The hexokinase 2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae

The hexokinase 2-dependent glucose signal transduction pathway ofSaccharomyces cerevisiae

Fernando Moreno *, Pilar HerreroDepartamento de Bioqu|¤mica y Biolog|¤a Molecular, Universidad de Oviedo, Edi¢cio Santiago Gasco¤n, Campus del Cristo, 33006 Oviedo, Spain

Received 16 August 2001; accepted 7 December 2001

First published online 23 January 2002

Abstract

Sugars, predominantly glucose, evoke a variety of responses in Saccharomyces cerevisiae. These responses are elicited through a complexnetwork of regulatory mechanisms that transduce the signal of presence of external glucose to their final intracellular targets. The HXK2gene, encoding hexokinase 2 (Hxk2), the enzyme that initiates glucose metabolism, is highly expressed during growth in glucose and plays apivotal role in the control of the expression of numerous genes, including itself. The mechanism of this autocontrol of expression is notcompletely understood. Hxk2 is found both in the nucleus and in the cytoplasm of S. cerevisiae ; the nuclear localization is dependent on thepresence of a stretch of amino acids located from lysine-6 to methionine-15. Although serine-14, within this stretch, can be phosphorylatedin the absence of glucose, it is still unsettled whether this phosphorylation plays a role in the cellular localization of Hxk2. The elucidation ofthe mechanism of transport of Hxk2 to and from the nucleus, the influence of the oligomeric state of the protein on the nuclear transportand the fine mechanism of regulation of transcription of HXK2 are among the important unanswered questions in relation with theregulatory role of Hxk2. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rightsreserved.

Keywords: Hexokinase 2; Glucose sensing; Glucose repression; Glucose induction; Transcriptional control; Saccharomyces cerevisiae

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842. Hxk2 and glucose signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

2.1. The glucose-phosphorylating enzymes of S. cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . 842.2. Hxk2 can enter the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852.3. Hxk2 participates both in glucose repression and in induction of gene expression . . . . 85

3. Integration of the glucose induction and repression pathways . . . . . . . . . . . . . . . . . . . . . . 863.1. The glucose sensors of the pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863.2. The central repressors of the transduction pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 863.3. Med8, a further regulatory element connected with Hxk2 . . . . . . . . . . . . . . . . . . . . . 87

4. A glucose-phosphorylating enzyme is also involved in mammalian glucose signallingpathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

5. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

0168-6445 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 4 5 ( 0 1 ) 0 0 0 7 7 - 8

* Corresponding author. Tel. : +34 (98) 5103567; Fax: +34 (98) 5103157. E-mail address: [email protected] (F. Moreno).

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www.fems-microbiology.org

1. Introduction

Adaptation to changes in the environment is critical forthe survival of all organisms, therefore along evolution,systems of di¡erent complexity have been selected tomeet this demand. Changes in external medium generatesignals that are transduced to the interior of the cell, wherethey provoke changes in gene expression and protein ac-tivity which result in an adequate cellular response to var-iations in the extracellular environment.

The ability to grow in di¡erent media exhibited by theyeast Saccharomyces cerevisiae is due to its capacity tosense and respond to changes in the availability of nu-trients. The transduction pathways of the nutritional sig-nals are induced by speci¢c nutrients, and in general thesepathways bring about changes in gene expression [1],mRNA stability [2] and post-translational modi¢cations[3^7]. Among sugars, glucose is likely the major signallingnutrient for S. cerevisiae, as well as the carbon and energysource used preferentially. Present knowledge concerningfactors required to transmit the glucose signal from theenvironment to the cell nucleus is mainly derived fromstudies with mutants a¡ected in di¡erent regulatory cir-cuits governed by glucose [8]. These studies have charac-terized, in di¡erent detail, several signal transduction path-ways that allow the yeast to perceive the level of glucose inthe medium and initiate the appropriate metabolic re-sponse [9,10]. Many of these responses involve alterationsin gene expression, and the majority of these alterationsoccur at the level of mRNA transcription, a phenomenonknown as glucose or catabolite repression [11^14]. Thegenes a¡ected by this process include among others thoseinvolved in utilization of alternative carbon sources, glu-coneogenesis, the glyoxylate and Krebs cycles, respiration,and peroxisomal functions. Glucose also induces expres-sion of genes required for its own utilization, like the genesencoding several glycolytic enzymes and glucose transport-ers [15]. In addition, glucose acts in yeast as a ‘growthhormone’, regulating several aspects of cell growth, metab-olism and development [16,17].

