramatic effects on the regulation of carbon metabolism and on many other properties of yeast cells. Several sensing andsignalling pathways are involved. For many years attention has focussed on the main glucose-repression pathway which is responsible forthe downregulation of respiration, gluconeogenesis and the transport and catabolic capacity of alternative sugars during growth onglucose. The hexokinase 2- dependent glucose-sensing mechanism of this pathway is not well understood but the downstream part of thepathway has been elucidated in great detail. Two putative glucose sensors, the Snf3 and Rgt2 non-transporting glucose carrier homologs,control the expression of many functional glucose carriers. Recently, several new components of this glucose-induction pathway have beenidentified. The Ras-cAMP pathway controls a wide variety of cellular properties in correlation with cellular proliferation. Glucose is apotent activator of cAMP synthesis. In this case glucose sensing is carried out by two systems, a G-protein-coupled receptor system and astill elusive glucose-phosphorylation-dependent system. The understanding of glucose sensing and signalling in yeast has made dramaticadvances in recent years and has become a strong paradigm for the elucidation of nutrient-sensing mechanisms in other eukaryoticorganisms. - 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Glucose sensing; Signal transduction; G-protein-coupled receptor; cAMP; Saccharomyces cerevisiae

1. Introduction

In the free-living micro-organisms’ constantly changingenvironment nutrient availability is the major factor con-trolling growth and development. For yeasts, like formany other micro-organisms, glucose is the preferred car-bon and energy source. It is therefore not surprising thatglucose is an important primary messenger molecule, sig-nalling optimal growth conditions to the cellular machin-ery. Accordingly, glucose also a¡ects many of the yeasts’commercially important traits such as growth rate, fer-mentation capacity and stress resistance. Together withits genetic amenability as a unicellular eukaryote, thishas stimulated the thorough characterization of a varietyof glucose-signalling pathways in Saccharomyces cerevisi-ae. Whereas downstream components and their function-

ing have often been clari¢ed in great detail, elucidation ofthe initial glucose-sensing and -activation mechanisms hasproven to be more di⁄cult. This is largely due to thesugars’ apostrophe dual function as a nutrient and signal-ling molecule, and the intertwining of the molecular basisof the two functions. Recently, however, substantial prog-ress has been made with the identi¢cation of several pro-teins with an apparently speci¢c function in glucose sens-ing. In higher multicellular organisms similar mechanismsmight be involved in the vital control of glucose homeo-stasis.

2. Stationary phase, respiration and fermentation

Unicellular free-living organisms like yeasts in generalhave adapted very well to constantly changing environ-mental conditions. More speci¢cally, they have developedmechanisms to respond to extreme variations in nutrientavailability by modulating their growth and metabolism.The most dramatic e¡ect in micro-organisms is observedupon nutrient starvation. Micro-organisms are able to sur-vive long periods of starvation by drastically decreasingtheir metabolic activity upon growth and cell cycle arrest,

1567-1356 / 02 / $22.00 - 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 0 4 6 - 6

* Corresponding author. Tel. : +32 (16) 321507; +32 (16) 321500(secr.) ; Fax: +32 (16) 321979.

E-mail address: [email protected] (J.M. Thevelein).

1 Present address: Department of Genetics, Harvard Medical Schooland Department of Molecular Biology, Massachusetts General Hospital,Boston, MA 02114, USA.

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www.fems-microbiology.org

combined with a range of physiological and often alsomorphological changes. These ‘stationary-phase cells’ arealso characterized by their high tolerance to heat and oth-er stress conditions and to cell wall-degrading enzymes. Awide variety of genes involved in stress resistance is in-duced and the reserve carbohydrate glycogen as well asthe stress protectant and reserve carbohydrate trehaloseaccumulate to high levels. Although yeasts are basicallyunicellular fungi, nutrient limitation can also cause a dras-tic morphogenetic switch in diploid cells, resulting in pseu-dohyphal growth. This morphology resembles that of the¢lamentous fungi and reminds of the yeasts’ derivationfrom multicellular ancestors. Such ¢lamentous growth oc-curs for instance when fermentable sugars are availablebut nitrogen is lacking, presumably enabling the yeast toactively search for a nitrogen source.Yeast cells are not only able to detect the mere presence

or absence of nutrients, depending on the carbon sourceavailable, they display totally di¡erent metabolic modes.Glucose-sensitive yeasts like S. cerevisiae and Schizosac-charomyces pombe prefer fermentation over respirationeven under aerobic conditions. In these yeasts, synthesisof key enzymes of respiratory sugar dissimilation is re-pressed by the ample presence of rapidly-fermentable sug-ars, such as glucose or fructose. Although, per mole ofsugar, alcoholic fermentation yields fewer ATP equivalentsthan respiration, it can proceed at much higher rates. Thisenables these yeasts to compete e¡ectively for survival,especially because the ethanol produced during fermenta-tion inhibits growth of competing micro-organisms. Thisethanol can subsequently aerobically be used as a non-fermentable carbon source resulting in a complete use ofall available carbon. In the presence of oxygen, cells areable to respire and generate ATP from non-fermentablecarbon sources by mitochondrial oxidative phosphoryla-tion. Cells that use non-fermentable carbon sources growmuch slower than fermenting cells. In addition, they dis-play several features which are similar to those of station-ary-phase cells, such as high expression levels of genesinvolved in stress resistance and accumulation of reservecarbohydrates.The addition of glucose to cells growing on non-fer-

mentable carbon sources or to stationary-phase cells trig-gers a wide variety of regulatory processes directed to-wards the exclusive and optimal utilization of thepreferred carbon source. Glycolysis is activated and glu-cose is almost completely converted into ethanol and car-bon dioxide. While glucose in£ux and the £ow throughglycolysis are stimulated, gluconeogenesis is inhibited. Inaddition, there is a drastic increase in growth rate which ispreceded by a characteristic upshift in ribosomal RNAand protein synthesis. Genes encoding enzymes involvedin the uptake and metabolization of alternative carbonsources and gene products involved in stress resistanceare repressed. Reserve carbohydrates are mobilized.Yeast cells use both positive and negative control mech-

anisms to regulate enzyme levels and activities in order toaccomplish this drastic metabolic switch. Enzyme levelsare regulated at the stage of gene transcription (repressionand induction), mRNA stability, translation and proteinstability, while enzyme activities are regulated post-tran-scriptionally by allosteric and covalent activation and in-hibition. Most of these processes are a¡ected either di-rectly or indirectly by speci¢c glucose sensing and signaltransduction pathways.

3. Glucose-signalling pathways

The major downregulating e¡ect of glucose takes placeat the transcriptional level. One class of genes repressed byglucose encodes proteins involved in respiration (Krebscycle and electron transport chain proteins), gluconeogen-esis and the glyoxylate cycle. Another important class en-codes proteins that are speci¢cally involved in the uptakeand metabolization steps of alternative carbon sources,such as the GAL, SUC and MAL genes and genes in-volved in utilization of ethanol, lactate and glycerol.Also, high-a⁄nity glucose transport is repressed by highlevels of glucose. Several families of genes involved in theuse of other carbon sources are under control of family-speci¢c inducers enabling a co-ordinated regulation oftheir expression. In the presence of glucose, the family-speci¢c inducers as well as the individual genes are subjectto repression. Finally, also a large group of STRE (stressresponse element)-controlled genes encoding proteins pri-marily involved in the yeasts’ response to various stressesare repressed by glucose.

3.1. Glucose repression by the main glucose-repressionpathway

Not all glucose-repressible genes are repressed in thesame way but isolation and characterization of repressionand derepression mutants has identi¢ed a general glucose-repression machinery involved in the regulation of expres-sion of a large number of glucose-repressed genes. As il-lustrated in Fig. 1A, its central components are the Mig1transcriptional repressor complex, the Snf1-protein kinasecomplex and protein phosphatase 1.Mig1 is a DNA-binding zinc-¢nger protein that recruits

the general co-repressor proteins Ssn6 and Tup1 to exertrepression of diverse gene families and their family-speci¢ctranscriptional inducer genes [1]. Essential for the functionof Mig1 in glucose repression is its glucose-regulated sub-cellular localization. In the presence of high levels of glu-cose, Mig1 rapidly moves into the nucleus, where it bindsto the promoters of glucose-repressible genes. When thecells are deprived of glucose, Mig1 is rapidly transportedback to the cytoplasm [2]. In addition to Mig1 otherDNA-binding proteins (such as its homolog Mig2) areinvolved in glucose repression.

