2002 review-rolland et al glucose-sensing and -signalling mechanisms in

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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 for

the downregulation of respiration, gluconeogenesis and the transport and catabolic capacity of alternative sugars during growth on

glucose. The hexokinase 2- dependent glucose-sensing mechanism of this pathway is not well understood but the downstream part of the

pathway 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 been

identified. The Ras-cAMP pathway controls a wide variety of cellular properties in correlation with cellular proliferation. Glucose is a

potent activator of cAMP synthesis. In this case glucose sensing is carried out by two systems, a G-protein-coupled receptor system and a

still elusive glucose-phosphorylation-dependent system. The understanding of glucose sensing and signalling in yeast has made dramatic

advances in recent years and has become a strong paradigm for the elucidation of nutrient-sensing mechanisms in other eukaryotic

organisms. 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 changing

environment nutrient availability is the major factor con-

trolling growth and development. For yeasts, like for

many other micro-organisms, glucose is the preferred car-

bon and energy source. It is therefore not surprising that

glucose is an important primary messenger molecule, sig-

nalling optimal growth conditions to the cellular machin-

ery. Accordingly, glucose also aects 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, this

has stimulated the thorough characterization of a variety

of glucose-signalling pathways in Saccharomyces cerevisi-

ae. Whereas downstream components and their function-

ing have often been claried in great detail, elucidation of

the initial glucose-sensing and -activation mechanisms has

proven to be more dicult. This is largely due to the

sugars apostrophe dual function as a nutrient and signal-

ling molecule, and the intertwining of the molecular basis

of the two functions. Recently, however, substantial prog-

ress has been made with the identication of several pro-

teins with an apparently specic function in glucose sens-

ing. In higher multicellular organisms similar mechanisms

might be involved in the vital control of glucose homeo-

stasis.

2. Stationary phase, respiration and fermentation

Unicellular free-living organisms like yeasts in general

have adapted very well to constantly changing environ-

mental conditions. More specically, they have developed

mechanisms to respond to extreme variations in nutrient

availability by modulating their growth and metabolism.

The most dramatic eect in micro-organisms is observed

upon nutrient starvation. Micro-organisms are able to sur-

vive long periods of starvation by drastically decreasing

their 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 56 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 School

and Department of Molecular Biology, Massachusetts General Hospital,

Boston, MA 02114, USA.

FEMS Yeast Research 2 (2002) 183^201

www.fems-microbiology.org


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combined with a range of physiological and often also

morphological changes. These stationary-phase cells are

also characterized by their high tolerance to heat and oth-

er stress conditions and to cell wall-degrading enzymes. A

wide variety of genes involved in stress resistance is in-

duced and the reserve carbohydrate glycogen as well as

the stress protectant and reserve carbohydrate trehaloseaccumulate to high levels. Although yeasts are basically

unicellular 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 derivation

from multicellular ancestors. Such lamentous growth oc-

curs for instance when fermentable sugars are available

but nitrogen is lacking, presumably enabling the yeast to

actively search for a nitrogen source.

Yeast cells are not only able to detect the mere presence

or absence of nutrients, depending on the carbon source

available, they display totally dierent metabolic modes.Glucose-sensitive yeasts like S. cerevisiae and Schizosac-

charomyces pombe prefer fermentation over respiration

even under aerobic conditions. In these yeasts, synthesis

of 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 of

sugar, alcoholic fermentation yields fewer ATP equivalents

than respiration, it can proceed at much higher rates. This

enables these yeasts to compete eectively for survival,

especially because the ethanol produced during fermenta-

tion inhibits growth of competing micro-organisms. This

ethanol 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 are

able to respire and generate ATP from non-fermentable

carbon sources by mitochondrial oxidative phosphoryla-

tion. Cells that use non-fermentable carbon sources grow

much 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 genes

involved in stress resistance and accumulation of reserve

carbohydrates.

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 the

preferred carbon source. Glycolysis is activated and glu-

cose is almost completely converted into ethanol and car-

bon dioxide. While glucose inux and the ow through

glycolysis are stimulated, gluconeogenesis is inhibited. In

addition, there is a drastic increase in growth rate which is

preceded by a characteristic upshift in ribosomal RNA

and protein synthesis. Genes encoding enzymes involved

in the uptake and metabolization of alternative carbon

sources and gene products involved in stress resistance

are repressed. Reserve carbohydrates are mobilized.

Yeast cells use both positive and negative control mech-

anisms to regulate enzyme levels and activities in order to

accomplish this drastic metabolic switch. Enzyme levels

are regulated at the stage of gene transcription (repression

and induction), mRNA stability, translation and protein

stability, while enzyme activities are regulated post-tran-

scriptionally by allosteric and covalent activation and in-

hibition. Most of these processes are aected either di-rectly or indirectly by specic glucose sensing and signal

transduction pathways.

3. Glucose-signalling pathways

The major downregulating eect of glucose takes place

at the transcriptional level. One class of genes repressed by

glucose encodes proteins involved in respiration (Krebs

cycle and electron transport chain proteins), gluconeogen-

esis and the glyoxylate cycle. Another important class en-

codes proteins that are specically 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-anity glucose transport is repressed by high

levels of glucose. Several families of genes involved in the

use of other carbon sources are under control of family-

specic inducers enabling a co-ordinated regulation of

their expression. In the presence of glucose, the family-

specic inducers as well as the individual genes are subject

to repression. Finally, also a large group of STRE (stress

response element)-controlled genes encoding proteins pri-

marily involved in the yeasts response to various stresses

are repressed by glucose.

3.1. Glucose repression by the main glucose-repression

pathway

Not all glucose-repressible genes are repressed in the

same way but isolation and characterization of repression

and derepression mutants has identied 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 Mig1

transcriptional repressor complex, the Snf1-protein kinase

complex and protein phosphatase 1.Mig1 is a DNA-binding zinc-nger protein that recruits

the general co-repressor proteins Ssn6 and Tup1 to exert

repression of diverse gene families and their family-specic

transcriptional inducer genes [1]. Essential for the function

of 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 binds

to the promoters of glucose-repressible genes. When the

cells are deprived of glucose, Mig1 is rapidly transported

back to the cytoplasm [2]. In addition to Mig1 other

DNA-binding proteins (such as its homolog Mig2) are

involved in glucose repression.

F. Rolland et al./ FEMS Yeast Research 2 (2002) 183 201184


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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 probably

phosphorylates and thereby causes translocation of Mig1

to the cytoplasm [2^4]. The Snf1 Ser/Thr kinase is associ-

ated with an activating subunit and three scaolding pro-

teins in high-molecular-mass complexes. The activating

subunit 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 specicity

to the kinase complex [8], possibly through regulation of

its 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 the

observed alterations in protein interactions within the

complex, the Snf1 regulatory domain auto-inhibits the cat-

alytic domain in glucose-grown cells. In the absence of

glucose, however, the Snf4-activating subunit binds to

the Snf1 regulatory domain, counteracting the auto-inhib-

itory interaction and thereby enabling Mig1 phosphoryla-

tion and its translocation to the cytoplasm [5]. The glucose

signal 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 as

well as (Mig1-dependent) expression of the two zinc-clus-

ter-activator proteins Cat8 and Sip4 which are involved in

the induction of gluconeogenic genes through carbon

source-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 that

activates Snf1 by phosphorylating a conserved Thr kinase

phosphorylation 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-specic regulatory subunit Reg1/

Hex2 targets its activity to the activated Snf1 kinase do-

main, presumably dephosphorylating Snf1 or another

component of the complex and facilitating the return to

the auto-inhibited state [16^18]. Hence, although the glu-

cose signal most likely inhibits the initial phosphorylation

of Snf1, it may also activate Reg1-Glc7 phosphatase 1

function.

3.2. Glucose induction

Yeast cells growing on glucose obtain their energymainly through fermentation. Since fermentation is a rel-

atively inecient way of generating energy, a high glyco-

lytic ux is essential. Yeast cells are able to increase their

glycolytic capacity by the induction of a large number of

glycolytic genes. In addition, glucose-uptake capacity is

increased through the induction of several glucose-trans-

porter-encoding HXT genes. Separate signal transduction

pathways and mechanisms seem to be involved.

In the presence of rapidly fermentable sugars yeast gly-

colysis is fully activated. Glucose causes a fast increase

and transient overshoot in glycolytic intermediates and

mutant studies have shown that increased levels of dier-

Fig. 1. The main glucose-repression pathway. A : Simplied schematic

representation of mediators and targets of the main catabolite-repression

pathway. Repression is exerted by the complex Mig1/Ssn6/Tup1 on dif-

ferent gene families including family-specic transcriptional activators

such 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 and

the activating subunit Snf4 has a negative eect on the activity of the

repression complex. During growth on glucose, Snf1 activity is inhibited

by dierent upstream regulators which include the hexose kinases and

the 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 conformational

change of the Snf1-protein kinase complex.

F. Rolland et al./ FEMS Yeast Research 2 (2002) 183 201 185


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ent metabolites trigger the induction of glycolytic genes

[19]. Dierent 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 ecient

way. How these metabolic signals are transmitted is still

unclear but in several genes sequence elements have beendened that are responsible for sugar-induced expression

and dierent DNA-binding factors have been identied

that are required for high-level expression of glycolytic

genes. 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 regulator

of transcription that binds to the CTTCC motif which is

conserved in most glycolytic genes [20,21]. The glycolytic

pathway 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 activation

of 6-phosphofructo-2-kinase (PF2K) which catalyzes syn-thesis of fructose-2,6-bisP, one of the allosteric regulators

in glycolysis [19]. In addition, rapid inactivation of gluco-

neogenesis is required for an ecient start-up of glycolysis.

S. cerevisiaecontains a whole series of hexose transport-

ers homologues (Hxt1-17, Gal2, Snf3 and Rgt2), all dis-

playing dierent substrate anities and expression pat-

terns [22^24]. Depending on the amount of glucose

present in the medium, specic transporters are expressed.

The mechanisms involved in the expression of the appro-

priate transporters and their post-translational modica-

tion have recently become more clear. High-anity trans-

porters like Hxt6 and Hxt7 are highly expressed on non-

fermentable carbon-sources and repressed by high levels ofglucose, whereas transporters with low anity, such as

Hxt1 and Hxt3, are induced by the presence of a high

concentration of glucose. The transporters with intermedi-

ate anity for glucose like Hxt2 and Hxt4, on the other

hand, are induced by low levels of glucose and repressed

by high levels of glucose. As shown in Fig. 2, both the

intermediate and the low-anity transporters are re-

pressed by Rgt1 in the absence of glucose. Rgt1 is a

zinc-nger-containing DNA-binding protein that, like

Mig1, recruits the Ssn6 repressor to the promoters of spe-

cic genes [25]. Low amounts of glucose inhibit Rgt1-re-

pressor function, resulting in derepression ofHXTexpres-sion. This inhibition requires the presence of the Grr1

protein [26]. This protein is part of a multiprotein SCF

complex containing the Skp1, Cdc53 and Cdc34 proteins

and the F-box Grr1 protein [27^29]. SCF complexes direct

protein ubiquitination and dier in their F-box-containing

component which is thought to recruit specic substrates

to the complex. Subsequent ubiquitination then marks

the substrate for degradation [30]. Glucose derepression

apparently involves ubiquitin-mediated proteolysis but it

is not clear whether the SCF complex directly modies

Rgt1. The HXT2 and HXT4 genes which encode trans-

porters with intermediate anity for glucose are repressed

by high glucose levels. This repression is mediated by the

Mig1 main glucose-repression pathway [26]. Also, repres-

sion of the high-anity transporter HXT6 is, at least in

part, mediated by the main glucose-repression pathway

[24]. However, in contrast to other glucose-repressed

genes, maintenance of HXT6repression is strictly depen-

dent on Snf3 [31]. Expression of HXT1, encoding a low-anity transporter is further induced by high glucose lev-

els. Besides the Grr1-Rgt1-dependent pathway, this also

involves 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 unidentied transcriptional activator or, alter-

natively, an additional Ssn6-dependent repression mecha-

nism that is inactivated by high levels of glucose. Full

induction ofHXT1 expression at high glucose concentra-

tions, however, does require Rgt1. Rgt1 apparently can be

converted into an activator of HXT1 expression under

these 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, glucose

transport is also subject to extensive post-translational

regulation [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 ATP

is catalyzed by the enzyme adenylate cyclase and cAMP

activates 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). In

derepressed yeast cells (growing on a non-fermentable car-

bon source or in stationary phase) rapidly-fermentable

sugars, and especially glucose, trigger a rapid, transient

increase in the cAMP level, initiating a PKA phosphory-

lation cascade. Also, intracellular acidication is able to

trigger a pronounced increase in the cAMP level. Like in

higher eukaryotes, yeast adenylate cyclase activity is con-

trolled by G-proteins. Remarkably, in S. cerevisiae the

two small G-proteins Ras1 and Ras2 are essential for ade-nylate cyclase activity. They therefore have been thought

for many years to act as functional equivalents of the

mammalian heterotrimeric GK-proteins of adenylate cy-

clase. Recently, however, a G-protein-coupled receptor

(GPCR) system has been identied that specically 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 has

been proposed as a link between nutrient availability and

cell 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-induced

activation of adenylate cyclase is repressed by glucose and

therefore considered not to be operative during growth onglucose. This appears to conne the physiological role of

this pathway to the short period of transition from the

derepressed state to the repressed state by means of a

cAMP-triggered protein phosphorylation cascade. Most

of the downstream targets of PKA identied are enzymes

involved 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 stimulates

growth and proliferation [36]. In their natural environ-ment, yeast cells experience long periods of nutrient star-

vation, alternating with very short periods of nutrient

abundance. Under such conditions, fast recovery from sta-

tionary phase and initiation of fermentation clearly oer a

selective advantage [35].

Initiation of fermentation also coincides with a loss of

stress resistance. Two multicopy suppressors of the snf1

defect, 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 to

STREs 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 genes

which are dependent on Msn2 and Msn4 for induction

upon sugar depletion was found to be repressed by

cAMP [40,41]. Moreover, PKA activity was shown to bedispensable in a strain lacking Msn2 and Msn4, indicating

that Msn2/4-dependent gene expression actually accounts

for many of the pleiotropic eects of PKA. PKA appar-

ently regulates processes such as glycogen accumulation

and stress response as well as growth by suppression of

Msn2/4-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 control

of stress resistance is supported by the isolation of mu-

tants decient in fermentation-induced loss of stress resis-tance (l). The l1 mutant carries a point mutation in the

CYR1/CDC35 gene [44], encoding adenylate cyclase, con-

sistent with the previous isolation of stress-resistant ade-

nylate cyclase mutants [45]. Interestingly, the l1 mutant

still displays wild-type growth and fermentation rates, as

opposed to other mutants with reduced activity of the

cAMP pathway. The l2 mutation was identied in the

gene encoding the GPCR Gpr1, which is specically in-

volved in glucose activation of cAMP synthesis [46]. In-

terestingly, a positive correlation was reported between

activity of the PKA pathway and longevity [47].

The observation that cAMP synthesis is apparently only

Fig. 2. Regulation of HXTtransporter 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 SNF3

are repressed by Mig1 via the main glucose-repression pathway. In addition, Snf3 is involved in a second pathway leading to the high-glucose-induced

repression ofHXT6.



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activated by rapidly-fermentable sugars and not by other

nutrients, and the glucose-repressible character of the ac-

tivation mechanism, seem to argue against a role for

cAMP-signalling in growth control by nutrients. Bcy1 v

mutants with attenuated catalytic subunits have indeed

been shown to respond appropriately to nutritional stress

conditions, even in the absence of adenylate cyclase [48].

This indicates that cAMP-independent mechanisms exist

for 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 signal

is 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 dierent 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 to

the cAMP pathway the intra- and extracellular glucose-sensing process is apparently able to sustain activation of the pathway separately. Detection of

other nutrients seems to be triggered by specic transporters such as Gap1 for amino acids and Mep2 for ammonium.



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sources) are able to trigger similar eects on the PKA

targets when re-added to cells starved for such a nutrient.

These eects are independent of activation of cAMP syn-

thesis but are still dependent on the free catalytic Tpk

subunits (Fig. 3B). Since these eects require the presence

of both a fermentable carbon source and a complete

growth medium for sustained activation, the signalling

pathway involved has been called the fermentable-growth-medium-induced (FGM) pathway [34,49]. The

FGM pathway controls PKA targets during growth on

glucose through the Sch9-protein kinase [50].

Often dierent mechanisms and signal transduction

pathways collaborate to control enzyme levels and activ-

ities. The extreme glucose sensitivity of the gluconeogenic

enzymes for example is mediated by glucose-induced allo-

steric inhibition, covalent modication and protein degra-

dation [51^53] as well as transcriptional repression and

accelerated mRNA degradation [54^56]. The combination

of 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 carbon

sources, such as maltose and galactose, repression of

gene expression is preceded by rapid glucose-induced in-

activation and degradation [57].

4. Glucose-sensing mechanisms

The dramatic eects of glucose on growth and metabo-

lism clearly support a hormone-like function for this sugar

in yeast cells. However, since it is also taken up and me-

tabolized as a nutrient, glucose can be detected by the cells

in many more ways than is the case for classical primary

messenger molecules [58]. Although cells could use the

activity of a component of the existing metabolic machin-

ery or the level of one or more glucose catabolites to

detect its presence (and metabolization), the lack of spec-

icity of such a system could have stimulated the develop-

ment of more specic sensors as illustrated in Fig. 4. Re-ceptors could have evolved from existing glucose-binding

proteins such as transporters or kinases (with or without

maintenance of the catalytic activity) or members of more

classical receptor families could have been recruited and

modied (or used originally) to gather specic information

on the nutritional status in the environment.

There is now much evidence that yeast uses a whole

range of such sensing mechanisms to ne-tune growth

and metabolic activity to the amount and quality of the

sugars available. For genes encoding glycolytic enzymes

and requiring glucose for full expression, induction by

glucose was shown to depend on the accumulation of in-termediary metabolites. For some genes, an increase in the

level of hexose-6-phosphates is required while for others

induction is triggered by glycolytic three-carbon metabo-

lites [19,59,60]. Also, for glucose sensing and signalling in

pancreatic L-cells a more extensive metabolization of the

sugar is required since it appears that the actual trigger for

insulin release is the ATP produced in glycolysis and res-

piration. An increase in the ATP:ADP ratio inhibits ATP-

sensitive K-channels. Membrane depolarization then ac-

tivates voltage-gated Ca2-channels, triggering a rise in

intracellular Ca2 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 specic 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 the

sensor 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), either

glucose-6-phosphate or a downstream metabolite.



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deletion suppresses the ranose growth defect of an snf3

mutant as well as the glucose fermentation defect of snf3

rgt2 double mutants through increased and unregulated

expression of the HXT2, HXT3 and HXT4 hexose trans-

porter genes. Deletion of STD1 cannot suppress the fer-

mentation defect but specically increases HXTexpression

in the presence of low glucose concentrations. Std1 andMth1 apparently act through distinct pathways and, like

Snf3 and Rgt2, respond to dierent levels of glucose. Std1

was shown to act upstream of the Snf1 kinase, both for

derepression of SUC2 and high-anity transporter gene

expression and repression of HXT1 expression, whereas

Mth1 mediates repression via an Snf1-independent path-

way [72]. The mutant forms ofMTH1 (HTR1, DGT1 and

BPC1) apparently block transduction of the Snf3- and

Rgt2-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 periphery

and 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 Snf3

overexpression blocks the ability of Std1 to induce

SUC2 expression suggest that the glucose sensors and

the Std1 and Mth1 proteins act antagonistically, with the

sensors being required for HXT induction and the Std1

and Mth1 proteins being required for their repression

[72]. Possibly, Snf3 and Rgt2 inhibit the negative (repres-

sing) eects of Mth1 and Std1 by sequestering them at the

plasma membrane [73].

The observation that Rgt2 and Snf3 alone do not sus-

tain transport of glucose to enable growth, (not even when

overexpressed) and isolation of the dominant RGT2-1 andSNF3-1 mutations in the fth cytoplasmic loop, have led

to the hypothesis that Snf3 and Rgt2 function as classical

signal receptors, in which binding of the extracellular li-

gand, in this case glucose, causes a conformational change

in a cytoplasmic domain [66]. Consistently, the hxt1-7v

strain, which is decient in glucose uptake was reported

to 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 full

induction ofHXT expression [26]. Phosphorylation of the

sugar 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 strain

may be sucient to enable signalling.

The use of transporter-like proteins as nutrient sensors

may be a more common strategy in eukaryotic cells. The

conserved sequence motif in the C-termini of Rgt2 and

Snf3 is also present in the C-terminal extension of Rag4

from Kluyveromyces lactis. Rag4 was shown to control

expression of the Rag1 glucose transporter [81] and may

function both as a high- and low-anity glucose sensor

[82]. In Neurospora crassa, the transporter homolog and

glucose 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 permeases

and contains an N-terminal cytoplasmic tail, was shown

to act as an amino acid sensor controlling amino acid

permease gene expression. In Arabidopsis thaliana, evi-

dence exists for specic Hxk-independent sucrose sensing

and transporter homologs have been proposed to act as

sucrose 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 transporter

Mep2 in nitrogen sensing for control of pseudohyphal

growth in yeast has been reported [86]. Also, for Gap1,

evidence for a role in amino acid sensing for control of the

PKA- 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 nutrient

transporters with a sensing function might also be present.

Recently, a regulatory function for mammalian Glut1 in

glucose-induced activation of ERK (MAPK)-signallinghas been suggested [87].

4.2. Glucose repression: the Hxk glucose sensor

A second mechanism controlling expression of sugar

transporters is the main glucose-repression pathway. In

the presence of glucose, high-anity glucose transport is

repressed together with a broad range of other genes in-

volved in the utilization of alternative carbon sources. In

addition, also Hxk1 and glucokinase (Glk1) are repressed

by glucose. Both high-anity transporters and sugar ki-

nases appear to be involved at least to some extent in

triggering 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 that

specic glucose transporters or pairs of them could play a

role in triggering glucose-induced regulatory responses

[34,78,88,89]. The requirement of a signicant amount of

glucose (s 20 mM) to fully trigger these signalling path-

ways was thought to indicate the specic involvement of

low-anity transport. Experiments with yeast strains ex-

pressing individual transporters showed that triggering ofglucose repression is not dependent on a specic hexose

transporter protein but rather correlates with the glucose

uptake activity of the cells and with glycolytic ux [90,91].

HXK2was identied as one of the rst genes involved in

glucose repression [92^94] but whether its requirement for

signalling simply reects the need for glucose phosphory-

lation or involves a separate regulatory function for the

Hxk2 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 repression

and it was proposed that this kinase was a bifunctional

enzyme with catalytic and regulatory domains for glucose



10/19

repression [95,96]. However, this could not be conrmed

in later experiments; in a large number of mutant Hxk

strains a good correlation between glucose repression

and residual phosphorylating capacity of the mutated

Hxk was observed [97]. A similar correlation was observed

with a series of Hxk1^Hxk2 hybrid constructs [98]. Also,

when Hxk1 was removed in addition to Hxk2, glucoserepression further diminished. Stable overexpression

showed that Hxk1 was also capable of mediating glucose

repression at least to a certain extent [98]. Interestingly, no

further metabolization beyond the sugar phosphorylation

step 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 was

used to isolate glucose-repression mutants which, as op-

posed to wild-type cells, are able to grow on ranose in itspresence [92]. Overexpression of GLK1 did not restore

glucose repression in a Hxk mutant [98] indicating that

glucose phosphorylation by itself is not sucient to trigger

glucose repression. These results supported the idea of a

specic function of the Hxk proteins in the activation

mechanism of glucose repression. More recently, new

data on the dierential requirement of the sugar kinases

in short- and long-term glucose and fructose repression

and the complex transcriptional regulation of the kinases

themselves has put the predominant role of Hxk2 in a new

light. Catabolite repression was shown to involve two dis-

tinct mechanisms: an initial rapid response is mediated

through any kinase able to phosphorylate the sugar, in-cluding Glk1, while long-term repression specically re-

quires Hxk2 for repression by glucose and either Hxk1

or Hxk2 for repression by fructose [100,101]. Both

HXK1 and GLK1 are repressed upon addition of glucose

or fructose but fructose repression ofHXK1 is only tran-

sient. This is consistent with the preference of Hxk1 for

fructose as a substrate and its requirement for long-term

fructose repression [101]. Apparently, activation of catab-

olite repression is controlled by a complex interregulatory

network, involving all three sugar kinases and the mecha-

nisms and pathways controlling their expression. In this

way not only the main glucose-repression pathway itselfbut also cAMP signalling indirectly aects catabolite re-

pression [101]. Consistently, rapid repression of the gluco-

neogenic genes FBP1 and PCK1 by very low levels of

glucose was shown to be triggered in the presence of any

one of the three kinases, whereas in the presence of high

glucose levels repression was mediated specically by the

Hxk2-dependent Mig1-repression mechanism [55]. It was

proposed thatHXK2gene expression could act as a sensor

for the glucose concentration in the medium [102]. Also,

more recently, novel alleles of Hxk2 have been isolated

that have distinct eects on catalytic activity and catabo-

lite repression ofSUC2 [46,103,104] and long- and short-

term phases of catabolite repression could be dissected

[103]. The lack of correlation between in vitro catalytic

activity of Hxk, in vivo sugar phosphate accumulation

and the establishment of catabolite repression again sug-

gested that the production of sugar phosphate is not the

only role of Hxk in repression but that also a regulatory

signalling site of the protein may be required (Fig. 5A).For galactokinase a clear distinction between the catalytic

function and the regulatory function in induction ofGAL

gene expression was made [105]. A similar situation might

apply to Hxk2. Structure^function analysis of Hxk2 more

specically suggests that the establishment of catabolite

repression is dependent on the onset of the phosphoryl

transfer reaction on Hxk and is probably related to the

stable 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 and

extensive mutant analysis seems to uncouple regulatory

and 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 a

putative glucose sensor have been identied, it still re-

mains to be elucidated what the actual glucose signal is

that triggers glucose repression. Since the rate of glucose

transport and phosphorylation correlate well with the level

of 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 some

sort of sensor for sugar phosphorylation and metabolic

activity (Fig. 5B). One model proposes a signalling role

for these nucleotides in triggering glucose repression based

on 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]. Mammalian

AMPK is involved in the cellular response to a variety

of stresses, like heat shock and nutrient starvation. Inacti-

vation of a number of biosynthetic enzymes under these

conditions ensures better conservation of cellular ATP[10,108]. Likewise, since it is responsible for triggering

derepression, Snf1 is involved indirectly in the generation

of ATP by enabling the cells to metabolize alternative

carbon sources in the absence of fermentable amounts of

glucose. 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, the

AMP level is repleted, resulting in a high AMP/ATP ratio

which could then activate Snf1 and relieve repression.

Thus, in this model the triggering signal for repression is



11/19

generated by the metabolism of glucose, consistent with

the predominant role of Hxk2 in both glucose phosphor-

ylation during fermentative growth and glucose repression.

However, although an increase in the AMP/ATP ratio was

observed when repressed cells are shifted to low-glucose

medium [10], AMP and ATP levels during growth on

glycerol and glucose appear to be very similar [109]. In

addition, this model does not t with the indications for

a separate regulatory function for Hxk. Snf-related protein

kinase (SnRK) signalling is also conserved in plants. PlantSnf1 homologs have been shown to complement yeast snf1

mutants and are proposed to act as global regulators of

carbon metabolism in plants [110]. As in yeast, plant

SnRKs are not directly activated by AMP although

AMP seems to inhibit their dephosphorylation [111]. In-

terestingly, glucose-6-P was reported to negatively regulate

a plant SnRK [112].

Yeast Hxk2 has recently been shown to have a role in

regulating the phosphorylation status of the regulatory

subunit of protein phosphatase 1 Reg1/Hex2. Reg1 is

phosphorylated in response to glucose limitation in an

Snf1-dependent way and dephosphorylated by Glc7when glucose is present. Phosphorylation of Reg1 by

Snf1 appears to stimulate both Glc7 activity in promoting

closure of the Snf1 complex and release of Reg1-Glc7

from the kinase complex. Hxk2 either stimulates binding

and/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 in

the cell nucleus [113] and that this nuclear localization,

which is dependent on a specic internal nuclear localiza-

tion sequence, is necessary for glucose-repression signal-

ling [114]. Furthermore, the Hxk2 protein was shown to

participate 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 aect nuclear

targetting [114]. Phosphorylation and the concomitant in-

crease in glucose anity of monomeric Hxk could providea mechanism to optimize glucose utilization at low con-

centrations [115], but although protein phosphatase 1 is

involved in dephosphorylation of the Hxk2 monomer

[113,116] seemingly contradictory results were obtained

as to whether this phosphorylation/dephosphorylation is

involved in signalling [97,113,114].

4.3. cAMP signalling: a dual sensing system

Experiments with hexose kinase and other glycolysis

mutants showed that transport and phosphorylation but

no 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 activation

reaction: the increase in the level of glucose-6-P after ad-

dition of dierent glucose concentrations did not show a

good correlation with the increase in the cAMP level.

From the increase in the cAMP level after addition of

dierent extracellular glucose concentrations an apparent

Ka for the activation mechanism of about 25 mM was

deduced, tting with the Km of what was believed to be

the low-anity glucose transporter system but diering by

at least one order of magnitude from the Km values of the

three 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 the

regulatory function can be closely associated with the catalytic activity, neither the substrates glucose and ATP nor the products glucose-6-phosphate

and 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 dierent components of the Snf1 kinase complex.



12/19

the primary triggering reaction was situated at the level of

transport and phosphorylation, possibly even transport-

associated phosphorylation. Glucose-induced cAMP-sig-

nalling is indeed dependent on transport of the sugar

but not on any specic glucose transporter. Also, the

transporter homologs and putative glucose sensors Rgt2

and Snf3 are not directly involved in this glucose-sensingprocess [58]. The role of sugar transport is apparently

limited to the provision of a sucient amount of substrate.

Although glucose-6-P does not seem to be the metabolic

messenger for activation, in Hxk mutants a clear correla-

tion was always observed between catalytic activity and

the triggering of cAMP signalling [46,103]. Apparently,

the role of yeast Hxk in sugar-induced activation of

cAMP signalling is closely connected to the catalytic func-

tion of the enzyme. How glucose phosphorylation is

coupled to the control of cAMP synthesis is still unclear.

Basal activity of the cAMP pathway is essential for via-

bility and this makes it dicult 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 are

signal transmitters in the pathway leading from glucose

to adenylate cyclase [118]. Subsequently also Cdc25, the

Ras-GEF, was shown to be involved in glucose-induced

activation of cAMP synthesis [119^121]. Glucose therefore

appeared to be a direct or indirect stimulator of Cdc25.

Recently, the possible involvement of the Ras proteins in

glucose signalling was investigated more directly [122]. In-

tracellular acidication, another stimulator of in vivo

cAMP synthesis, but not glucose, caused an increase inthe GTP/GDP ratio on the Ras proteins. Stimulation of

cAMP synthesis by glucose was shown to be dependent on

another G-protein, Gpa2. The GPA2 gene was originally

cloned as a yeast homolog of mammalian heterotrimeric

Ga-proteins and was already implicated in cAMP signal-

ling. However, although overexpression of the gene clearly

aected cAMP levels, no eect was observed in a gpa2v

strain on glucose-induced cAMP signalling [123,124]. This

was later shown to be due to interference with the eect of

intracellular acidication caused by the addition of glu-

cose. The increase in cAMP observed after addition of

100 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 the

control of the basal cAMP level. Moreover, although de-

letion ofGPA2 confers to some extent the typical pheno-

type associated with a reduced level of cAMP, the function

of Gpa2 appears to be limited mainly to the stimulation of

cAMP synthesis during the transition from respirative

growth 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, like

Gpa2, was shown to be specically required for glucose

activation of the cAMP pathway during the transition to

growth on glucose and a gpr1v mutant could be rescued

by the constitutively activated GPA2val132 allele [46]. Ap-

parently, Gpr1 and Gpa2 constitute a glucose-sensing

GPCR system for activation of the cAMP pathway (Fig.3). This not only brings the yeast adenylate cyclase system

back in line with the mammalian system of adenylate cy-

clase control, it also appears to be the rst example of a

GPCR system activated by a nutrient in eukaryotic cells.

S. pombe contains a similar glucose-sensing GPCR system

for activation of cAMP synthesis (consisting of the GK-

protein gpa2 and the putative glucose receptor git3) and

also 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-inducedcAMP accumulation, GPR1 was also isolated as a mutant

allele (l2) in a screen for mutants decient in fermenta-

tion-induced loss (l) of heat resistance [46]. In a similar

screen, 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) domain

and was indeed shown to negatively regulate glucose acti-

vation of the cAMP pathway through direct inhibition of

Gpa2. Consistent with its homology to other RGS pro-

teins, Rgs2 acts as a stimulator of the GTPase activity of

Gpa2. It remains to be shown, however, that Gpa2 indeed

acts as the signal transducer from glucose to adenylatecyclase. The fact that deletion of GPA2 is lethal in the

absence of Ras2 is consistent with a role for Gpa2 as

stimulator of adenylate cyclase [130].

A mutation in the catalytic domain of adenylate cyclase

(cyr1met1876) has been identied that specically aects glu-

cose- and acidication-induced cAMP signalling and not

the basal cAMP level [131]. This lcr1 (lack of cAMP re-

sponse) mutation not only abolishes the cAMP signal but

also 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 overactivating

eect 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 lcr1

mutation only results in a delay in glucose-induced

changes in PKA targets associated with the adaptation

to growth on glucose, and does not aect the typical var-

iations of PKA-controlled phenotypic properties during

diauxic 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 dierentiation is induced

in diploid cells in response to nitrogen starvation in the



13/19

presence of a fermentable carbon source and is mediated

both by the pheromone-responsive MAPK cascade and

the 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 is

an element of the nitrogen-sensing machinery that regu-

lates pseudohyphal dierentiation by modulating cAMPlevels [133]. However, genetic and physiological studies

on pseudohyphal growth recently conrmed that the

Gpr1^Gpa2 GPCR system is activated by glucose. Be-

cause of the fact that GPR1expression is induced by nitro-

gen starvation, it is proposed that the receptor acts as a

dual sensor for both abundant carbon and nitrogen star-

vation [134]. Obviously, the demonstration that Gpr1 itself

binds glucose and acts as a real glucose receptor is an

important issue.

As mentioned before, elucidation of the exact mecha-

nisms of glucose sensing is often complicated because of

the requirement for partial metabolism of the glucose.This is also the case for the glucose-induced activation

mechanism of cAMP synthesis and the involvement of

the Gpr1^Gpa2 GPCR system. In spite of this, an actual

glucose-sensing function for Gpr1 has recently become

more 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 GPCR

system but also on transport and phosphorylation of the

sugar (Fig. 6A). We showed that it is possible to uncouple

the GPCR-dependent sensing process from the glucose

phosphorylation. 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 aect the cAMP level by itself but

apparently fullls the glucose phosphorylation require-

ment for activation of the cAMP pathway by glucose,

which in the Hxt null strain cannot be transported into

the cell. Using this set-up it was shown that the GPCRGpr1 or at least the glucose-sensing mechanism that is

dependent on it, specically responds to extracellular glu-

cose (and also sucrose, but not fructose or other sugars)

with low apparent anity. This is consistent with the fact

that yeast cells switch metabolism to the fermentative

mode only at glucose concentrations of at least 20 mM.

Interestingly, the presence of the constitutively active

GPA2val132 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 as

5 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. In

conclusion, the two essential requirements for glucose-in-

duced activation of cAMP synthesis can be fullled sepa-

rately. It remains unclear at what point the two require-

ments are integrated. Apparently, glucose phosphorylation

is required in some way to make adenylate cyclase respon-

sive to activation by the GPCR system. Since no increase

in the GTP/GDP ratio of Ras is observed after addition of

glucose, it seems unlikely that the hexose kinase-dependent

sensing system acts through Cdc25-Ras2. The kinases

might also act directly on adenylate cyclase, possibly re-

leasing inhibition of catalytic activity by the N-terminalregulatory domain.

Fig. 6. Fulllment 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 fullled by addition of a low level of maltose which is transported

and hydrolyzed by a specic system consisting of the maltose transporter (MalT) and maltase (MalS).



14/19

The demonstration that the Gpr1^Gpa2 GPCR system

is responsible for glucose control of the cAMP pathway

has brought up again the question as to what is the actual

function of Ras-dependent control of adenylate cyclase in

yeast [35]. Ras mutants exist which seem to be specically

aected in signal transduction. Strains carrying the

ras2ser318 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 decient in post-translational modication

of Ras are specically decient in cAMP signalling [136].

Many results indicating a role for the Cdc25-Ras system in

glucose-induced cAMP signalling could possibly be ex-

plained by their requirement for localization of adenylate

cyclase to the plasma membrane. Consistent with such a

role for Cdc25 is the recent evidence of direct binding of

Cdc25 to adenylate cyclase through an SH3 domain. This

binding might promote an ecient assembly of the ade-

nylate cyclase complex [137]. Proper membrane localiza-tion of the adenylate cyclase complex might be essential

for optimal interaction with and activation by the Gpr1^

Gpa2 system. The main function of the Ras proteins

might 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 the

cAMP level is clearly essential, especially under less-favor-

able conditions that require slow growth and high stress

resistance. The association with Ras might increase the

responsiveness of adenylate cyclase to stimulation by the

GPCR system when it is activated by a high level of glu-

cose in the medium.

FGM signalling still occurs in hxk1vhxk2vglk1 andgpr1v 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-sensing

systems might be sucient for FGM signalling while they

are 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 eects require a sig-nal transduction mechanism. Allosteric activation and in-

hibition is exerted by metabolic intermediates of glucose

catabolism. Allosteric regulation has been studied rst

with 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 be

fructose-2,6-bisP and fructose-1,6-bisP, controlling two of

its 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 the

reverse 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 PYK1

does not change glycolytic ux signicantly [139] and mu-

tant studies of PF2Ks did not reveal an essential role for

fructose-2,6-bis-P in the regulation of carbon uxes in

yeast cells [140]. Apparently, these enzymes do not cata-lyze rate-limiting steps in glycolysis and allosteric eects

appear to control metabolite homeostasis rather then met-

abolic uxes. Metabolic control analysis indeed pointed to

sugar uptake as the major ux-controlling step in glycol-

ysis [141]. The control coecient of glucose transport was

calculated to be signicantly higher then that of PFK. The

high level of control by transport over growth and glyco-

lytic ux has also been conrmed in an hxt null strain

expressing a single transporter [91]. More recently, the

trehalose-6-phosphate synthase subunit of the trehalose

synthase complex was found to control in some way the

entry of glucose into glycolysis [88]. While in mammaliancells glucose-6-P is the most important allosteric inhibitor

of the hexose kinases, in yeast cells this function appears

to be exerted by trehalose-6-phosphate [142]. In addition,

there is evidence for the possible involvement of the Tps1

protein itself in controlling glycolytic ux [143,144]. Prop-

er control of glucose inux into glycolysis is required for a

wide range of glucose-signalling eects in yeast, as was

demonstrated with the tps1v mutant which is unable to

synthesize trehalose-6-phosphate and therefore shows a

severe deregulation of glycolysis after addition of glucose

[145]. This indicates that the research on glucose-sensing

mechanisms cannot be seen as separate from that on glu-

cose metabolism, and that for every mutant aected inglucose signalling, investigation of possible interference

with glucose metabolism is of paramount importance.

5. Conclusions and perspectives

The preference ofS. cerevisiae for glucose as a carbon

and energy source is reected by the variety of glucose-

sensing and -signalling mechanisms ensuring its optimal

use. Nutrient-sensing and -signalling mechanisms must

have evolved early in evolution and might be at the origin

of the sophisticated hormone- and growth factor-inducedsignal transduction pathways best known from research

on mammalian cells. The glucose-sensing mechanisms in

yeast are therefore an excellent model system for studying

signal transduction in general. Glucose is also the prime

carbon 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 for

maintenance of sugar homeostasis [58]. In mammals glu-

cose serves as the blood sugar and maintenance of the

glucose concentration within narrow limits is controlled

by a complex interplay of several endocrine and neural

glucostatic systems that direct its uptake and release



15/19

[146]. In addition, glucose also plays a more direct role in

transcriptional 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 aects many aspects of

growth, metabolism and development throughout the

whole plant life cycle [148,149]. It is therefore likely thathigher eukaryotes are equally well supplied with glucose-

sensing and -signalling mechanisms. One striking example

of 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 with

increased resistance to (oxidative) stress and the stress-re-

sistance transcription factors Msn2 and Msn4 were indeed

shown to be required for life-span extension in Sch9 and

adenylate cyclase mutants [150]. Another report empha-

sized the requirement of NAD (and its regulation of the

silencing 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 eects

on longevity as caloric restriction (growth on low glucose),

conrming the role of this GPCR system in the sensing of

high glucose concentrations. Sch9 shows most similarity to

Akt/PKB. This protein kinase is involved in a signalling

pathway controlled by an insulin receptor-like protein and

regulating carbon metabolism, stress resistance and lon-

gevity in Caenorhabditis elegans [151]. Since also human

Akt/PKB is involved in insulin signalling, translocation of

glucose 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) may

have been conserved in all eukaryotic organisms [150].

Interestingly, also yeast Snf1 in addition to cellular energy

utilization was reported to control aging, and conserved

homologs in other organisms might have similar eects.

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 similar

specic sensing mechanisms as in yeast are also present

in higher eukaryotes. An abundance of results seems to

point at a central role for Hxk in eukaryotic glucose sens-ing. Although structure^function analysis and mutagenesis

have enabled separation of catalytic and regulatory activ-

ity to some extent, more detailed analysis will be required

to elucidate the actual Hxk- sensing and -signalling mech-

anism. Subcellular localization might be an important fac-

tor in Hxk regulatory function. In addition, more specic

sensors might be involved in higher eukaryotic glucose

sensing. As mentioned above, glucose transporter-like pro-

teins might have tissue- or cell-specic regulatory func-

tions in mammals and plants. Finally, classical receptor

families may be involved, as shown by the example of

yeast Gpr1. The elucidation of the yeast glucose-sensing

GPCR system obviously tempts to speculate that amongst

the hundreds of eukaryotic orphan receptors a subfamily

of nutrient sensors is waiting to be discovered.

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