Although several of the genes implicated in the path-ways that control glucose repression and induction havebeen identi¢ed [9], a complete mechanistic picture of thephenomenon is not yet available. In particular, the posi-tion of each factor in the signalling cascade and the inter-actions among them are still not well known. However, inthe last few years important advances in this ¢eld havebeen made [9,10]. We consider in this review the presentknowledge about an important factor in the signal trans-duction pathway, namely hexokinase 2 (Hxk2), a proteinthat in addition to its classical metabolic role plays animportant role in glucose signalling.

2. Hxk2 and glucose signalling

2.1. The glucose-phosphorylating enzymes of S. cerevisiae

In S. cerevisiae glucose metabolism starts by phosphor-ylation of the sugar at C6. Three enzymes, namely Hxk1,Hxk2 and glucokinase (Glk1) (encoded respectively by theHXK1, HXK2 and GLK1 genes) can catalyze this ¢rst ir-reversible step in the intracellular metabolism of glucose.However, Northern analysis showed that when glucose isused as carbon source, only the HXK2 gene is highly ex-pressed. In contrast, the expression of the HXK1 andGLK1 genes is only important when the culture mediumcontains non-fermentable carbon sources or galactose [18].These facts have been con¢rmed by exploring the meta-bolic and genetic control of gene expression on a genomicscale [19]. These ¢ndings indicate that in wild-type S. ce-revisiae, only HXK2 is important for glucose phosphory-lation in vivo. Either Hxk1 or Glk1 support growth ofmutants in which Hxk2 is absent [13], indicating that theseproteins are able to act in vivo, and that their irrelevancefor glucose metabolism is due to the poor expression ofthe genes which encode them.

What makes Hxk2 a special protein is not only that it isthe enzyme responsible for the phosphorylation of glucose,but the fact that it is also implicated in glucose repression.It was found that both point [20] and null mutations [21]in the HXK2 gene blocked glucose repression of certaingenes. Since the glucose-phosphorylating activity in thecorresponding extracts was reduced, the idea that therewas a correlation between the glucose-phosphorylating ac-tivity of Hxk2 and glucose repression appeared as a veryattractive one [22,23]. However, this idea was shaken bythe following ¢ndings: (i) when the GLK1 gene is overex-pressed in a hxk1/hxk2 double-null mutant the trans-formed strains are still insensitive to glucose repression,even though a three-fold increase of phosphorylating ac-tivity is achieved [23]; (ii) glucose repression is not linearlyrelieved with decreasing kinase activity, indicating thatsugar kinase activity and sugar signalling are mediatedat least in part through separated domains of Hxk2[24,25]; (iii) mutant alleles with low catalytic activitywere still fully functional in glucose signalling [26]. Inthis context it is also interesting to point out that earlyglucose repression of the SUC2 gene (a glucose repressionreporter gene) does not require speci¢cally Hxk2 [27] andthat Hxk2 is only necessary for the long-term glucose re-sponse [28]. The correlation between glucose-phosphoryla-tion activity of Hxk2 and glucose repression appears lesslikely at present.

The dual role of hexokinase as a metabolic enzyme andas a regulatory protein, may be not so unique in sugarmetabolism. Recently, it has been found that yeast galac-tokinase (Gal1), has also a dual function: it functions asan enzyme in the phosphorylation of galactose and as atranscriptional regulator. It has been found recently that

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in the presence of galactose and ATP, Gal1 activates thetranscriptional factor Gal4 by direct binding to the Gal4inhibitor Gal80 [29].