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Transcription of the glucose-repressible genes in dere-pressing conditions is dependent on the Snf1-protein ki-nase complex. In the absence of glucose Snf1 probablyphosphorylates and thereby causes translocation of Mig1to the cytoplasm [2^4]. The Snf1 Ser/Thr kinase is associ-ated with an activating subunit and three sca¡olding pro-teins in high-molecular-mass complexes. The activatingsubunit Snf4 (Cat3) is required for Snf1 activity [5,6],

while the Sip1, Sip2 and Gal83 proteins maintain associa-tion of Snf4 with the Snf1 kinase [7] and confer speci¢cityto the kinase complex [8], possibly through regulation ofits subcellular localization [9].Snf1 kinase activity is inhibited by glucose and stimu-

lated when glucose is limiting [6,10]. Activation of the ki-nase in response to glucose limitation is apparently accom-panied by a conformational change of the kinase complex[11] (Fig. 1B). According to the model derived from theobserved alterations in protein interactions within thecomplex, the Snf1 regulatory domain auto-inhibits the cat-alytic domain in glucose-grown cells. In the absence ofglucose, however, the Snf4-activating subunit binds tothe Snf1 regulatory domain, counteracting the auto-inhib-itory interaction and thereby enabling Mig1 phosphoryla-tion and its translocation to the cytoplasm [5]. The glucosesignal apparently regulates (inhibits) the Snf1^Snf4 inter-action, thereby stimulating auto-inhibition of the kinase.The kinase is then unable to inhibit Mig1-mediated repres-sion [11]. Snf1 has also been shown to regulate activity aswell as (Mig1-dependent) expression of the two zinc-clus-ter-activator proteins Cat8 and Sip4 which are involved inthe induction of gluconeogenic genes through carbonsource-responsive promoter elements [8,12^14].Snf1 kinase activity itself also appears to be regulated

by phosphorylation and dephosphorylation. Several ex-periments suggest the existence of a protein kinase thatactivates Snf1 by phosphorylating a conserved Thr kinasephosphorylation site in the activation loop [6,10,15,16].Protein phosphatase 1 (Glc7) has been shown to act an-tagonistically to Snf1 in glucose repression. This phospha-tase is involved in the control of a variety of processes andits glucose-repression-speci¢c regulatory subunit Reg1/Hex2 targets its activity to the activated Snf1 kinase do-main, presumably dephosphorylating Snf1 or anothercomponent of the complex and facilitating the return tothe auto-inhibited state [16^18]. Hence, although the glu-cose signal most likely inhibits the initial phosphorylationof Snf1, it may also activate Reg1-Glc7 phosphatase 1function.

3.2. Glucose induction

Yeast cells growing on glucose obtain their energymainly through fermentation. Since fermentation is a rel-atively ine⁄cient way of generating energy, a high glyco-lytic £ux is essential. Yeast cells are able to increase theirglycolytic capacity by the induction of a large number ofglycolytic genes. In addition, glucose-uptake capacity isincreased through the induction of several glucose-trans-porter-encoding HXT genes. Separate signal transductionpathways and mechanisms seem to be involved.In the presence of rapidly fermentable sugars yeast gly-

colysis is fully activated. Glucose causes a fast increaseand transient overshoot in glycolytic intermediates andmutant studies have shown that increased levels of di¡er-

Fig. 1. The main glucose-repression pathway. A: Simpli¢ed schematicrepresentation of mediators and targets of the main catabolite-repressionpathway. Repression is exerted by the complex Mig1/Ssn6/Tup1 on dif-ferent gene families including family-speci¢c transcriptional activatorssuch as Gal4 (galactose utilization), MalR (maltose utilization), Hap4(respiratory genes) and Cat8 (gluconeogenic genes). The Snf1 kinase as-sociated with one of the regulatory subunits Sip1, Sip2 or Gal83 andthe activating subunit Snf4 has a negative e¡ect on the activity of therepression complex. During growth on glucose, Snf1 activity is inhibitedby di¡erent upstream regulators which include the hexose kinases andthe Glc7 phosphatase. The Snf1 kinase complex is also required for acti-vation of Sip4 which is required in concert with Cat8 for the derepres-sion of the gluconeogenic genes. B: Glucose-induced conformationalchange of the Snf1-protein kinase complex.

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ent metabolites trigger the induction of glycolytic genes[19]. Di¡erent glycolytic intermediates seem to act as sig-nalling molecules or ‘metabolic messengers’ to adapt gly-colytic activity to the presence of varying amounts of sug-ars in a very complex but highly controlled and e⁄cientway. How these metabolic signals are transmitted is stillunclear but in several genes sequence elements have beende¢ned that are responsible for sugar-induced expressionand di¡erent DNA-binding factors have been identi¢edthat are required for high-level expression of glycolyticgenes. The Gcr1 protein, for instance, seems to be of cen-tral importance for the coordinated regulation of glyco-lytic gene expression. It is a trans-acting positive regulatorof transcription that binds to the CTTCC motif which isconserved in most glycolytic genes [20,21]. The glycolyticpathway is also subject to extensive post-translational al-losteric and covalent regulation. An increase in the glu-cose-6-P level, for example, also triggers rapid activationof 6-phosphofructo-2-kinase (PF2K) which catalyzes syn-thesis of fructose-2,6-bisP, one of the allosteric regulatorsin glycolysis [19]. In addition, rapid inactivation of gluco-neogenesis is required for an e⁄cient start-up of glycolysis.

S. cerevisiae contains a whole series of hexose transport-ers homologues (Hxt1-17, Gal2, Snf3 and Rgt2), all dis-playing di¡erent substrate a⁄nities and expression pat-terns [22^24]. Depending on the amount of glucosepresent in the medium, speci¢c transporters are expressed.The mechanisms involved in the expression of the appro-priate transporters and their post-translational modi¢ca-tion have recently become more clear. High-a⁄nity trans-porters like Hxt6 and Hxt7 are highly expressed on non-fermentable carbon-sources and repressed by high levels ofglucose, whereas transporters with low a⁄nity, such asHxt1 and Hxt3, are induced by the presence of a highconcentration of glucose. The transporters with intermedi-ate a⁄nity for glucose like Hxt2 and Hxt4, on the otherhand, are induced by low levels of glucose and repressedby high levels of glucose. As shown in Fig. 2, both theintermediate and the low-a⁄nity transporters are re-pressed by Rgt1 in the absence of glucose. Rgt1 is azinc-¢nger-containing DNA-binding protein that, likeMig1, recruits the Ssn6 repressor to the promoters of spe-ci¢c genes [25]. Low amounts of glucose inhibit Rgt1-re-pressor function, resulting in derepression of HXT expres-sion. This inhibition requires the presence of the Grr1protein [26]. This protein is part of a multiprotein SCFcomplex containing the Skp1, Cdc53 and Cdc34 proteinsand the F-box Grr1 protein [27^29]. SCF complexes directprotein ubiquitination and di¡er in their F-box-containingcomponent which is thought to recruit speci¢c substratesto the complex. Subsequent ubiquitination then ‘marks’the substrate for degradation [30]. Glucose derepressionapparently involves ubiquitin-mediated proteolysis but itis not clear whether the SCF complex directly modi¢esRgt1. The HXT2 and HXT4 genes which encode trans-porters with intermediate a⁄nity for glucose are repressed

by high glucose levels. This repression is mediated by theMig1 main glucose-repression pathway [26]. Also, repres-sion of the high-a⁄nity transporter HXT6 is, at least inpart, mediated by the main glucose-repression pathway[24]. However, in contrast to other glucose-repressedgenes, maintenance of HXT6 repression is strictly depen-dent on Snf3 [31]. Expression of HXT1, encoding a low-a⁄nity transporter is further induced by high glucose lev-els. Besides the Grr1-Rgt1-dependent pathway, this alsoinvolves another mechanism, that shares some compo-nents with the main glucose-repression pathway [26].This induction is independent of Rgt1 and apparently re-quires a yet unidenti¢ed transcriptional activator or, alter-natively, an additional Ssn6-dependent repression mecha-nism that is inactivated by high levels of glucose. Fullinduction of HXT1 expression at high glucose concentra-tions, however, does require Rgt1. Rgt1 apparently can beconverted into an activator of HXT1 expression underthese conditions. Interestingly, Grr1 is required for bothlow-glucose-induced inactivation and high-glucose-in-duced conversion of Rgt1 [25]. In addition to glucose-con-centration-dependent induction and repression, glucosetransport is also subject to extensive post-translationalregulation [24].

3.3. The Ras-cAMP pathway

A major glucose-signalling pathway involved in post-translational regulation by phosphorylation is the Ras-cAMP pathway (Fig. 3A). Synthesis of cAMP from ATPis catalyzed by the enzyme adenylate cyclase and cAMPactivates cAMP-dependent protein kinase A (PKA) bybinding to its regulatory subunits (encoded by BCY1),thereby releasing and activating the catalytic protein ki-nase subunits (encoded by TPK1, TPK2 and TPK3). Inderepressed yeast cells (growing on a non-fermentable car-bon source or in stationary phase) rapidly-fermentablesugars, and especially glucose, trigger a rapid, transientincrease in the cAMP level, initiating a PKA phosphory-lation cascade. Also, intracellular acidi¢cation is able totrigger a pronounced increase in the cAMP level. Like inhigher eukaryotes, yeast adenylate cyclase activity is con-trolled by G-proteins. Remarkably, in S. cerevisiae thetwo small G-proteins Ras1 and Ras2 are essential for ade-nylate cyclase activity. They therefore have been thoughtfor many years to act as functional equivalents of themammalian heterotrimeric GK-proteins of adenylate cy-clase. Recently, however, a G-protein-coupled receptor(GPCR) system has been identi¢ed that speci¢cally con-trols glucose-induced activation of cAMP synthesis.In S. cerevisiae cAMP signalling plays a central role in

the control of metabolism, stress resistance and prolifera-tion. Translational control of Cln3 synthesis by PKA hasbeen proposed as a link between nutrient availability andcell cycle control [32,33]. Indeed, several phenotypic prop-erties controlled by PKA are indicative of high PKA ac-

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tivity during fast growth on glucose and low activity dur-ing growth on non-fermentable carbon sources and in sta-tionary phase [34,35]. However, there is no clear correla-tion with basal cAMP levels. Moreover, glucose-inducedactivation of adenylate cyclase is repressed by glucose andtherefore considered not to be operative during growth onglucose. This appears to con¢ne the physiological role ofthis pathway to the short period of transition from thederepressed state to the repressed state by means of acAMP-triggered protein phosphorylation cascade. Mostof the downstream targets of PKA identi¢ed are enzymesinvolved in intermediary metabolism and carbon metabo-lism in particular, consistent with a role for cAMP signal-ling in stimulation of fermentation. In addition to activa-tion of enzymes involved in energy metabolism, glucose-induced activation of protein synthesis through PKA-de-pendent induction of ribosomal protein genes stimulatesgrowth and proliferation [36]. In their natural environ-ment, yeast cells experience long periods of nutrient star-vation, alternating with very short periods of nutrientabundance. Under such conditions, fast recovery from sta-tionary phase and initiation of fermentation clearly o¡er aselective advantage [35].Initiation of fermentation also coincides with a loss of

stress resistance. Two multicopy suppressors of the snf1defect, Msn2 and Msn4, appear to mediate glucose repres-sion of stress resistance by the cAMP-PKA pathway.These zinc-¢nger proteins act as positive transcription fac-tors in the general stress-response pathway by binding toSTREs in the promoters of stress-regulated genes [37,38].

Nuclear localization of Msn2 and Msn4 is regulated an-tagonistically by stress conditions and PKA activity [39].Consistently, a large number of STRE-controlled geneswhich are dependent on Msn2 and Msn4 for inductionupon sugar depletion was found to be repressed bycAMP [40,41]. Moreover, PKA activity was shown to bedispensable in a strain lacking Msn2 and Msn4, indicatingthat Msn2/4-dependent gene expression actually accountsfor many of the pleiotropic e¡ects of PKA. PKA appar-ently regulates processes such as glycogen accumulationand stress response as well as growth by suppression ofMsn2/4-gene expression [42]. Interestingly, also the rapa-mycin-sensitive TOR-signalling pathway was shown to in-hibit expression of carbon-source-regulated genes by se-questration of Msn2 and Msn4 in the cytoplasm [43].The central role of the cAMP-PKA pathway in the controlof stress resistance is supported by the isolation of mu-tants de¢cient in fermentation-induced loss of stress resis-tance (¢l). The ¢l1 mutant carries a point mutation in theCYR1/CDC35 gene [44], encoding adenylate cyclase, con-sistent with the previous isolation of stress-resistant ade-nylate cyclase mutants [45]. Interestingly, the ¢l1 mutantstill displays wild-type growth and fermentation rates, asopposed to other mutants with reduced activity of thecAMP pathway. The ¢l2 mutation was identi¢ed in thegene encoding the GPCR Gpr1, which is speci¢cally in-volved in glucose activation of cAMP synthesis [46]. In-terestingly, a positive correlation was reported betweenactivity of the PKA pathway and longevity [47].The observation that cAMP synthesis is apparently only

Fig. 2. Regulation of HXT transporter gene expression in response to glucose. In the absence of glucose, Rgt1-represses transcription of HXT1-4. Lowamounts of glucose inhibit the Rgt1-repressing activity, a process triggered by Snf3 via Grr1-mediated ubiquitination. At high concentrations of glucose,Rgt2 triggers HXT1 expression. This involves Grr1-dependent conversion of Rgt1 into a transcriptional activator and another mechanism in which sev-eral components of the main glucose-repression pathway are involved. The Snf3- and Rgt2-mediated derepression of the HXT genes also involves se-questering at the plasma membrane of the transcriptional repressors Mth1 and Std1. At high glucose concentrations HXT2, HXT4, HXT6 and SNF3are repressed by Mig1 via the main glucose-repression pathway. In addition, Snf3 is involved in a second pathway leading to the high-glucose-inducedrepression of HXT6.

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activated by rapidly-fermentable sugars and not by othernutrients, and the glucose-repressible character of the ac-tivation mechanism, seem to argue against a role forcAMP-signalling in growth control by nutrients. Bcy1 v

mutants with attenuated catalytic subunits have indeed

been shown to respond appropriately to nutritional stressconditions, even in the absence of adenylate cyclase [48].This indicates that cAMP-independent mechanisms existfor regulation of these responses. Interestingly, in the pres-ence of glucose, other essential nutrients (such as N, S or P

Fig. 3. Control of PKA activity in yeast. A: Activation of the cAMP pathway occurs when glucose is added to cells growing on non-fermentable car-bon sources or to stationary phase cells. Glucose is detected via a dual sensing process: an intracellular glucose-sensing process involving the hexose ki-nases following transport of the glucose, and the extracellular glucose detection system involving the Gpr1^Gpa2 GPCR system. How the glucose signalis transmitted to adenylate cyclase is still unknown but a possible involvement of the Ras proteins and their regulators Cdc25 and the Ira proteins can-not be excluded. B: The FGM pathway integrates the availability of di¡erent nutrients including the fermentable carbon source. It supports mainte-nance of high PKA activity during growth on glucose via a cAMP-independent signalling cascade that involves the Sch9-protein kinase. In contrast tothe cAMP pathway the intra- and extracellular glucose-sensing process is apparently able to sustain activation of the pathway separately. Detection ofother nutrients seems to be triggered by speci¢c transporters such as Gap1 for amino acids and Mep2 for ammonium.

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sources) are able to trigger similar e¡ects on the PKAtargets when re-added to cells starved for such a nutrient.These e¡ects are independent of activation of cAMP syn-thesis but are still dependent on the free catalytic Tpksubunits (Fig. 3B). Since these e¡ects require the presenceof both a fermentable carbon source and a completegrowth medium for sustained activation, the signallingpathway involved has been called the ‘fermentable-growth-medium-induced (FGM) pathway’ [34,49]. TheFGM pathway controls PKA targets during growth onglucose through the Sch9-protein kinase [50].Often di¡erent mechanisms and signal transduction

pathways collaborate to control enzyme levels and activ-ities. The extreme glucose sensitivity of the gluconeogenicenzymes for example is mediated by glucose-induced allo-steric inhibition, covalent modi¢cation and protein degra-dation [51^53] as well as transcriptional repression andaccelerated mRNA degradation [54^56]. The combinationof these mechanisms ensures the rapid decrease in gluco-neogenic enzyme levels when yeast cells switch to glyco-lytic metabolism. Also, in the case of enzymes and perme-ases involved in the metabolism of alternative carbonsources, such as maltose and galactose, repression ofgene expression is preceded by rapid glucose-induced in-activation and degradation [57].

4. Glucose-sensing mechanisms

The dramatic e¡ects of glucose on growth and metabo-lism clearly support a hormone-like function for this sugarin yeast cells. However, since it is also taken up and me-

tabolized as a nutrient, glucose can be detected by the cellsin many more ways than is the case for classical primarymessenger molecules [58]. Although cells could use theactivity of a component of the existing metabolic machin-ery or the level of one or more glucose catabolites todetect its presence (and metabolization), the lack of spec-i¢city of such a system could have stimulated the develop-ment of more speci¢c sensors as illustrated in Fig. 4. Re-ceptors could have evolved from existing glucose-bindingproteins such as transporters or kinases (with or withoutmaintenance of the catalytic activity) or members of moreclassical receptor families could have been recruited andmodi¢ed (or used originally) to gather speci¢c informationon the nutritional status in the environment.There is now much evidence that yeast uses a whole

range of such sensing mechanisms to ¢ne-tune growthand metabolic activity to the amount and quality of thesugars available. For genes encoding glycolytic enzymesand requiring glucose for full expression, induction byglucose was shown to depend on the accumulation of in-termediary metabolites. For some genes, an increase in thelevel of hexose-6-phosphates is required while for othersinduction is triggered by glycolytic three-carbon metabo-lites [19,59,60]. Also, for glucose sensing and signalling inpancreatic L-cells a more extensive metabolization of thesugar is required since it appears that the actual trigger forinsulin release is the ATP produced in glycolysis and res-piration. An increase in the ATP:ADP ratio inhibits ATP-sensitive Kþ-channels. Membrane depolarization then ac-tivates voltage-gated Ca2þ-channels, triggering a rise inintracellular Ca2þ which stimulates fusion of insulin stor-age vesicles with the plasma membrane. Many glucose-

Fig. 4. Possible mechanisms for glucose sensing. Glucose can be detected by speci¢c glucose receptors in the plasma membrane (a), by an active glucosetransporter (b) or transporter homologs that developed into a glucose sensor (c). When the glucose-sensing mechanism is dependent on metabolism thesensor can be a hexose kinase homolog that developed into a regulatory protein with weak or no catalytic activity (d) or an active glucose-phosphory-lating enzyme in which the catalytic and regulatory functions are closely related (e). Finally, the glucose signal can be a metabolic messenger (f), eitherglucose-6-phosphate or a downstream metabolite.

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induced e¡ects studied in yeast, however, require onlypartial metabolization of the sugar. This makes it possibleto distinguish clearly between the regulatory function ofglucose and its nutrient function: most glucose-inducedsignal transduction pathways apparently require no me-tabolization beyond the sugar kinase step for their activa-tion. This also points at a central role for transport and/orphosphorylation in the sensing process. The use of non- orpartially-metabolizable sugar analogues is a very usefultool to determine to which extent the sugar has to bemetabolized to trigger a response.Interestingly, sugar transport and phosphorylation

themselves are subject to complex regulation by glucosethrough di¡erent signal transduction mechanisms. Re-cently, research on the regulation of expression of glucosetransporter genes has contributed signi¢cantly to ourunderstanding of the glucose-sensing process. Two trans-porter homologs, Rgt2 and Snf3, have been proposed tofunction as sensors or receptors of extracellular glucose forinduction of HXT expression. Research on the glucose-sensing mechanisms involved in catabolite repression, onthe other hand, has focussed from the very beginning onthe involvement of the sugar kinases and more speci¢callyon a predominant role for hexokinase 2 (Hxk2) as an‘intracellular’ sensor. Finally, work in our laboratory hasrevealed the involvement of a GPCR system in concertwith a glucose phosphorylation-dependent mechanismfor glucose-induced activation of cAMP-synthesis.

4.1. The Rgt2 and Snf3 glucose sensors

Rgt2 and Snf3 are two unusual members of the hexosetransporter family. They have only limited sequence sim-ilarities to the other hexose transporter homologs and pos-sess long C-terminal cytoplasmic tails. The SNF3 gene wasoriginally identi¢ed in a screen for mutants de¢cient in theutilization of the trisaccharide ra⁄nose [61,62], based onthe inability to derepress the invertase-encoding SUC2gene. Snf3 (sucrose non-fermenting) mutants in additionare unable to grow fermentatively on low concentrationsof glucose or fructose and kinetic analysis showed that theSNF3 gene is required for high-a⁄nity glucose transport[63]. Sequence homology with mammalian glucose trans-porters [64] supported the idea of a function as high-a⁄n-ity glucose transporter. The RGT2 gene was cloned as adominant mutant allele (RGT2-1) that bypasses the re-quirement of Snf3 for growth on low concentrations ofglucose by restoring high-a⁄nity transport [65,66]. Morerecent results indicate that Snf3 and Rgt2 do not directlysupport catabolic sugar transport [31,67] but rather act asextracellular glucose sensors, involved in the regulation ofexpression of catabolic hexose transporter genes (Fig. 2).Snf3 was found to be required for induction of transcrip-tion of the HXT2, HXT3 and HXT4 genes by low levels ofglucose, suggesting that snf3 mutants are defective in high-a⁄nity transport because of de¢cient expression of the

high-a⁄nity transporter-encoding genes [26]. Rgt2 is re-quired for maximal induction of HXT1 expression byhigh concentrations of glucose [66]. Interestingly, a dom-inant mutation in RGT2 was identi¢ed that causes consti-tutive glucose-independent expression of the HXT1 gene[66]. The mutation results in the substitution of arginine231 into lysine. This residue is located at the start of the¢fth cytoplasmic loop of the protein in a highly conservedregion within the transporter superfamily. When intro-duced in SNF3 (Arg229Lys), the mutation causes similare¡ects, resulting in constitutive expression of HXT2. Theseresults suggest that Rgt2 senses high extracellular glucoseconcentrations, while Snf3 senses low glucose concentra-tions. The fact that RGT2 is expressed constitutively at alow level while SNF3 is glucose-repressed is also consistentwith a role as respectively low- and high-a⁄nity glucosesensors, although the observation that the dominant mu-tant RGT2-1 allele restores high-a⁄nity glucose uptake ina snf3 mutant strain suggests some overlap in the targetgenes. To date, little is known about the actual Snf3 andRgt2 glucose-sensing mechanism and the nature of thesignal they transmit.The molecular structure of Rgt2 and Snf3 is distinct

from that of most other glucose-transporter proteins, es-pecially in the long carboxy-terminal extension that is be-lieved to be located in the cytoplasm [64]. Deletion anal-ysis of Snf3 showed that this carboxy-terminal extension isindeed required for Snf3-dependent expression of the high-a⁄nity transporter genes. The Snf3 and Rgt2 C-terminaltails are relatively dissimilar except for a sequence motifthat occurs twice in the Snf3 tail and once in the tail ofRgt2. This motif is essential for their signalling function[68^70]. Although the dominant mutations suggest the in-volvement of the conserved arginine residue in the ¢fthcytoplasmic loop in the glucose-sensing process, the car-boxy-terminal tails indeed appear to be the actual signal-ling domains. Transplantation of the Snf3-tail onto theHxt1 and Hxt2 glucose transporters was suggested to con-vert these transporters into functional glucose sensors ableto generate the signal for glucose-induced HXT gene ex-pression [68]. Moreover, even when expressed as a solubleprotein, the cytoplasmic Snf3 tail was still found to signal[70,71]. This obviously has important implications for theway in which the sensors function.Two proteins, Std1 and Mth1, have been shown to in-

teract with the tails of the glucose sensors and geneticanalysis suggests that they are involved in transductionof the glucose signal to regulate invertase and hexosetransporter gene expression [72^74]. STD1 (MSN3) wasoriginally isolated as a multicopy suppressor of the snf(sucrose non-fermenting) phenotype of an snf4 mutantby a partial relief of SUC2 repression [75,76]. Its MTH1homolog is allelic to the genes HTR1, DGT1 and BPC1,for which previously dominant mutant alleles have beenisolated [73,74,77]. These dominant mutations were shownto cause severely impaired glucose uptake [78^80]. MTH1