2.2. Hxk2 can enter the nucleus

If Hxk2 plays a role as a transcriptional regulator, oneshould expect that under certain conditions, the enzymewill be present in the cell nucleus. Several results usingdi¡erent approaches support such a localization: (i)Hxk2 was localized in isolated nuclei by speci¢c antibodiesand concomitantly hexokinase activity was found in thepreparation [30] ; (ii) expression of a Hxk2^GFP fusionrevealed that a fraction of the total Hxk2 was present inthe nucleus, making it unlikely that the previous ¢ndingwas due to cross-contamination during subcellular frac-tionation [30]; (iii) a determinant for the nuclear localiza-tion of Hxk2p has been characterized as an internal se-quence located between lysine-6 and methionine-15(KKPQARKGSM), that has been named nuclear localiza-tion sequence (NLS) [31]. Elimination of this sequenceabolishes both nuclear localization of Hxk2 and glucoserepression of SUC2, HXK1 and GLK1 [31,32]. Therefore,Hxk2 complies with the conditions needed to participatedirectly in the control of the transcription of several genes.In yeast cells grown in the absence of glucose, Hxk2 isphosphorylated at serine-14 [33]. This phosphorylationcatalyzed by a still-unknown kinase has several consequen-ces : (i) Hxk2 becomes sensitive to inhibition by free ATP[34,35] thus allowing the cell to integrate the intracellularATP concentration into the control of glucose phosphor-ylation; (ii) autophosphorylation at serine-157 is stimu-lated, a reaction which results in inhibition of the Hxk2enzymatic activity [33,34,36,37]. How the new conforma-tion of the diphosphoenzyme may a¡ect glucose signallingis unknown at the moment; (iii) the equilibrium between

the two isoforms of Hxk2, a monomer and a dimmer [38^40], is shifted to the monomeric form [34,41].

In the presence of glucose, the regulatory protein Reg1targets the protein phosphatase 1 Glc7, to dephosphory-late serine-14 in Hxk2 [42] and displaces the equilibriumtowards the homodimeric form [34,41]. Since serine-14,which can be phosphorylated in vivo, is within the NLSsequence, it was not illogical to think that the state ofphosphorylation of this residue could regulate NLS func-tion. However, the available results have not allowed anunequivocal validation of this idea. It has been reportedthat the expression of a mutant gene HXK2(S14A) in ahxk1/hxk2 double mutant strain allowed nuclear localiza-tion of the mutant protein and that this one restored glu-cose repression of the SUC2 gene [31,43]. However, an-other report showed that both glucose repression of SUC2and glucose-induced expression of glucose transporterswere impaired in cells which expressed the mutated protein[41]. The experiments were carried out in two di¡erentgenetic backgrounds and the discrepancy between the re-sults could be related to di¡erences in the amount of hexo-kinase being produced in the di¡erent transformed strains.

2.3. Hxk2 participates both in glucose repression andin induction of gene expression

The amount of glucose-phosphorylating enzymes in S.cerevisiae is mainly regulated at the transcriptional level bymechanisms not completely elucidated [18]. In the case ofHXK2, DNA^protein complexes have been described[44,45] that involve two nucleotide sequences (downstreamrepressing sequences, DRSs) located within the coding re-gion of the gene. The regulatory factors that operatethrough these DRSs repress HXK2 transcription underconditions of sugar limitation or when ethanol is used asa carbon source [18]. Transcription of the GLK1 gene is

Table 1DNA sequence of regulatory regions in the HXK1, GLK1, HXK2 and SUC2 promoters

Important sequence elements are indicated. They include: for all the genes, UASSUC -like regulatory sequences for HXK1, GLK1 and HXK2 (DRS1),ERA regulatory sequences, and for SUC2 promoter, one Mig1p-binding site overlapping the ¢rst UASSUC regulatory sequence.