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deletion suppresses the ra⁄nose growth defect of an snf3mutant as well as the glucose fermentation defect of snf3rgt2 double mutants through increased and unregulatedexpression of the HXT2, HXT3 and HXT4 hexose trans-porter genes. Deletion of STD1 cannot suppress the fer-mentation defect but speci¢cally increases HXT expressionin the presence of low glucose concentrations. Std1 andMth1 apparently act through distinct pathways and, likeSnf3 and Rgt2, respond to di¡erent levels of glucose. Std1was shown to act upstream of the Snf1 kinase, both forderepression of SUC2 and high-a⁄nity transporter geneexpression and repression of HXT1 expression, whereasMth1 mediates repression via an Snf1-independent path-way [72]. The mutant forms of MTH1 (HTR1, DGT1 andBPC1) apparently block transduction of the Snf3- andRgt2-mediated glucose signals upstream of the Rgt1 re-pressor [73,74]. Studies with green £uorescent protein fu-sions indicated that Std1 is localized in the cell peripheryand the cell nucleus, supporting the idea that it may trans-duce signals from the plasma membrane to the nucleus[72]. The HXT expression data and the fact that Snf3overexpression blocks the ability of Std1 to induceSUC2 expression suggest that the glucose sensors andthe Std1 and Mth1 proteins act antagonistically, with thesensors being required for HXT induction and the Std1and Mth1 proteins being required for their repression[72]. Possibly, Snf3 and Rgt2 inhibit the negative (repres-sing) e¡ects of Mth1 and Std1 by sequestering them at theplasma membrane [73].The observation that Rgt2 and Snf3 alone do not sus-

tain transport of glucose to enable growth, (not even whenoverexpressed) and isolation of the dominant RGT2-1 andSNF3-1 mutations in the ¢fth cytoplasmic loop, have ledto the hypothesis that Snf3 and Rgt2 function as classicalsignal receptors, in which binding of the extracellular li-gand, in this case glucose, causes a conformational changein a cytoplasmic domain [66]. Consistently, the hxt1-7vstrain, which is de¢cient in glucose uptake was reportedto exhibit normal glucose induction of HXT1 and HXT2(as measured by fusions of their promoter to LacZ) [68].However, Hxk2 was shown to be partially required for fullinduction of HXT expression [26]. Phosphorylation of thesugar might be important for wild-type Rgt2- and Snf3-mediated signalling and the small amounts of glucose thatare still taken up and phosphorylated in an hxt1-7v strainmay be su⁄cient to enable signalling.The use of transporter-like proteins as nutrient sensors

may be a more common strategy in eukaryotic cells. Theconserved sequence motif in the C-termini of Rgt2 andSnf3 is also present in the C-terminal extension of Rag4from Kluyveromyces lactis. Rag4 was shown to controlexpression of the Rag1 glucose transporter [81] and mayfunction both as a high- and low-a⁄nity glucose sensor[82]. In Neurospora crassa, the transporter homolog andglucose sensor Rco3 contains a C-terminal extension sim-ilar to that of Snf3 and Rgt2 [83]. Also, the yeast Ssy1

protein, which is homologous to amino acid permeasesand contains an N-terminal cytoplasmic tail, was shownto act as an amino acid sensor controlling amino acidpermease gene expression. In Arabidopsis thaliana, evi-dence exists for speci¢c Hxk-independent sucrose sensingand transporter homologs have been proposed to act assucrose sensors [84,85]. On the other hand, functional nu-trient transporters might also play a role in nutrient sens-ing. Evidence for a role of the ammonium transporterMep2 in nitrogen sensing for control of pseudohyphalgrowth in yeast has been reported [86]. Also, for Gap1,evidence for a role in amino acid sensing for control of thePKA- and FGM-pathways targets has been obtained re-cently (Donaton, M.C.V., Holsbeeks, I., Lagatie, O.,Crauwels, M., Winderickx, J. and Thevelein, J.M., unpub-lished results) (see Fig. 3B). In higher eukaryotes nutrienttransporters with a sensing function might also be present.Recently, a regulatory function for mammalian Glut1 inglucose-induced activation of ERK (MAPK)-signallinghas been suggested [87].

4.2. Glucose repression: the Hxk glucose sensor

A second mechanism controlling expression of sugartransporters is the main glucose-repression pathway. Inthe presence of glucose, high-a⁄nity glucose transport isrepressed together with a broad range of other genes in-volved in the utilization of alternative carbon sources. Inaddition, also Hxk1 and glucokinase (Glk1) are repressedby glucose. Both high-a⁄nity transporters and sugar ki-nases appear to be involved at least to some extent intriggering their own repression, since activation of theglucose-repression mechanism requires uptake and subse-quent phosphorylation of the sugar.In a variety of conditions the level of glucose repression

was found to correlate well with the level of glucose-trans-port activity [78^80] and it has often been speculated thatspeci¢c glucose transporters or pairs of them could play arole in triggering glucose-induced regulatory responses[34,78,88,89]. The requirement of a signi¢cant amount ofglucose (s 20 mM) to fully trigger these signalling path-ways was thought to indicate the speci¢c involvement oflow-a⁄nity transport. Experiments with yeast strains ex-pressing individual transporters showed that triggering ofglucose repression is not dependent on a speci¢c hexosetransporter protein but rather correlates with the glucoseuptake activity of the cells and with glycolytic £ux [90,91].

HXK2 was identi¢ed as one of the ¢rst genes involved inglucose repression [92^94] but whether its requirement forsignalling simply re£ects the need for glucose phosphory-lation or involves a separate regulatory function for theHxk2 protein has been and still is a matter of debate.Early experiments suggested that of the three sugar ki-nases Hxk2 played a unique role in glucose repressionand it was proposed that this kinase was a bifunctionalenzyme with catalytic and regulatory domains for glucose

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repression [95,96]. However, this could not be con¢rmedin later experiments; in a large number of mutant Hxkstrains a good correlation between glucose repressionand residual phosphorylating capacity of the mutatedHxk was observed [97]. A similar correlation was observedwith a series of Hxk1^Hxk2 hybrid constructs [98]. Also,when Hxk1 was removed in addition to Hxk2, glucoserepression further diminished. Stable overexpressionshowed that Hxk1 was also capable of mediating glucoserepression at least to a certain extent [98]. Interestingly, nofurther metabolization beyond the sugar phosphorylationstep appears to be necessary for triggering glucose repres-sion since phosphoglucoisomerase mutants with only 1%residual isomerase activity still showed normal glucose re-pression [98]. In addition, 2-deoxyglucose, which is trans-ported and phosphorylated but not further metabolized,also triggers repression [99]. This glucose analogue wasused to isolate glucose-repression mutants which, as op-posed to wild-type cells, are able to grow on ra⁄nose in itspresence [92]. Overexpression of GLK1 did not restoreglucose repression in a Hxk mutant [98] indicating thatglucose phosphorylation by itself is not su⁄cient to triggerglucose repression. These results supported the idea of aspeci¢c function of the Hxk proteins in the activationmechanism of glucose repression. More recently, newdata on the di¡erential requirement of the sugar kinasesin short- and long-term glucose and fructose repressionand the complex transcriptional regulation of the kinasesthemselves has put the predominant role of Hxk2 in a newlight. Catabolite repression was shown to involve two dis-tinct mechanisms: an initial rapid response is mediatedthrough any kinase able to phosphorylate the sugar, in-cluding Glk1, while long-term repression speci¢cally re-quires Hxk2 for repression by glucose and either Hxk1or Hxk2 for repression by fructose [100,101]. BothHXK1 and GLK1 are repressed upon addition of glucoseor fructose but fructose repression of HXK1 is only tran-sient. This is consistent with the preference of Hxk1 forfructose as a substrate and its requirement for long-termfructose repression [101]. Apparently, activation of catab-olite repression is controlled by a complex interregulatorynetwork, involving all three sugar kinases and the mecha-nisms and pathways controlling their expression. In thisway not only the main glucose-repression pathway itselfbut also cAMP signalling indirectly a¡ects catabolite re-pression [101]. Consistently, rapid repression of the gluco-neogenic genes FBP1 and PCK1 by very low levels ofglucose was shown to be triggered in the presence of anyone of the three kinases, whereas in the presence of highglucose levels repression was mediated speci¢cally by theHxk2-dependent Mig1-repression mechanism [55]. It wasproposed that HXK2 gene expression could act as a sensorfor the glucose concentration in the medium [102]. Also,more recently, novel alleles of Hxk2 have been isolatedthat have distinct e¡ects on catalytic activity and catabo-lite repression of SUC2 [46,103,104] and long- and short-

term phases of catabolite repression could be dissected[103]. The lack of correlation between in vitro catalyticactivity of Hxk, in vivo sugar phosphate accumulationand the establishment of catabolite repression again sug-gested that the production of sugar phosphate is not theonly role of Hxk in repression but that also a regulatorysignalling site of the protein may be required (Fig. 5A).For galactokinase a clear distinction between the catalyticfunction and the regulatory function in induction of GALgene expression was made [105]. A similar situation mightapply to Hxk2. Structure^function analysis of Hxk2 morespeci¢cally suggests that the establishment of cataboliterepression is dependent on the onset of the phosphoryltransfer reaction on Hxk and is probably related to thestable formation of a transition intermediate and concom-itant conformational changes within the enzyme [46]. Also,in plants, Hxk is proposed to be a glucose sensor andextensive mutant analysis seems to uncouple regulatoryand catalytic activity (Moore and Sheen; pers. comm).Although the core components of the main glucose-re-

pression pathway and the important role of Hxk2 as aputative glucose sensor have been identi¢ed, it still re-mains to be elucidated what the actual glucose signal isthat triggers glucose repression. Since the rate of glucosetransport and phosphorylation correlate well with the levelof glucose repression, glucose-6-P or other initial glyco-lytic metabolites have often been proposed to be the trig-gering molecules. Interestingly, also ATP, the second sub-strate for the sugar kinases during sugar phosphorylation,has recently been implicated in the triggering reaction (re-viewed by [106]). ATP and ADP, respectively, are the sub-strate and product of the phosphorylation reaction. There-fore the AMP/ATP ratio could in principle act as somesort of sensor for sugar phosphorylation and metabolicactivity (Fig. 5B). One model proposes a signalling rolefor these nucleotides in triggering glucose repression basedon the fact that the three components of the Snf1 kinase(Snf1, Snf4 and the Sip proteins) are similar to the sub-units of the functionally related mammalian AMP-acti-vated protein kinase (AMPK) [6,107]. MammalianAMPK is involved in the cellular response to a varietyof stresses, like heat shock and nutrient starvation. Inacti-vation of a number of biosynthetic enzymes under theseconditions ensures better conservation of cellular ATP[10,108]. Likewise, since it is responsible for triggeringderepression, Snf1 is involved indirectly in the generationof ATP by enabling the cells to metabolize alternativecarbon sources in the absence of fermentable amounts ofglucose. Although it has been shown that Snf1 is not di-rectly activated by AMP [6,107], a good correlation be-tween Snf1 activity and the AMP/ATP ratio was reported[10]. In glucose-growing cells, ATP generation by glycol-ysis depletes AMP. When the glucose is exhausted, theAMP level is repleted, resulting in a high AMP/ATP ratiowhich could then activate Snf1 and relieve repression.Thus, in this model the triggering signal for repression is

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generated by the metabolism of glucose, consistent withthe predominant role of Hxk2 in both glucose phosphor-ylation during fermentative growth and glucose repression.However, although an increase in the AMP/ATP ratio wasobserved when repressed cells are shifted to low-glucosemedium [10], AMP and ATP levels during growth onglycerol and glucose appear to be very similar [109]. Inaddition, this model does not ¢t with the indications fora separate regulatory function for Hxk. Snf-related proteinkinase (SnRK) signalling is also conserved in plants. PlantSnf1 homologs have been shown to complement yeast snf1mutants and are proposed to act as global regulators ofcarbon metabolism in plants [110]. As in yeast, plantSnRKs are not directly activated by AMP althoughAMP seems to inhibit their dephosphorylation [111]. In-terestingly, glucose-6-P was reported to negatively regulatea plant SnRK [112].Yeast Hxk2 has recently been shown to have a role in

regulating the phosphorylation status of the regulatorysubunit of protein phosphatase 1 Reg1/Hex2. Reg1 isphosphorylated in response to glucose limitation in anSnf1-dependent way and dephosphorylated by Glc7when glucose is present. Phosphorylation of Reg1 bySnf1 appears to stimulate both Glc7 activity in promotingclosure of the Snf1 complex and release of Reg1-Glc7from the kinase complex. Hxk2 either stimulates bindingand/or phosphorylation of Reg1 or inhibits dephosphory-lation of Reg1 by Glc7 [18].Other recent data suggest that the Hxk2 protein might

have an even more direct role in signalling to the repres-sion machinery. It was found that Hxk2 resides partly inthe cell nucleus [113] and that this nuclear localization,which is dependent on a speci¢c internal nuclear localiza-tion sequence, is necessary for glucose-repression signal-

ling [114]. Furthermore, the Hxk2 protein was shown toparticipate in regulatory DNA^protein complexes with cis-acting regulatory elements of the SUC2 promoter [114].Hxk2 therefore might be involved in transducing the glu-cose signal by interacting directly with transcriptional fac-tors controlling the expression of glucose-repressed genes.Phosphorylation at Ser-15, which also shifts the dimeric^monomeric equilibrium, does not seem to a¡ect nucleartargetting [114]. Phosphorylation and the concomitant in-crease in glucose a⁄nity of monomeric Hxk could providea mechanism to optimize glucose utilization at low con-centrations [115], but although protein phosphatase 1 isinvolved in dephosphorylation of the Hxk2 monomer[113,116] seemingly contradictory results were obtainedas to whether this phosphorylation/dephosphorylation isinvolved in signalling [97,113,114].

4.3. cAMP signalling: a dual sensing system

Experiments with hexose kinase and other glycolysismutants showed that transport and phosphorylation butno further metabolization of the sugar is required to acti-vate cAMP synthesis by glucose [117]. However, glucose-6-P does not appear to be the trigger of the activationreaction: the increase in the level of glucose-6-P after ad-dition of di¡erent glucose concentrations did not show agood correlation with the increase in the cAMP level.From the increase in the cAMP level after addition ofdi¡erent extracellular glucose concentrations an apparentKa for the activation mechanism of about 25 mM wasdeduced, ¢tting with the Km of what was believed to bethe low-a⁄nity glucose transporter system but di¡ering byat least one order of magnitude from the Km values of thethree hexose kinases. Together, these results suggested that

Fig. 5. Role of Hxk in the main glucose-repression pathway. A: Model in which a regulatory signaling function is associated with Hxk. Although theregulatory function can be closely associated with the catalytic activity, neither the substrates glucose and ATP nor the products glucose-6-phosphateand ADP act as metabolic messengers. B: Model based on metabolic messenger function of nucleotides. Glycolysis changes the ADP/ATP and AMP/ATP ratios. Changes in the nucleotide levels may act as a sensor of metabolic activity and exert a signalling function in triggering glucose repression.This model is based on the similarity between mammalian AMPK and the di¡erent components of the Snf1 kinase complex.

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the primary triggering reaction was situated at the level oftransport and phosphorylation, possibly even transport-associated phosphorylation. Glucose-induced cAMP-sig-nalling is indeed dependent on transport of the sugarbut not on any speci¢c glucose transporter. Also, thetransporter homologs and putative glucose sensors Rgt2and Snf3 are not directly involved in this glucose-sensingprocess [58]. The role of sugar transport is apparentlylimited to the provision of a su⁄cient amount of substrate.Although glucose-6-P does not seem to be the metabolicmessenger for activation, in Hxk mutants a clear correla-tion was always observed between catalytic activity andthe triggering of cAMP signalling [46,103]. Apparently,the role of yeast Hxk in sugar-induced activation ofcAMP signalling is closely connected to the catalytic func-tion of the enzyme. How glucose phosphorylation iscoupled to the control of cAMP synthesis is still unclear.Basal activity of the cAMP pathway is essential for via-bility and this makes it di⁄cult to study the activationmechanism.It was proposed that the Ras proteins are not only