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repressed in media with glucose but is high upon glucosedepletion [46]. This regulation is achieved through thecombinatorial e¡ect of three regulatory sequences of theGLK1 promoter: a STRE sequence (stress-responsive ele-ment) [47,48], an ethanol repression autoregulation(ERA)/TA box) element [49], and a Gcr1-binding site[50^52]. The transcription of HXK1 is also repressed inthe presence of glucose by regulatory factors that operatethrough an ERA element; upon glucose depletion, a quickinduction of transcription, through several STRE ele-ments, has been observed (F. Moreno, unpublished re-sults). Hxk2 is involved both in the glucose-induced re-pression of the HXK1 and GLK1 genes and in theglucose-induced expression of the HXK2 gene [32]. Hxk1also acts in this regulatory system as an inhibiting factorfor the expression of the GLK1 and HXK2 genes [32].Further experimental evidences, derived from a hxk2 mu-tant expressing a truncated version of Hxk2 unable toenter the nucleus, showed that a nuclear localization ofHxk2 is necessary for glucose-induced repression of theHXK1 and GLK1 genes, for glucose-induced expressionof HXK2 gene and for glucose repression of the SUC2gene [31,32]. Hxk2 participates in DNA^protein com-plexes with cis-acting regulatory elements of the SUC2gene, which contain the heptameric motif (C/A)-(G/A)GAAAT [31]. Interestingly, the sequences of bothDRSs in the HXK2 gene include the stretches CGGAAATand AAGAAAT, which are also found in the sequence ofan UAS element of SUC2 gene [45]. These sequences arealso found in the promoters of the HXK1 and GLK1 genesoverlapping with ERA motifs that participate in theHxk2-regulated transcription [31,53]. Thus, as can beseen in Table 1, the HXK1, GLK1, HXK2 and SUC2 geneshave regulatory elements with the consensus sequence(C/A)(G/A)(G/A)AAAT.

These ¢ndings suggest a mechanism of gene regulationwhereby in the presence of glucose the product of theHXK2 gene, normally resident in the cytosol, is translo-cated to the nucleus where it impairs the activation oftranscription by UASSUC-like heptameric motif containedin the SUC2, HXK1 and GLK1 promoters and hinders theblocking of transcription by DRSHXK2.

The induction by low levels of glucose of the expressionof HXT2^4 genes that encode certain glucose transportersis signi¢cantly reduced in hxk2 mutants [54]. This maypartially account for the observation that a mutant unableto phosphorylate glucose (hxk1 hxk2 glk1), lacks high-af-¢nity glucose transport [55,56], and makes less likely theconclusions of previous kinetic analyses of glucose trans-port in glucose kinase mutants (hxk1 hxk2 glk1), namelythat the hexose kinases interact directly with hexose trans-porter proteins and modulate their a⁄nity for glucose[57,58]. Control of the transporters at the level of tran-scription is also supported by the fact that the sole hexo-kinase of Kluyveromyces lactis (encoded by RAG5) is es-sential for glucose-induced transcription of the RAG1gene, which encodes a low-a⁄nity glucose transporter

[59]. Hxk2 is also required for full induction of HXT1expression by high levels of glucose, suggesting that theprotein is involved in the Rgt1-independent glucose induc-tion mechanism that operates on HXT1 [54].

3. Integration of the glucose induction andrepression pathways

The facts described indicate that Hxk2 is a factor bothof the repression and induction pathways triggered byglucose in yeast. Although these pathways seem to sharesome components and may respond to the same primarysignal(s) derived from glucose, the connection between theinduction and the repression pathways is not completelyunderstood at present.

We describe in the following paragraphs some of theelements of these pathways.

3.1. The glucose sensors of the pathway

Glucose is sensed by the receptors proteins Snf3 andRgt2, members of a family of hexose transport proteinsthat in S. cerevisiae consist of Hxt1 to Hxt17, Snf3, Rgt2and Gal2 [15,60]. Snf3 and Rgt2 are structurally distinctfrom the other 18 members of this family in yeast by thepresence of a large, hydrophilic C-terminal domain [61].Several lines of evidence indicate that these C-terminaltails are essential for glucose sensing and signal transduc-tion: (i) deletion of the tail domain reduces Snf3 function[62,63]; (ii) fusion of the tail domain to Hxt1 or Hxt2confers glucose-sensing ability to those proteins [63]; and(iii) expression of the Snf3 tail domain by itself can sup-press defects in glucose transport observed in a snf3 strain[64]. Snf3 and Rgt2 proteins do not actually transporthexoses themselves [65] but control hexose transport byregulating the expression of high- and low-a⁄nity trans-porters [61]. Thus, the yeast glucose sensors may haveevolved from a glucose transporter that changed the trans-porter domain into a glucose-binding domain able totransmit information about extracellular glucose concen-tration to the interior of the cell [66,67].

Glucose transport and metabolism are not absolutelyrequired for glucose signalling, as shown by the fact thata dominant mutation in RGT2 could produce changes ingene expression in the absence of glucose [61].

3.2. The central repressors of the transduction pathway

Two major repressors speci¢c for the glucose signaltransduction pathway have been identi¢ed: Mig1 andRgt1. Mig1 is a Cys2His2 zinc-¢nger protein that repressesthe transcription of several genes in the presence of highlevels of glucose by recruiting the co-repressors Ssn6^Tup1to glucose-repressed genes [68,69]. Rgt1, a Cys6 zinc-clus-ter protein, represses the transcription of several HXTgenes when glucose is absent by direct binding to the cor-

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responding promoters and recruitment of the co-repressorsSsn6^Tup1 [70].

Mig1 binds to the promoters of numerous glucose-re-pressed genes through the consensus sequence T(C/G)-(C/T)GGGG, but it also requires an AT-rich region 5Pof the GC box [71^73]. The subcellular localization ofMig1 is regulated by glucose: Mig1 is imported into thenucleus when glucose is present and transported to thecytoplasm when cells are glucose-limited [74]. This regu-lated movement of Mig1 appears to be due to phosphor-ylation: in derepressed cells Mig1 is both phosphorylatedand translocated to the cytosol [74]. Mig1 contains a nu-clear export signal that is phosphorylated by Snf1 uponglucose exhaustion, causing it to be recognized by thenuclear exportin Msn5 and carried out of the nucleusinto the cytoplasm [75]. The protein phosphatase thatacts on the phosphorylated Mig1 protein has been identi-¢ed as the protein phosphatase 1 encoded by the essentialgene GLC7. Like its mammalian counterpart [76], Glc7 isregulated by interaction with many distinct regulatory tar-geting subunits [77]. In S. cerevisiae, genetic studies to-gether with two-hybrid analyses have implicated theGlc7-binding protein Reg1 in the regulation of the glucoserepression pathway [78,79] and demonstrated a direct in-teraction of Reg1 with Hxk2 and Snf1, suggesting thatReg1 targets Glc7 to dephosphorylate both Hxk2 andSnf1 in vivo [42,78,80,81].

Rgt1 plays two central roles in glucose induction ofgene expression. It is required for repression in the absenceof glucose and for maximal induction of the HXT1 gene athigh glucose concentrations. Grr1p is required both foractivation of Rgt1 repressor function in response to lowlevels of glucose and for conversion of Rgt1 from a re-pressor to an activator by high levels of glucose. Hxk2

also appears to be involved in the process because inhxk2 mutants, a reduction or increase in glucose-inductionof HXT genes expression was observed [70,82].

3.3. Med8, a further regulatory element connected withHxk2

In a search to identify new factors required for expres-sion of SUC2 gene, a protein, Med8, was identi¢ed whichspeci¢cally binds both to the DRSs of the HXK2 gene andto the upstream activating sequences of the SUC2 gene[83,84]. Because Med8 has been described as a subunitof the Srb/mediator complex interacting with the carboxyterminal domain of the RNA polymerase II [85,86], itsrole could be to act as a coupling factor that links activat-ing and repressing transcription complexes to the RNApolymerase II holoenzyme transcriptional machinery.Co-precipitation experiments, two-hybrid assays and gel-mobility analyses with puri¢ed proteins have been used toinitiate the generation of protein-interaction maps of fac-tors involved in the glucose signalling pathway of S. cere-visiae. This approach has resulted in the identi¢cation ofinteractions of Hxk2 with Med8 and Mig1 (F. Moreno,unpublished results); the three proteins found interactingtogether in a cluster, may be part of a protein^DNA com-plex involved in the regulation of glucose repression. Apossible model of how Hxk2 and Mig1 repress transcrip-tion of the SUC2 gene is shown in Fig. 1.