essential for maintaining a basal level of cAMP by sustain-ing basal adenylate cyclase activity, but in addition aresignal transmitters in the pathway leading from glucoseto adenylate cyclase [118]. Subsequently also Cdc25, theRas-GEF, was shown to be involved in glucose-inducedactivation of cAMP synthesis [119^121]. Glucose thereforeappeared to be a direct or indirect stimulator of Cdc25.Recently, the possible involvement of the Ras proteins inglucose signalling was investigated more directly [122]. In-tracellular acidi¢cation, another stimulator of in vivocAMP synthesis, but not glucose, caused an increase inthe GTP/GDP ratio on the Ras proteins. Stimulation ofcAMP synthesis by glucose was shown to be dependent onanother G-protein, Gpa2. The GPA2 gene was originallycloned as a yeast homolog of mammalian heterotrimericGa-proteins and was already implicated in cAMP signal-ling. However, although overexpression of the gene clearlya¡ected cAMP levels, no e¡ect was observed in a gpa2vstrain on glucose-induced cAMP signalling [123,124]. Thiswas later shown to be due to interference with the e¡ect ofintracellular acidi¢cation caused by the addition of glu-cose. The increase in cAMP observed after addition of100 mM glucose shortly after pre-addition of 5 mM glu-cose was entirely dependent on the presence of Gpa2 [122].Gpa2 does not seem to play an important role in thecontrol of the basal cAMP level. Moreover, although de-letion of GPA2 confers to some extent the typical pheno-type associated with a reduced level of cAMP, the functionof Gpa2 appears to be limited mainly to the stimulation ofcAMP synthesis during the transition from respirativegrowth on a non-fermentable carbon source to fermenta-tive growth on glucose [122].Using the two-hybrid screen and Gpa2 as bait, a frag-

ment of a putative GPCR, Gpr1, was isolated [46,125,126]. Surprisingly, Plc1 (phospholipase C) appears to be

required for this interaction [127]. The GPCR Gpr1, likeGpa2, was shown to be speci¢cally required for glucoseactivation of the cAMP pathway during the transition togrowth on glucose and a gpr1v mutant could be rescuedby the constitutively activated GPA2val132 allele [46]. Ap-parently, Gpr1 and Gpa2 constitute a glucose-sensingGPCR system for activation of the cAMP pathway (Fig.3). This not only brings the yeast adenylate cyclase systemback in line with the mammalian system of adenylate cy-clase control, it also appears to be the ¢rst example of aGPCR system activated by a nutrient in eukaryotic cells.S. pombe contains a similar glucose-sensing GPCR systemfor activation of cAMP synthesis (consisting of the GK-protein gpa2 and the putative glucose receptor git3) andalso Candida albicans contains a Gpr1 homolog with ex-tensive similarity to its S. cerevisiae counterpart, suggest-ing the existence of a new GPCR family involved in glu-cose sensing [128].Consistent with its requirement for glucose-induced

cAMP accumulation, GPR1 was also isolated as a mutantallele (¢l2) in a screen for mutants de¢cient in fermenta-tion-induced loss (¢l) of heat resistance [46]. In a similarscreen, the RGS2 gene was isolated as a multi-copy sup-pressor of glucose-induced loss of heat resistance [129].RGS2 encodes a protein with a typical conserved RGS(regulator of heterotrimeric G-protein signalling) domainand was indeed shown to negatively regulate glucose acti-vation of the cAMP pathway through direct inhibition ofGpa2. Consistent with its homology to other RGS pro-teins, Rgs2 acts as a stimulator of the GTPase activity ofGpa2. It remains to be shown, however, that Gpa2 indeedacts as the signal transducer from glucose to adenylatecyclase. The fact that deletion of GPA2 is lethal in theabsence of Ras2 is consistent with a role for Gpa2 asstimulator of adenylate cyclase [130].A mutation in the catalytic domain of adenylate cyclase

(cyr1met1876) has been identi¢ed that speci¢cally a¡ects glu-cose- and acidi¢cation-induced cAMP signalling and notthe basal cAMP level [131]. This lcr1 (lack of cAMP re-sponse) mutation not only abolishes the cAMP signal butalso the transient increase in the basal cAMP level ob-served during the lag phase of growth on glucose [132].In addition, it appears to counteract the overactivatinge¡ect of both the RAS2val19- and GPA2val132- dominantalleles, supporting the theory that Gpa2 indeed acts up-stream of adenylate cyclase. The observation that elimina-tion of glucose activation of cAMP-synthesis by the lcr1mutation only results in a delay in glucose-inducedchanges in PKA targets associated with the adaptationto growth on glucose, and does not a¡ect the typical var-iations of PKA-controlled phenotypic properties duringdiauxic growth, supports the idea of an alternative path-way responsible for glucose signalling during growth.Gpa2 was also found to be required for pseudohyphal

growth [130,133]. Pseudohyphal di¡erentiation is inducedin diploid cells in response to nitrogen starvation in the

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presence of a fermentable carbon source and is mediatedboth by the pheromone-responsive MAPK cascade andthe cAMP pathway. Consistent with the fact that the con-stitutively active GPA2val132 allele stimulates ¢lamentation,even on nitrogen-rich media, it was proposed that Gpa2 isan element of the nitrogen-sensing machinery that regu-lates pseudohyphal di¡erentiation by modulating cAMPlevels [133]. However, genetic and physiological studieson pseudohyphal growth recently con¢rmed that theGpr1^Gpa2 GPCR system is activated by glucose. Be-cause of the fact that GPR1 expression is induced by nitro-gen starvation, it is proposed that the receptor acts as adual sensor for both abundant carbon and nitrogen star-vation [134]. Obviously, the demonstration that Gpr1 itselfbinds glucose and acts as a real glucose receptor is animportant issue.As mentioned before, elucidation of the exact mecha-

nisms of glucose sensing is often complicated because ofthe requirement for partial metabolism of the glucose.This is also the case for the glucose-induced activationmechanism of cAMP synthesis and the involvement ofthe Gpr1^Gpa2 GPCR system. In spite of this, an actualglucose-sensing function for Gpr1 has recently becomemore apparent with the demonstration that Gpr1 is essen-tial for the sensing of extracellular glucose. The glucose-induced cAMP signal is not only dependent on the GPCRsystem but also on transport and phosphorylation of thesugar (Fig. 6A). We showed that it is possible to uncouplethe GPCR-dependent sensing process from the glucosephosphorylation. For this purpose a method was estab-lished allowing independent investigation of the two re-quirements based on the observation that the absence ofthe glucose-induced cAMP signal can be restored in the

Hxt null strain by pre-addition of a low concentration(0.025% or 0.7 mM) of maltose (Fig. 6B). This concentra-tion of maltose does not a¡ect the cAMP level by itself butapparently ful¢lls the glucose phosphorylation require-ment for activation of the cAMP pathway by glucose,which in the Hxt null strain cannot be transported intothe cell. Using this set-up it was shown that the GPCRGpr1 or at least the glucose-sensing mechanism that isdependent on it, speci¢cally responds to extracellular glu-cose (and also sucrose, but not fructose or other sugars)with low apparent a⁄nity. This is consistent with the factthat yeast cells switch metabolism to the fermentativemode only at glucose concentrations of at least 20 mM.Interestingly, the presence of the constitutively activeGPA2val132 allele increases the fructose-induced cAMP sig-nal to the same intensity as the glucose signal in trans-porter wild-type cells and enables concentrations as low as5 mM glucose to fully activate the pathway. This is con-sistent with the fact that in such a strain activation of thepathway is only dependent on phosphorylation of the sug-ar, since the GPCR system is constitutively activated. Inconclusion, the two essential requirements for glucose-in-duced activation of cAMP synthesis can be ful¢lled sepa-rately. It remains unclear at what point the two require-ments are integrated. Apparently, glucose phosphorylationis required in some way to make adenylate cyclase respon-sive to activation by the GPCR system. Since no increasein the GTP/GDP ratio of Ras is observed after addition ofglucose, it seems unlikely that the hexose kinase-dependentsensing system acts through Cdc25-Ras2. The kinasesmight also act directly on adenylate cyclase, possibly re-leasing inhibition of catalytic activity by the N-terminalregulatory domain.

Fig. 6. Ful¢llment of the glucose phosphorylation requirement for cAMP signalling. A: The relationship between the intracellular glucose phosphoryla-tion process and the Gpr1/Gpa2 dependent extracellular glucose detection system in a wild-type strain. B: In a strain without functional glucose trans-porters (Hxt), the glucose phosphorylation requirement for cAMP signalling can be ful¢lled by addition of a low level of maltose which is transportedand hydrolyzed by a speci¢c system consisting of the maltose transporter (MalT) and maltase (MalS).

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The demonstration that the Gpr1^Gpa2 GPCR systemis responsible for glucose control of the cAMP pathwayhas brought up again the question as to what is the actualfunction of Ras-dependent control of adenylate cyclase inyeast [35]. Ras mutants exist which seem to be speci¢callya¡ected in signal transduction. Strains carrying theras2ser318 allele as their sole RAS gene, for example, dis-play normal steady-state levels of cAMP, while the glu-cose-induced cAMP signal is totally absent [135]. Also,several mutants de¢cient in post-translational modi¢cationof Ras are speci¢cally de¢cient in cAMP signalling [136].Many results indicating a role for the Cdc25-Ras system inglucose-induced cAMP signalling could possibly be ex-plained by their requirement for localization of adenylatecyclase to the plasma membrane. Consistent with such arole for Cdc25 is the recent evidence of direct binding ofCdc25 to adenylate cyclase through an SH3 domain. Thisbinding might promote an e⁄cient assembly of the ade-nylate cyclase complex [137]. Proper membrane localiza-tion of the adenylate cyclase complex might be essentialfor optimal interaction with and activation by the Gpr1^Gpa2 system. The main function of the Ras proteinsmight therefore be to control basal adenylate cyclase ac-tivity [35]. S. cerevisiae cells indeed have a very high ca-pacity to synthesize cAMP and a strict control of thecAMP level is clearly essential, especially under less-favor-able conditions that require slow growth and high stressresistance. The association with Ras might increase theresponsiveness of adenylate cyclase to stimulation by theGPCR system when it is activated by a high level of glu-cose in the medium.FGM signalling still occurs in hxk1vhxk2vglk1 and

gpr1v or gpa2v strains, possibly pointing to a totally dif-ferent glucose-sensing system for FGM signalling com-pared to cAMP signalling. However, recent results indi-cate that the presence of one of the two glucose-sensingsystems might be su⁄cient for FGM signalling while theyare both required for glucose activation of cAMP signal-ling (Donaton, M., Winderickx, J. and Thevelein, J.M.,unpublished results).

4.4. Allosteric regulation

Not all glucose-induced regulatory e¡ects require a sig-nal transduction mechanism. Allosteric activation and in-hibition is exerted by metabolic intermediates of glucosecatabolism. Allosteric regulation has been studied ¢rstwith respect to the control of glycolysis, which was the¢rst metabolic pathway to be discovered and elucidated.The main allosteric regulators of glycolysis appeared to befructose-2,6-bisP and fructose-1,6-bisP, controlling two ofits irreversible steps, catalyzed respectively by phospho-fructokinase (PFK) and pyruvate kinase (PYK). Fruc-tose-2,6-bisP not only activates PFK, but in addition in-hibits fructose-1,6-bis-phosphatase, which catalyzes thereverse reaction in gluconeogenesis. The product of the

PFK reaction, fructose-1,6-bis-phosphate, in turn allosteri-cally activates PYK more downstream in glycolysis [138].However, enhanced expression of both PFK1 and PYK1does not change glycolytic £ux signi¢cantly [139] and mu-tant studies of PF2Ks did not reveal an essential role forfructose-2,6-bis-P in the regulation of carbon £uxes inyeast cells [140]. Apparently, these enzymes do not cata-lyze rate-limiting steps in glycolysis and allosteric e¡ectsappear to control metabolite homeostasis rather then met-abolic £uxes. Metabolic control analysis indeed pointed tosugar uptake as the major £ux-controlling step in glycol-ysis [141]. The control coe⁄cient of glucose transport wascalculated to be signi¢cantly higher then that of PFK. Thehigh level of control by transport over growth and glyco-lytic £ux has also been con¢rmed in an hxt null strainexpressing a single transporter [91]. More recently, thetrehalose-6-phosphate synthase subunit of the trehalosesynthase complex was found to control in some way theentry of glucose into glycolysis [88]. While in mammaliancells glucose-6-P is the most important allosteric inhibitorof the hexose kinases, in yeast cells this function appearsto be exerted by trehalose-6-phosphate [142]. In addition,there is evidence for the possible involvement of the Tps1protein itself in controlling glycolytic £ux [143,144]. Prop-er control of glucose in£ux into glycolysis is required for awide range of glucose-signalling e¡ects in yeast, as wasdemonstrated with the tps1v mutant which is unable tosynthesize trehalose-6-phosphate and therefore shows asevere deregulation of glycolysis after addition of glucose[145]. This indicates that the research on glucose-sensingmechanisms cannot be seen as separate from that on glu-cose metabolism, and that for every mutant a¡ected inglucose signalling, investigation of possible interferencewith glucose metabolism is of paramount importance.

5. Conclusions and perspectives

The preference of S. cerevisiae for glucose as a carbonand energy source is re£ected by the variety of glucose-sensing and -signalling mechanisms ensuring its optimaluse. Nutrient-sensing and -signalling mechanisms musthave evolved early in evolution and might be at the originof the sophisticated hormone- and growth factor-inducedsignal transduction pathways best known from researchon mammalian cells. The glucose-sensing mechanisms inyeast are therefore an excellent model system for studyingsignal transduction in general. Glucose is also the primecarbon and energy source in higher multicellular organ-isms and it is becoming clear that glucose-sensing and -signalling in these organisms is of vital importance formaintenance of sugar homeostasis [58]. In mammals glu-cose serves as the blood sugar and maintenance of theglucose concentration within narrow limits is controlledby a complex interplay of several endocrine and neuralglucostatic systems that direct its uptake and release

FEMSYR 1457 5-6-02


[146]. In addition, glucose also plays a more direct role intranscriptional regulation [147]. In plants, sugars like su-crose, glucose and fructose are the main products of pho-tosynthesis and the primary carbon source for respiration.Sugar-sensing and -signalling a¡ects many aspects ofgrowth, metabolism and development throughout thewhole plant life cycle [148,149]. It is therefore likely thathigher eukaryotes are equally well supplied with glucose-sensing and -signalling mechanisms. One striking exampleof an apparently conserved signalling mechanism in eu-karyotes is involved in the control of life span. Yeast lon-gevity was shown to be regulated by PKA (adenylate cy-clase) and Sch9. Longevity is often associated withincreased resistance to (oxidative) stress and the stress-re-sistance transcription factors Msn2 and Msn4 were indeedshown to be required for life-span extension in Sch9 andadenylate cyclase mutants [150]. Another report empha-sized the requirement of NAD (and its regulation of thesilencing protein Sir2) for life-span extension by caloricrestriction and the involvement of the cAMP-PKA path-way in this process independently of stress resistance [47].Interestingly, deletion of Gpr1 or Gpa2 had similar e¡ectson longevity as caloric restriction (growth on low glucose),con¢rming the role of this GPCR system in the sensing ofhigh glucose concentrations. Sch9 shows most similarity toAkt/PKB. This protein kinase is involved in a signallingpathway controlled by an insulin receptor-like protein andregulating carbon metabolism, stress resistance and lon-gevity in Caenorhabditis elegans [151]. Since also humanAkt/PKB is involved in insulin signalling, translocation ofglucose transporters, apoptosis and cellular proliferation,an ancient (glucose) signalling mechanism that coordin-ately regulates metabolism, stress resistance and longevity(enabling survival over long periods of starvation) mayhave been conserved in all eukaryotic organisms [150].Interestingly, also yeast Snf1 in addition to cellular energyutilization was reported to control aging, and conservedhomologs in other organisms might have similar e¡ects.Since glucose signalling appears to be fundamental to cel-lular and organismal function and therefore widespread(and in some cases conserved), it is likely that similarspeci¢c sensing mechanisms as in yeast are also presentin higher eukaryotes. An abundance of results seems topoint at a central role for Hxk in eukaryotic glucose sens-ing. Although structure^function analysis and mutagenesishave enabled separation of catalytic and regulatory activ-ity to some extent, more detailed analysis will be requiredto elucidate the actual Hxk- sensing and -signalling mech-anism. Subcellular localization might be an important fac-tor in Hxk regulatory function. In addition, more speci¢csensors might be involved in higher eukaryotic glucosesensing. As mentioned above, glucose transporter-like pro-teins might have tissue- or cell-speci¢c regulatory func-tions in mammals and plants. Finally, classical receptorfamilies may be involved, as shown by the example ofyeast Gpr1. The elucidation of the yeast glucose-sensing

GPCR system obviously tempts to speculate that amongstthe hundreds of eukaryotic orphan receptors a subfamilyof nutrient sensors is waiting to be discovered.

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Glucose-sensing and -Signalling Mechanisms in Yeast

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