4. A glucose-phosphorylating enzyme is also involved inmammalian glucose signalling pathways

The glucose-sensing pathway of S. cerevisiae has certain

Fig. 1. A schematic model of the participation of Hxk2 in transcriptional regulation. The case of the regulation of SUC2 is shown. In high-glucose me-dia Mig1 is present in the nucleus and binds to a Mig1-binding site that overlaps with one of the two Med8-binding sequences in the SUC2 promoter.Nuclear Hxk2 may interact through di¡erent protein domains both with Mig1 and Med8, impairing Med8 function and facilitating the repression oftranscription by Mig1. In the absence of glucose, Mig1 is phosphorylated by the Snf1 protein kinase complex and translocated to the cytosol. HXK2 isexpressed at low levels or not at all ; Med8 binds to both Med8-binding sequences in the promoter, recruits the basal transcription machinery and tran-scription occurs. RNA polymerase II (RNAPII) represents in the picture the transcription machinery.

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similarities with the mammalian pathway controlling theglucose-induced expression of insulin in the pancreaticL-cell.

In most mammalian glucose-sensitive tissues, glucoseentry is mediated through speci¢c glucose transporters,such as Glut2p, in the liver and L-cells, and Glut4p, aninsulin-sensitive transporter, in adipocytes and muscle. Ithas been recently suggested that the large intracytoplasmicloop of Glut2p in the liver and L-cells could also play arole in the transmission of a signal [87,88], and it can bethus considered that the Glut2p function in L-cells hassimilarities with the function of the Snf3/Rgt2-sensor sys-tem of yeast.

Glucose regulates insulin production in pancreaticL-cells by stimulating the transcription of the insulingene. This e¡ect is mediated through a transcription factor(PDX1), which binds to speci¢c regulatory elements withinthe human insulin gene promoter and to the promoter ofseveral genes expressed preferentially in the L-cell, includ-ing those encoding Glut2p and glucokinase (GlkBp) [89].Glucose activates PDX1 by facilitating the phosphoryla-tion of a cytoplasmic form of the factor that translocatesto the nucleus [90] through a mechanism reminiscent ofthat described for Mig1 in yeast cells. Glucokinase fromL-cells and liver that has also been proposed to act as aglucose-sensor molecule [91,92] has a double cytosolic-nu-clear localization that is regulated by the nuclear proteinGkrp and by the glucose concentration [93^95]. Thus,mammalian glucokinase and yeast Hxk2 have a similarsubcellular distribution and both are necessary for theappropriate regulation of a network of glucose-responsivegenes. Interestingly, the expression of the GlkBp gene en-coding glucokinase in a hxk2 yeast mutant strain restoresboth the processes of glucose induction and glucose re-pression, showing that it acts similarly to its yeast counter-part [96].

5. Conclusions and perspectives

The results available at this moment clearly show theHxk2 as an important participant in the complex regula-tory system that mediates glucose repression and inductionin yeast. However, a series of questions concerning thissystem are still unanswered and some of them speci¢callypertain to Hxk2. We summarize brie£y some of these.Concerning the dual cytoplasmic-nuclear localization ofHxk2, it has not been established if the protein is in thenucleus in the phosphorylated or unphosphorylated form;also, it remains to be established how the transport to thenucleus is achieved. Moreover, the fate of the protein afterglucose withdrawal has not been studied. Another impor-tant unsolved question is that of the possible in£uence ofthe monomer^dimer equilibrium of the protein on thetransport. Up to now there are no results available con-cerning this point.

Another question that needs study is the analysis of thetranscriptional regulation of the HXK2 gene. The fact thatHxk2 itself participates in this regulation raises the questionof how the autoactivation of the transcription is controlled.

A ¢nal question is that of the eventual participation ofsome metabolites in the processes regulated by Hxk2.Although up to now largely ignored, it may well be quiteimportant and deserves consideration.

Acknowledgements

We are grateful to Juana M. Gancedo for many scien-ti¢c comments and to C. Gancedo for critical reading ofthe manuscript. The work from this laboratory was sup-ported by Grants PB97-1213-C02-02 and BMC2001-1690-C0202 from the Direccio¤n General de Investigacio¤n(DGI), Ministerio de Ciencia y Tecnolog|¤a.

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The hexokinase 2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae