The early steps of glucose signalling in yeast

R E V I E W A R T I C L E

The early stepsofglucose signalling inyeastJuana M. Gancedo

Department of Metabolism and Cell Signalling, Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, Madrid, Spain

Correspondence: Juana M. Gancedo,

Department of Metabolism and Cell

Signalling, Instituto de Investigaciones

Biomedicas Alberto Sols, CSIC-UAM, Madrid,

Spain. Tel: 134 91 585 4433; fax: 134 91

585 4401; e-mail: [email protected]

Received 29 October 2007; revised 25 March

2008; accepted 22 April 2008.

First published online June 2008.

DOI:10.1111/j.1574-6976.2008.00117.x

Editor: Martin Kupiec

Keywords

glucose signalling; sensing elements; yeast.

Abstract

In the presence of glucose, yeast undergoes an important remodelling of its

metabolism. There are changes in the concentration of intracellular metabolites

and in the stability of proteins and mRNAs; modifications occur in the activity of

enzymes as well as in the rate of transcription of a large number of genes, some of

the genes being induced while others are repressed. Diverse combinations of input

signals are required for glucose regulation of gene expression and of other cellular

processes. This review focuses on the early elements in glucose signalling and

discusses their relevance for the regulation of specific processes. Glucose sensing

involves the plasma membrane proteins Snf3, Rgt2 and Gpr1 and the glucose-

phosphorylating enzyme Hxk2, as well as other regulatory elements whose

functions are still incompletely understood. The similarities and differences in the

way in which yeasts and mammalian cells respond to glucose are also examined.

It is shown that in Saccharomyces cerevisiae, sensing systems for other nutrients

share some of the characteristics of the glucose-sensing pathways.

Introduction

Adaptation to changes in the environment is a key process for

the successful survival of organisms. The response of the cells

to these changes is mediated by a large variety of signalling

pathways that may be sorted into a few basic types, among

which two are prominent. In one of them the first element in

the pathway is a protein in the plasma membrane, able to

bind a nutrient or a hormone and to adopt a new conforma-

tion that activates a cascade of reactions, affecting metabolic

or regulatory enzymes, transcription factors, etc. (Forsberg &

Ljungdahl, 2001a; Kroeze et al., 2003; Holsbeeks et al., 2004;

Mascher et al., 2006; Monge et al., 2006; Hao et al., 2007). The

second type of pathway depends on the uptake of the nutrient

and, in most cases, on its metabolism, which causes changes

in the concentration of intracellular metabolites with a

regulatory function. The metabolites may, in turn, interact

with different kinds of proteins and modify their activity, their

stability and/or their localization. The regulated proteins can

have a variety of functions: they may have enzymatic activity,

they may bind other proteins and they are often able to bind

specific DNA regions and to modulate the transcription rate

of the corresponding genes (Sellick & Reece, 2005).

An interesting example of the response of a cell to a change

in the environment is the response of Saccharomyces cerevisiae

to the presence of glucose. The mechanisms involved are

particularly complex, as this sugar is not only a preferential

carbon source for the organism but also a molecule that

affects yeast physiology at many levels. In the presence of

glucose, there are changes in the concentration of intracellular

metabolites (Kresnowati et al., 2006), modifications and

eventually degradation of some enzymes (Serrano, 1983;

Gancedo & Gancedo, 1997; Portela & Moreno, 2006), and

alterations in the stability of a number of mRNAs (Mercado

et al., 1994; Cereghino & Scheffler, 1996; Scheffler et al., 1998;

Yin et al., 2003; Kresnowati et al., 2006). In addition, the

transcription of different genes is either induced or repressed

(Gancedo, 1998; Johnston, 1999; Wang et al., 2004). This

large remodelling of metabolism results in an increase in the

rate of growth of the yeast (Johnston et al., 1979).

This review focuses on the early elements involved in

glucose signalling in yeasts and on their relevance for the

regulation of specific processes. There is a special emphasis

on the role of the plasma membrane glucose-sensing pro-

teins Snf3, Rgt2 and Gpr1, and of Hxk2 in S. cerevisiae,

but reference is made to what is known for other species at

the end of each section. Signalling involving intracellular

metabolites, and other regulatory elements, such as the TOR

(target of rapamycin) pathway or phospholipase C, are also

discussed. The main targets for glucose signals considered

are the genes repressed or induced by glucose, and the

proteins activated, inactivated or degraded in the presence

FEMS Microbiol Rev 32 (2008) 673–704 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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of glucose. Most data correspond to S. cerevisiae but

reference to other yeast species is made, as far as information

is available. The review also addresses briefly the question of

how yeast senses the presence of other nutrients, and

discusses elements shared between the sensing pathways

used by different nutrients. Finally, similarities and differ-

ences between the glucose-sensing systems of yeast and

mammalian cells will be highlighted.

Sensing elements

Plasma membrane glucose-sensing proteins

Any yeast protein able to bind glucose may, in principle, play

a role in a glucose-signalling pathway. In the plasma mem-

brane of S. cerevisiae, there is a large family of at least 20

glucose transporters (Wieczorke et al., 1999), the most

relevant being Hxt1 and Hxt3, with a low affinity for glucose

and high transport capacity, and Hxt2, Hxt4 and Hxt7,

with a high affinity and low transport capacity (Boles &

Hollenberg, 1997). When glucose repression of the genes

MAL2, SUC2 and GAL1 was measured in mutants expressing

different sets of glucose transporters, it was found that the

rate of glucose transport determined the strength of repres-

sion, but that no specific transporter was required for

repression to take place (Reifenberger et al., 1997). It has also

been shown that one of the pathways of glucose signalling,

involving cAMP, can be restored in an hxt1 to hxt6 null

mutant by constitutive expression of GAL2, which encodes a

galactose permease also able to transport glucose (Rolland

et al., 2000). It can be concluded therefore that the glucose

transporters do not play a direct role in glucose signalling.

Snf3 and Rgt2

Snf3 and Rgt2 are plasma membrane proteins, with 12

transmembrane domains, highly similar to the Hxt glucose

transporters (Neigeborn et al., 1986; Ozcan et al., 1996), but

unable to transport glucose themselves (Ozcan et al., 1998).

Because they are required for the induction by glucose of the

HXT genes (Ozcan et al., 1996), Snf3 and Rgt2 act likely as

receptors that sense external glucose, but their capacity to

bind glucose has not been demonstrated directly. Snf3

appears to be a sensor for low levels of glucose, as it is

needed for the induction of HXT genes by low glucose, while

Rgt2 would be a sensor for high levels of glucose, as it is

required for maximal induction of HXT1 by high glucose

(Ozcan et al., 1996). Snf3 and Rgt2, unlike the yeast glucose

transporters, have long C-terminal tails in the cytoplasm,

which play an important role in glucose signalling (Ozcan

et al., 1998; Dlugai et al., 2001), but are not an absolute

requirement, as shown by the fact that an overexpressed,

tail-less Rgt2 can be functional (Moriya & Johnston, 2004).

The replacement by a lysine of an arginine residue in

Snf3 or Rgt2, conserved in all glucose transporters and

situated in a cytoplasmic loop preceding the fifth transmem-

brane domain, allows total induction of HXT2 and partial

induction of HXT1 in the absence of glucose (Ozcan et al.,

1996). This suggests that the mutated receptors adopt a

conformation similar to that of the glucose-bound Snf3 and

Rgt2, independent of the carbon source present in the

medium.

The schematic diagram of the regulation of HXT genes by

Snf3/Rgt2, shown in Fig. 1, is based on the following

observations.

Fig. 1. Snf3/Rgt2-signalling pathway. In the absence of glucose a repressing complex, including Rgt1, Mth1/Std1, Cyc8 and Tup1, binds to the

promoters of the HXT genes, and blocks their transcription. When glucose (Glu) is present, it binds to the Snf3/Rgt2 sensors, thus activating the

membrane bound casein kinase I (Yck1/2). Activated Yck1/2 phosphorylates Mth1/Std1, bound to the C-terminal tails of Snf3 and Rgt2. Phosphorylated

Mth1/Std1 are recognized by the SCFGrr1 complex, which tags them, through ubiquitination, to be degraded by the proteasome. Removal of Mth1/Std1

allows the phosphorylation of Rgt1, which dissociates from the promoter, allowing derepression of the HXT genes.

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There is strong experimental evidence that Rgt2 interacts

with the membrane-bound, type I casein kinases Yck1 and

Yck2 and, upon binding glucose, activates them (Moriya &

Johnston, 2004); it is assumed that Snf3 acts in the same

way. The activated casein kinases phosphorylate the regula-

tory proteins Mth1 and Std1 (Moriya & Johnston, 2004),

and this reaction is facilitated by the recruitment of these

proteins to the C tails of Snf3 and Rgt2 (Schmidt et al., 1999;

Lafuente et al., 2000).

Mth1 and Std1 are paralogous proteins (Hubbard et al.,

1994), which interact with Rgt1 (Tomas-Cobos & Sanz,

2002; Lakshmanan et al., 2003), a transcriptional repressor

of genes induced by glucose (Flick et al., 2003; Kim et al.,

2003; Palomino et al., 2005; Belinchon & Gancedo, 2007a).

This interaction prevents the dissociation from the corre-

sponding promoters of a repressing complex formed by

Rgt1, Mth1/Std1 and the proteins Cyc8 and Tup1 (Polish

et al., 2005). Serine-rich sequences have been identified in

both Mth1 and Std1, which are possible targets for Yck1/

Yck2 (consensus target sequence SXXS). When the serine

residues are replaced by another amino acid, Mth1 and Std1

are converted into constitutive repressors, which are no

longer degraded in the presence of glucose (Moriya &

Johnston, 2004). This indicates that the phosphorylation of

Mth1 and Std1 by Yck1/Yck2 targets them for degradation.

In fact, Grr1, an F-box protein, component of an SCF

ubiquitin ligase complex, and required for the induction

of the HXT genes (Ozcan & Johnston, 1995), binds phos-

phorylated Mth1 or Std1, which are then ubiquitinated by

the SCFGrr1 complex, and degraded via the proteasome

(Spielewoy et al., 2004; Kim et al., 2006). Although both

Mth1 and Std1 are degraded in the presence of high glucose,

cellular levels of Mth1 decrease much more than those of

Std1. This is due to the fact that expression of MTH1 and

STD1 is regulated in a different way: while under these

conditions MTH1 is repressed, the repression of STD1 by

Rgt1 is relieved (Kim et al., 2006).

The DNA-binding capacity of Rgt1 is lost when Rgt1 is

hyperphosphorylated in the presence of glucose (Kim et al.,

2003). This phosphorylation allows an intramolecular inter-

action between a central region of Rgt1 and its DNA-

binding domain, thus preventing the binding of Rgt1 to

DNA (Polish et al., 2005). Because, in the absence of Mth1

and Std1, Rgt1 no longer acts as a repressor (Flick et al.,

2003; Lakshmanan et al., 2003), it has been proposed

that binding of Mth1 to Rgt1 prevents its phosphorylation

(Polish et al., 2005). Although Std1 is, to some degree,

functionally redundant with Mth1, it appears to play a

specific role that has not yet been clarified (Polish

et al., 2005).

It should be stressed that HXT1 expression at high glucose

concentrations is strongly reduced in an rgt1 mutant, and

therefore Rgt1 acts formally as a transcriptional activator

(Ozcan & Johnston, 1995). Moreover, a fusion protein lexA-

Rgt1 acts as an activator of the transcription of a lexO-lacZ

reporter gene in a glucose-grown yeast (Kim et al., 2006).

This contrasts with the observation that the binding of Rgt1

to the HXT1 promoter takes place only in the absence of

glucose (Mosley et al., 2003). A possible interpretation for

these, apparently contradictory, findings is that in yeast

grown in a high-glucose medium, phosphorylated Rgt1

activates the transcription of a putative gene encoding a

transcriptional activator of HXT1 or, less likely, inhibits the

expression of a gene encoding an HXT1 repressor.

Glucose sensing through receptors similar to Snf3/Rgt2 is

not restricted to Saccharomyces. In Kluyveromyces lactis, a

single membrane protein, Rag4, appears to play the same

role as Snf3/Rgt2 (Betina et al., 2001), and there is an Rgt1

orthologue, KlRgt1, that represses RAG1, a gene encoding a

high-capacity, low-affinity glucose transporter (Rolland

et al., 2006). A casein kinase 1, Rag8, has been found to bind

Rag4, and both Rag4 and Rag8 are required to relieve

repression by Rgt1, in the presence of high glucose,

although, in contrast with the situation in S. cerevisiae,

Rgt1 remains bound to the RAG1 promoter under derepres-

sing conditions (Rolland et al., 2006). Another difference

between S. cerevisiae and K. lactis is that KlRgt1 is not

required for maximal expression of RAG1 (Rolland et al.,

2006). The gene KLLA0F15928g encodes a protein similar to

Mth1/Std1 [Genolevures (Sherman et al., 2006)], but its role

in regulating RAG1 has not yet been ascertained. In Pichia

angusta (Hansenula polymorpha), a hexose transport homo-

logue, Gcr1, has been identified as being involved in glucose

repression (Stasyk et al., 2004); there is no information,

however, on other elements of the putative glucose-signal-

ling pathway. In Candida albicans, Hgt4 is the orthologue of

Snf3/Rgt2 and there are also proteins similar to Std1

(orf19.6173) and to Rgt1, which act like the corresponding

proteins of S. cerevisiae (Brown et al., 2006). On the other

hand, no obvious orthologues of the different regulatory

proteins appear to be present in Schizosaccharomyces pombe,

while in Yarrowia lipolytica, although gene YALI0B04796g

encodes a protein with some similarity to Mth1/Std1

[Genolevures (Sherman et al., 2006)], there are no clear

relatives of Snf3/Rgt2 or Rgt1.

Gpr1

Gpr1 is a plasma membrane protein, with seven transmem-

brane domains, coupled to the G protein Gpa2 (Yun et al.,

1997; Xue et al., 1998) and involved in the increase in cAMP

levels triggered by glucose (Yun et al., 1998; Kraakman et al.,

1999a; Rolland et al., 2002). The Gpr1-Gpa2 couple is

responsive to glucose and to sucrose but not to other sugars

such as fructose, 2-deoxyglucose or xylose; mannose acts

as an antagonist (Rolland et al., 2000). Unexpectedly, the


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system has a better affinity for sucrose than for glucose, as

the effector concentration for half-maximum response

(EC50) is about 0.5 mM for sucrose and 20 mM for glucose

(Lemaire et al., 2004). There is no direct evidence for the

binding of glucose to Gpr1. However, the fact that mutants

of Gpr1 have been obtained, deficient in glucose-induced,

but not in sucrose-induced, cAMP increase, strongly sug-

gests that there is a binding site for the sugars in Gpr1 and

that some mutations in this site, such as A640C, Q644C

or E648C, affect specifically the interaction with glucose

(Lemaire et al., 2004). G proteins are usually heterotrimeric

proteins consisting of a, b and g subunits, and ligand

binding to the G protein-coupled receptor stimulates a

GDP to GTP exchange in the Ga subunit and the release of

the GbGg dimer. Gpa2 behaves as a Ga subunit but no

canonical Gb and Gg subunits associate with it; instead

Gpa2 forms a complex with proteins with a different

structure, the kelch-repeat proteins Krh1/Gpb2 and Krh2/

Gpb1 (Harashima & Heitman, 2002; Batlle et al., 2003).

Gpa2 also interacts with the protein Rgs2 that stimulates the

intrinsic GTPase activity of Gpa2, facilitating the formation

of the inactive GDP-bound Gpa2 (Versele et al., 1999), but

there is no information on a possible regulation by glucose

of Rgs2 activity. It is thought that the binding of glucose to

Gpr1 directs the formation of the GTP-bound, active form

of Gpa2 (Fig. 2), and it has been shown that adenylate

cyclase, Cyr1, binds to GTP-Gpa2 but not to GDP-Gpa2

(Peeters et al., 2006). This mechanism explains the rapid

increase in cAMP levels observed when glucose is added

to glucose-deprived cells. Increased levels of cAMP cause

the activation of protein kinase A (PKA), as cAMP binds to

the regulatory subunit Bcy1 and dissociates it from the

alternative catalytic subunits Tpk1, Tpk2 or Tpk3, which

then become active (Toda et al., 1987a, b). Because PKA

phosphorylates a large number of proteins, it can mediate

many of the effects produced by glucose, as it will be

discussed below.

The regulatory role of the Krh1/Krh2 proteins is still

subject to debate. They act as negative regulators of PKA (Lu

& Hirsch, 2005) but two different mechanisms have been

proposed to explain their effect. In the absence of Krh1/2 the

interaction between Tpk1 and Bcy1 is reduced, as shown in a

two-hybrid assay (Peeters et al., 2006). Therefore, Krh1/2

could facilitate the association between the regulatory and

catalytic subunits of PKA. On the other hand, it has been

found that Krh1/2 bind to a conserved C-terminal domain

of Ira1 and Ira2, which function as GTPase-activating

proteins on GTP-bound Ras (Tanaka et al., 1990), and

stabilize them (Harashima et al., 2006). In the absence of

Krh1/2, Ira1 and Ira2 are more readily degraded and

elevated levels of Ras2-GTP lead to increased cAMP produc-

tion. Both mechanisms are not mutually exclusive and it

should be noted that the Gpr1-Gpa2 system is able to

regulate the activity of PKA in an adenylate cyclase mutant

where the system cannot control cAMP levels (Peeters et al.,

2006). In this mutant, both a constitutively active GPA2

allele and a deletion of KRH1/2 lower the concentration of

external cAMP required to allow yeast growth.

A particularly relevant substrate of PKA is the transcrip-

tional repressor Rgt1, as the hyperphosphorylation of Rgt1

triggered by glucose integrates glucose signals relayed

through Snf3/Rgt2 and Gpr1. It has been shown that after

Rgt1 is phosphorylated by the Tpk3 isoenzyme (or by a

Tpk3-dependent protein kinase), it dissociates from the

HXK2 promoter, thereby relieving repression of the gene

(Palomino et al., 2006). There is also direct evidence that the

serine residues from Rgt1 located in consensus sequences for

phosphorylation by PKA are required for the intramolecular

Fig. 2. Gpr1-signalling pathway. In the absence of glucose, Gpa2 and the Ras proteins are mainly in their GDP-bound, inactive form, their GTPase

activity being stimulated by Rgs2 and Ira1/2, respectively. Protein kinase A (Tpk) has a low activity, as Krh1/2 favors its binding to the regulatory subunit

Bcy1, and cAMP levels are low due to a reduced activity of adenylate cyclase (Cyr1). When glucose is present, it binds Gpr1, which then interacts with

Gpa2, facilitating the formation of the GTP-bound, active form of the protein. Glucose metabolism also causes an activation of the Ras proteins, possibly

due to inhibition of Ira1/2, and the activated Ras and Gpa2 stimulate together Cyr1 activity. In the presence of glucose, there is also a decrease in the

activity of Krh1/2, which destabilizes Ira1/2 and facilitates the dissociation of the Bcy1-Tpk complex.


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interaction that regulates the capacity of Rgt1 to bind DNA

(Kim & Johnston, 2006).

In S. pombe, the proteins git3 and Spgpa2 have been

identified as being involved in the activation of adenylate

cyclase triggered by glucose, thus being functionally similar

to Gpr1 and Gpa2 (Welton & Hoffman, 2000). However, the

putative glucose-binding protein git3 has only a partial

sequence homology with Gpr1 (Welton & Hoffman, 2000),

and Spgpa2, able to bind to an N-terminal domain of

adenylate cyclase (Ivey & Hoffman, 2005), is associated with

canonic Gb and Gg subunits (git5 and git11), which are

required for a normal response to glucose in S. pombe

(Landry & Hoffman, 2001).

In C. albicans, there are conflicting reports on the role of

the homologues of Gpr1 and Gpa2: in one case, they were

found to be required for a glucose-dependent increase in

cellular cAMP (Miwa et al., 2004), while in another it was

concluded that Gpr1 and Gpa2 did not mediate glucose-

induced cAMP signalling (Maidan et al., 2005). While there

is a functional Gpa2 protein in K. lactis, involved in cAMP

signalling (Savinon-Tejeda et al., 1996), the role of a protein

with homology to Gpr1, encoded by ORF KLLA0F24750g

[Genolevures (Sherman et al., 2006)], has not been in-

vestigated. No information on a cAMP-signalling pathway

in Y. lipolytica is available, although a gene search shows the

existence of ORF YALI0A09592g encoding a protein similar

to Gpa2 and of ORFs YALI0D13552g and YALI0F19426g

encoding proteins with some similarities to Gpr1 [Genolevures

(Sherman et al., 2006)].

Hxk2

Relevant intracellular proteins from S. cerevisiae able to bind

glucose are the hexose kinases Hxk1, Hxk2 and Glk1 (Lobo

& Maitra, 1977). It has long been known that in hxk2

mutant strains, glucose repression of some genes such as

SUC2 or GAL1 is very much reduced (Zimmermann &

Scheel, 1977; Entian, 1980). While it was initially proposed

that Hxk2 has a specific regulatory domain required for

glucose repression (Entian & Frohlich, 1984), later experi-

ments with strains expressing mutated forms of Hxk2

showed a parallelism between glucose repression and the

capacity of the strains to phosphorylate glucose, and sug-

gested that Hxk2 plays only a metabolic role (Ma et al.,

1989b; Rose et al., 1991). Although there have been more

recent reports on mutant forms of Hxk2 with reduced

catalytic activity but still functional in glucose signalling

(Kraakman et al., 1999b; Mayordomo & Sanz, 2001), there is

not yet evidence for a mutant form of Hxk2 devoid of

catalytic activity but maintaining its regulatory capacity.

New perspectives were provided when the intracellular

location of Hxk2 was investigated, using isolated nuclei and

specific antibodies or expressing an Hxk2-green fluorescent

protein (GFP) fusion. It was found that a small proportion

of Hxk2 is located within the nucleus (Herrero et al., 1998;

Randez-Gil et al., 1998a) and that under conditions where

Hxk2 does not enter the nucleus glucose repression

of the genes SUC2, HXK1 or GLK1 does not take place

(Herrero et al., 1998; Rodrıguez et al., 2001). These results

indicate a nonmetabolic role for Hxk2 that requires a

nuclear localization.

Another question to be solved is the identification of the

molecular target(s) for Hxk2. A first candidate was the

protein kinase Snf1, required for the expression of glucose-

repressed genes. When a high concentration of glucose is

available to yeast cells, Snf1 is inactivated (Carlson, 1999)

and it was assumed that this inactivation does not take place

in the absence of Hxk2 (Treitel et al., 1998), an assumption

consistent with the following observations. In an hxk2

mutant, glucose repression of some genes that require Snf1

to be transcribed is weak (Zimmermann & Scheel, 1977;

Entian & Zimmermann, 1980), and Snf1 phosphorylates the

repressor Mig1, even in the presence of high glucose (Treitel

et al., 1998; Ahuatzi et al., 2007). The interaction between

Snf1 and the regulatory protein Snf4, low at high glucose

and required to lift Snf1 autoinhibition, is considerably

increased in the absence of Hxk2 (Jiang & Carlson, 1996;

Sanz et al., 2000). REG1 overexpression partially suppresses

the effect of an hxk2 mutation on glucose repression (Sanz

et al., 2000), an observation suggesting that the presence of

Hxk2 facilitates the dephosphorylation, and subsequent

inactivation, of Snf1 by the protein phosphatase complex

Glc7-Reg1. However, while an interaction of Snf1 with Hxk2

was observed in extracts from cells grown in either high or

low glucose (Ahuatzi et al., 2007), no interaction was

observed, in a two-hybrid assay, between Hxk2 and Reg1 or

Glc7 (Sanz et al., 2000). Moreover, actual measurements of

the capacity of Snf1 to phosphorylate a peptide substrate in

vitro did not show an increased activity of Snf1 in extracts

from an hxk2 mutant grown on glucose (K. Hedbacker &

M. Carlson, pers. commun.). Therefore, it remains unclear

whether Hxk2 controls the intrinsic activity of Snf1, or only

its capacity to phosphorylate Mig1.

Other possible targets for Hxk2 have been uncovered

during the last years, suggesting a model for the Hxk2 role in

the control of transcription of some glucose-repressed genes

(Fig. 3). An interaction of Hxk2 with the protein Med8, a

subunit of the Srb/mediator complex (Myers et al., 1998;

Myers & Kornberg, 2000), has been observed, using two-

hybrid assays, coprecipitation experiments and gel mobility

analysis with purified proteins (de la Cera et al., 2002). This

interaction may be physiologically relevant, as Med8 binds

to regulatory elements in genes controlled by glucose, such

as those found in the promoter of the SUC2 gene and also in

the coding region of the HXK2 gene (Chaves et al., 1999;

Moreno-Herrero et al., 1999; Palomino et al., 2005). In the



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case of glucose-repressed genes, the function of the binding

of Hxk2 to Med8, which takes place in glucose growing cells,

could be to interfere with the recruitment of RNA polymer-

ase II by the mediator complex. However, for a glucose-

activated gene such as HXK2, it is harder to visualize how

the binding of Hxk2 to Med8 could facilitate transcription.

A possible explanation for the positive role played by Hxk2

in HXK2 transcription is that, in the presence of glucose,

Hxk2 sequesters the transcriptional repressor Rgt1, thus

helping to relieve its block on HXK2 transcription (Palomino

et al., 2006).

It has also been demonstrated, using different techniques,

that Hxk2 interacts directly with the transcriptional repres-

sor Mig1 and that the Lys6-Met15 decapeptide from Hxk2 is

needed for the interaction (Ahuatzi et al., 2004). The

interaction between Mig1 and Hxk2 is required for Hxk2 to

be retained within the nucleus and there is a correlation

between the level of Mig1 in the cell and the amount of

Hxk2 located in the nucleus (Ahuatzi et al., 2004). The fact

that a nuclear localization of Mig1 requires the presence of

high glucose in the culture medium (DeVit et al., 1997)

explains the observation that the partial nuclear localization

of Hxk2 is also glucose-dependent (Ahuatzi et al., 2004). It

should be noted that Mig1 does not need to form a complex

with Hxk2 to enter into the nucleus, as it is found in the

nucleus in an snf1 hxk2 double mutant (Tomas-Cobos &

Sanz, 2002; Ahuatzi et al., 2007). The binding of Hxk2 and

Mig1 in the nucleus suggests that the main role of Hxk2 in

glucose repression is to hinder the contact between Mig1

and Snf1, blocking phosphorylation of Mig1 and thus

maintaining its repressing capacity (Ahuatzi et al., 2004).

The interaction between Hxk2 and Mig1 would be espe-

cially important in avoiding cross-talk between different

signalling pathways, as it has been observed that in sodium-

stressed cells, growing in the presence of high glucose, Snf1

is activated but does not phosphorylate Mig1 (McCartney &

Schmidt, 2001).

It is likely, however, that Hxk2 plays some additional role,

as lack of Hxk2 has a stronger effect than lack of Mig1 on

relieving glucose repression of SUC2 (Entian, 1981; Neige-

born & Carlson, 1987; Nehlin & Ronne, 1990; Walsh et al.,

1991; Lutfiyya & Johnston, 1996; Salgado et al., 2002). This

could involve the protein Hxk2 itself or products of glucose

metabolism. Because, in the absence of Hxk2, Hxk1 is able

to support repression of invertase by fructose (De Winde

et al., 1996) and, when overexpressed, allows partial repres-

sion of invertase by glucose (Ma & Botstein, 1986), a

metabolic role for the glucose-phosphorylating enzymes

appears likely.

It has been reported recently (Sarma et al., 2007) that, at

high glucose and in the absence of Hxk2, about 50% of Mig1

remains in the nucleus, but excluded from the perinuclear

compartment. This observation contrasts with earlier ex-

periments showing that, in an hxk2 mutant, a Mig1-GFP

fusion protein remains in the cytoplasm in the presence of

glucose (DeVit et al., 1997; Ahuatzi et al., 2007), although it

was already indicated that, under these conditions, a small

amount of fluorescence was visible in the nuclei of some

cells (DeVit et al., 1997). In any case, it should be noted that

the localization of Mig1 in the nucleus is not sufficient to

cause repression, as shown by the fact that, in a mutant

lacking the nuclear exportin Msn5, GAL1 is derepressed

normally, although Mig1 always remains in the nucleus

(DeVit & Johnston, 1999). It appears that phosphorylation

of Mig1 by Snf1 is all that is needed to block its capacity to

repress gene transcription.

Fig. 3. Model for the role of Hxk2 in the nucleus in glucose signalling. During growth on glucose the repressing protein Mig1 is mainly in its

unphosphorylated form, due to the protein phosphatase activity of the Glc7-Reg1 complex, coupled with a low activity of the Snf1 complex. In these

conditions Mig1 has a nuclear localization and Hxk2 is partially retained within the nucleus through its binding to Mig1: this binding hinders the contact

between Mig1 and any active molecule of Snf1 in the nucleus and would thus reinforce the repressing capacity of the Mig1-Cyc8-Tup1 complex.

Nuclear Hxk2 is also able to bind Med8, a subunit of the Srb/mediator complex, and this binding may interfere with the capacity of RNA polymerase II

(RNA Pol II) to interact with the promoter of the regulated gene. When glucose is removed, the Snf1 complex is activated, Mig1 is phosphorylated and

both Mig1 and Hxk2 leave the nucleus. As Hxk2 dissociates from Med8, the mediator complex would be able to recruit RNA Pol II and transcription may

proceed. See text for details.


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A disputed point has been the role of the serine 14 of

Hxk2 in the capacity of the enzyme to mediate catabolite

repression of genes such as SUC2. This serine was called

Ser15 (corresponding to codon 15) until Hxk2 was se-

quenced and it was found that the N-terminal amino acid

is valine (corresponding to codon 2) and not methionine

(Behlke et al., 1998). Although serine 14 can be phosphory-

lated by PKA in vitro (Kriegel et al., 1994), PKA is not likely

to catalyze the reaction in vivo, because phosphorylation of

serine 14 is reduced under conditions where the cAMP-

dependent protein kinases are activated (Vojtek & Fraenkel,

1990). Different groups have looked at the consequences of

replacing serine 14 by an alanine residue and have reported

contradictory results. While some laboratories found that

the modified enzyme was still functional in glucose signal-

ling (Herrero et al., 1998; Mayordomo & Sanz, 2001),

another group reported that the mutation impaired the

functionality of Hxk2 and concluded that it is the phos-

phorylated Hxk2 that transmits the glucose signal (Randez-

Gil et al., 1998b). This, however, does not appear to agree

with the observation that the proportion of phosphorylated

Hxk2 is higher under derepressing than under repressing

conditions (Vojtek & Fraenkel, 1990; Randez-Gil et al.,

1998b). It has been suggested that the discrepancy between

the results of different groups investigating the effects of

Hxk2S14A could be due to variations in the amount of

mutated hexokinase synthesized in the diverse strains used

(Moreno & Herrero, 2002) and, in fact, the number of

copies of the mutated HXK2 gene was not the same in the

different experiments, as the gene was carried either in an

integrative plasmid (Randez-Gil et al., 1998b) or in centro-

meric (Mayordomo & Sanz, 2001) or multicopy (Herrero

et al., 1998) episomal plasmids. Taking into account that a

modified Hxk2 lacking the 14 N-terminal amino acids can

still mediate repression of SUC2 or induction of HXT1 by

glucose (Ma et al., 1989a; Mayordomo & Sanz, 2001), it can

be concluded that serine 14 is dispensable for glucose

signalling, and that a more relevant feature is the hexokinase

activity in the cell. The functionality of an Hxk2 lacking its

14 N-terminal amino acids would also indicate that, at least

under some circumstances, full glucose repression may take

place in a strain with an Hxk2 lacking the Lys6–Met15

decapeptide, although this decapeptide is required for

interaction with Mig1 (Ahuatzi et al., 2004).

For most other yeasts, there is little evidence for hexoki-

nases playing a nonmetabolic role in glucose repression. In

K. lactis there is a single hexokinase, Rag5, required for

glucose repression (Bar et al., 2003) and a glucokinase that

allows growth on glucose at a very slow rate (35 h doubling

time) in the absence of Rag5 (Kettner et al., 2007). Although

this makes it hard to distinguish between a metabolic and a

regulatory role for Rag5, it has been found that Rag5 is not

efficient for restoring glucose repression of invertase in hxk2

S. cerevisiae mutants (Prior et al., 1993; Petit & Gancedo,

1999). In P. angusta (H. polymorpha), a hexokinase,

HpHxk1, and a glucokinase, HpGlk1, have been identified

and any of them supports repression by glucose of maltase,

alcohol oxidase or catalase (Kramarenko et al., 2000).

However, although HpGlk1 allows growth on glucose of an

S. cerevisiae triple kinase mutant, in the S. cerevisiae

transformants synthesis of invertase and maltase remains

insensitive to glucose repression (Laht et al., 2002). Whereas

HpHxk1 has been cloned (Karp et al., 2003), its effect in

S. cerevisiae has not been reported. In S. pombe, two

hexokinases have been identified: SpHxk1 with low affinity

for glucose and much higher activity with fructose than with

glucose as a substrate, and SpHxk2, a kinetically conven-

tional hexokinase (Petit et al., 1996). As occurred with the

kinases from K. lactis and P. angusta, SpHxk1 could not

restore glucose repression of invertase in an S. cerevisiae

triple mutant (Petit et al., 1998) and repression in the

presence of SpHxk2 was only partial (Petit & Gancedo,

1999). In contrast, in a similar experiment, Hxk1 from

Y. lipolytica was fully effective (Petit & Gancedo, 1999) and

there is preliminary evidence that YlHxk1 is involved in

glucose repression of the LIP2 gene encoding an extracellu-

lar lipase in Y. lipolytica (Fickers et al., 2005). It has also been

reported that in mutants of Schwanniomyces occidentalis or

Pachysolen tannophilus, which lack a specific isoenzyme of

hexokinase, glucose repression is defective (McCann et al.,

1987; Wedlock & Thornton, 1989).

Signalling by intracellular metabolites

Because most glucose effects require glucose metabolism

(Belinchon & Gancedo, 2007a, b), it may be concluded that

binding of glucose to a receptor is usually not sufficient to

signal the presence of glucose in yeast. Although there have

been some reports on glucose signalling, independent of

glucose uptake (Liang & Gaber, 1996; Ozcan, 2002), they

should be considered with caution, because the hxt strain

used showed a residual growth on glucose, indicating that

glucose could still be transported and metabolized. While

growth could be completely blocked by adding antimycin to

the medium, the experiments on the signalling capacity of

glucose were performed in the absence of antimycin.

An intracellular metabolite that plays an important role

in glucose signalling is cAMP, as shown by the fact that many

of the transcriptional changes elicited by glucose can be

reproduced by activation of the GTP-binding proteins Ras2

or Gpa2. This can be done, in the absence of glucose, using

genes encoding constitutively active forms of Ras2 or Gpa2,

placed under the control of a conditional promoter (Wang

et al., 2004). Activation of either Ras2 or Gpa2 causes an

increase in cAMP levels (Toda et al., 1985; Nakafuku et al.,

1988), which stimulates the cAMP-dependent protein



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kinases Tpk1, Tpk2 and Tpk3 (Toda et al., 1987b). Surpris-

ingly, from the many potential substrates already identified

for the three isoenzymes Tpk1, Tpk2 and Tpk3 (thereafter

PKA) present in S. cerevisiae (Budovskaya et al., 2005;

Ptacek et al., 2005), there is none clearly related to the

regulation of important elements mediating glucose control,

such as the Snf1 kinase and the Glc7 phosphatase complexes

(Santangelo, 2006).

Glucose signalling through the Ras-cAMP system requires

the phosphorylation of the sugar (Rolland et al., 2000,

2001). It has been suggested that the metabolism of glucose

causes an inhibition of Ira1/Ira2, stimulators of the GTPase

activity of the Ras proteins, and therefore, increases the

GTP-loading of Ras1 and Ras2 (Colombo et al., 2004). GTP-

loaded Ras proteins in turn activate adenylate cyclase,

increasing the intracellular concentration of cAMP [see a

recent review (Santangelo, 2006) for details].

As indicated above, it is also possible to mimic the glucose

response, in the absence of the sugar. Although the changes

in transcript levels resulting from Ras2 or Gpa2 activation

are dependent on PKA, it has also been shown that most

glucose-responsive genes can also be regulated through a

PKA-independent pathway (Wang et al., 2004). Information

is not yet available on the elements of this regulatory

pathway, but it may start with changes in the concentra-

tion of some intracellular metabolite(s) other than cAMP

(Boles & Zimmermann, 1993; Goncalves et al., 1997).

Glucose-6-phosphate and fructose-1,6-bisphosphate may

appear as good candidates, as their concentrations are

strongly responsive to the presence of glucose (Banuelos

et al., 1977); however, there is no clear correlation between

the intracellular concentration of these hexose phosphates

and the degree of repression by different carbon sources

of the gluconeogenic enzyme fructose-1,6-bisphosphatase

(FbPase) (Rodrıguez & Gancedo, 1999; Belinchon & Gance-

do, 2003). Although studies on the induction of glycolytic

enzymes in a variety of mutants suggest that different

metabolites control the expression of different genes (Boles

& Zimmermann, 1993; Muller et al., 1995; Boles et al.,

1996), the identity of the regulatory metabolites has not

been established and their mechanism of action is not

known.

In mammalian cells, the AMP-dependent protein kinase,

a homologue of the protein kinase Snf1 from yeast, is

activated under stress conditions due to an increase in the

concentration of intracellular AMP (Hardie, 1999). In S.

cerevisiae, however, there is no evidence that AMP controls

Snf1, or even that the AMP concentration decreases in the

presence of glucose (Kresnowati et al., 2006). It has been

proposed that yeast could, in some unknown way, sense

the ‘glucose flux’ or the rate of glucose consumption and

adjust in this way the degree of repression of genes involved

in the utilization of alternative carbon sources (Bisson &

Kunathigan, 2003). However, there is evidence that glucose

repression of a gene such as SUC2 is not directly related to

the glucose flux (Meijer et al., 1998). The identification of

regulatory elements in pathways alternative to that con-

trolled by PKA therefore remains an important point that

requires further investigation.

An interesting regulatory metabolite that accumulates in

yeast cells metabolizing glucose is fructose-2,6-bisphosphate

(Lederer et al., 1981). It is worth noting that this compound

has been found to activate the phosphorylation of FbPase by

PKA in vitro (Gancedo et al., 1983), and is therefore likely

to control the phosphorylation and partial inactivation

of FbPase, which takes place on addition of glucose to

derepressed yeast cells (Mazon et al., 1982).

Other early elements in glucose signallingin S. cerevisiae

The TOR pathway

Although the TOR-signalling pathway has been investigated

mainly in relation to the nitrogen source in the medium

(Cooper, 2002), it may also mediate some of the yeast

responses to glucose (Hardwick et al., 1999). In the presence

of rapamycin, an inhibitor of Tor1 and Tor2, the transcript

levels of several genes induced by glucose decrease, while a

number of glucose-repressed genes are expressed at higher

levels (Hardwick et al., 1999). In the case of the low-affinity

glucose transporter Hxt1, rapamycin not only decreases the

levels of HXT1 mRNA (Hardwick et al., 1999; Tomas-Cobos

et al., 2005) but also causes instability and mislocalization of

Hxt1 (Schmelzle et al., 2004). It therefore appears that the

TOR pathway acts both on transcription and on posttran-

scriptional processes. It has been proposed that glucose

signalling through TOR takes place independently of the

TOR effectors Tap2 and Sit4, and is mediated by the Ras/

cAMP pathway (Schmelzle et al., 2004). It is not yet known,

however, whether the Tor proteins sense intracellular glu-

cose or some other indicator of carbon source availability.

The lack of Tor1 also impairs the downregulation by

glucose of different amino acid permeases (Peter et al.,

2006). In addition, in the absence of Tor1, mitochondrial

respiration is increased during growth on glucose. This

increase, which is not observed in glycerol-growing cells, is

due to an enhanced translation of mRNAs corresponding to

subunits of the oxidative phosphorylation complex encoded

by mit-DNA (Bonawitz et al., 2007).

14-3-3 proteins

The 14-3-3 proteins Bmh1/Bmh2, positive regulators of the

TOR kinase pathway (Bertram et al., 1998), affect some

processes regulated by glucose. In a double mutant

bmh1bmh2 catabolite inactivation of maltose permease is


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impaired (Mayordomo et al., 2003), glucose repression of

ADH2 is partially relieved (Dombek et al., 2004) and

induction of HXT1 by high glucose is prevented (Tomas-

Cobos et al., 2005). It has been shown that Bmh1 and Bmh2

interact with Reg1, a regulatory subunit of the type 1 protein

phosphatase complex (Dombek et al., 2004) and that the

complex Bmh1/2-Reg1 interacts with Grr1, a component of

the SCFGrr1 ubiquitination complex (Tomas-Cobos et al.,

2005). This would suggest that the Bmh proteins mediate

HXT1 induction by facilitating, in a Reg1-dependent pro-

cess, the interaction of Grr1 with Std1. The Bmh proteins

may also play a Reg1-independent role, possibly related to

the activation of the TOR pathway, as the bmh1bmh2 and

reg1 mutations release synergistically glucose repression of

ADH2 (Dombek et al., 2004). It has also been reported that,

in the presence of glucose, Bmh1/2 interacts with the protein

kinase Yak1 (Moriya et al., 2001); under these conditions,

Yak1 is phosphorylated by PKA (Zappacosta et al., 2002)

and is exported from the nucleus in a Bmh-dependent

process (Moriya et al., 2001). Upon glucose depletion Yak1

is activated, enters the nucleus and phosphorylates Pop2, a

subunit of the Ccr4-Not complex that mediates mRNA

deadenylation, causing cell-cycle arrest (Moriya et al.,

2001). Activated Yak1 also phosphorylates the transcription

inhibitor Crf1, causing a decrease in the transcription of the

ribosomal genes (Martin et al., 2004).

Phospholipase C

Another regulatory pathway controlled by glucose involves

phospholipase C (Plc1). Addition of glucose to starved yeast

cells activates Plc1 and thereby increases phosphatidylinosi-

tol turnover and the intracellular level of different species of

diacylglycerols (Coccetti et al., 1998). The fact that Plc1

interacts with the glucose sensor Gpr1 and its coupled G

protein Gpa2 (Ansari et al., 1999) suggests that activation of

Plc1 is mediated by glucose binding to Gpr1. This is further

supported by the observation that the Plc1-dependent,

glucose-triggered increase in free intracellular Ca21 requires

Gpr1 and Gpa2 (Tisi et al., 2002). It has also been observed

that the transient elevation of cytosolic calcium caused by

glucose requires the phosphorylation of the sugar (Tokes-

Fuzesi et al., 2002) and it has been suggested that the

intracellular calcium concentration is related to the ratio

glucose-1-P/glucose-6-P (Aiello et al., 2002). There is also

preliminary evidence for inositol triphosphate acting as a

mediator of glucose-induced calcium signalling (Tisi et al.,

2004). The primary target of the diacylglycerols formed

upon activation of Plc1 is the protein kinase C (PKC), and

PKC may be involved in the control of carbon metabolism.

This control, however, is complex because pkc1 mutants

show defects both in the induction of HXT genes by glucose

and in the derepression of SUC2, alcohol dehydrogenase

or glycerol kinase that takes place on glucose depletion

(Brandao et al., 2002; Salgado et al., 2002; Gomes et al.,

2005). For this function, PKC does not require the MAP kinase

cascade formed by the kinases Bck1, Mkk1/2 and Mpk1, but

may act by regulating the intracellular localization of DNA-

binding proteins such as the repressor Mig1 or the transcrip-

tion factor Adr1 (Salgado et al., 2002; Gomes et al., 2005).

Others

A different way for glucose to act, when it reaches the

100 mM range, is as a weak osmolyte, able to activate the

Sln1-dependent branch of the HOG pathway (Tomas-Cobos

et al., 2004). This activation has been shown to be absolutely

required for glucose to induce transcription of the HXT1

gene (Tomas-Cobos et al., 2004). Still other elements have

been shown to affect processes controlled by glucose, but

there have only been suggestions on their mode of action,

rather than clear evidence. The increased induction of

HXT1, which takes place in the absence of Cdc55, has been

attributed to interference of Cdc55 with the activity of the

protein kinases Yck1/2 that phosphorylate Std1 and Mth1,

negative regulators of HXT1 transcription (Tomas-Cobos

et al., 2005). The protein kinase Pho85 is required to control

the important remodelling of gene expression that takes

place when the glucose of the medium is depleted; in its

absence downregulation of glycolytic genes is defective,

induction of genes involved in gluconeogenesis and in the

glyoxylate cycle occurs before the glucose of the medium is

completely exhausted, and the expression of genes required

for proper mitochondrial function is impaired (Nishizawa

et al., 2004). The molecular mechanisms underlying these

effects are not known, but it is likely that the Pho85-

associated cyclins Pcl6 and Pcl7 are involved in the forma-

tion of functional mitochondria, as mutants lacking Pho85,

Pcl6 or Pcl7 are unable to grow on nonfermentable carbon

sources (Gilliquet & Berben, 1993; Lee et al., 2000). The

observation that in a mutant defective in Tps1, a subunit of

the trehalose synthase complex, glucose-induced effects are

impaired led Thevelein & Hohmann (1995) to conclude that

Tps1 is involved in the transduction of a glucose signal.

Although the inability of tps1 mutants to grow on glucose

complicated the interpretation of the earlier observations,

later work showed that Tps1 is dispensable for glucose

signalling (Rodrıguez & Gancedo, 1999).

Targets for glucose signals

In S. cerevisiae

Genes repressed by glucose

An important signal for glucose repression is an increase in

intracellular cAMP, which allows the activation of PKA. This



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is shown by the large number of genes for which the

transcription rate decreases on activation of Ras2 (Wang

et al., 2004). Under artificial conditions, this downregula-

tion of transcription can take place in the absence of glucose,

but is dependent on the presence of a protein kinase subject

to activation by cAMP. This shows that, for a number of

genes, the activation of PKA is sufficient to trigger transcrip-

tional repression. An analysis of the data of Wang et al.

(2004) shows, however, that not every gene repressed by

glucose responds in the same way to the activation of Ras2

and PKA, as illustrated by some selected cases shown in

Table 1. For some genes, cAMP is the main signal and

glucose repression in a tpkw bcy1 background is impaired or

absent. Representative examples are genes related to the

metabolism of trehalose or glycogen (TPS1, TPS2, GPH1),

glucose utilization (HXK1, GLK1, TKL2, GPD1), or ubiqui-

tination (COQ3, YJR036). In contrast, other genes do not

respond to the activation of Ras2, among them SUC2, one of

the model genes used to study repression by glucose. It

should be noted that genes involved in the same or related

metabolic pathways do not always behave in parallel. For

instance, considering genes encoding enzymes from the

gluconeogenic pathway or the glyoxylate cycle, it can be

observed that activation of Ras2 blocks transcription of

FBP1, while its effect on the transcription of PCK1, ICL1 or

MLS1 is weak or absent. This is consistent with results

showing that external cAMP has a stronger inhibitory effect

on derepression of Fbp1 than on derepression of Pck1 or

Icl1 (Zaragoza et al., 1999). Among genes involved in

respiratory metabolism, CYB2 and FOX2 are strongly re-

pressed when Ras2 is activated, while the response of COX6

is weak and CYC1 shows no response. For most glucose-

repressed genes signalling through cAMP is redundant with

another glucose-signalling pathway, as shown by the fact

that glucose repression takes place normally in a tpkw bcy1

strain. The nature of the corresponding signal has not been

yet elucidated, but it does not involve the plasma membrane

glucose sensors Snf3 or Rgt2, and depends on glucose

metabolism (Belinchon & Gancedo, 2007b). In fact, in

mutants where glycolytic flux is strongly reduced, glucose

repression is partially relieved (Gamo et al., 1994; Elbing

et al., 2004).

For the repression of some genes such as SUC2, HXK1 or

the GAL genes, the glucose-phosphorylating enzyme Hxk2

is specifically required (Zimmermann & Scheel, 1977; Enti-

an, 1980; Rodrıguez et al., 2001) (see section on Hxk2). On

the other hand, repression of ADH2 or FOX1 is not relieved

in an hxk2 mutant (Dombek et al., 1993; Stanway et al.,

1995) and relief is only partial for genes such as CYC1,

CYB2, GLK1 or GDH2 (Ma & Botstein, 1986; Brown et al.,

1995; Rodrıguez et al., 2001; Belinchon & Gancedo, 2007b).

Some genes such as FBP1, PCK1 or ICL1 are completely

repressed by glucose, even in a double mutant hxk1 hxk2

(Yin et al., 1996; Belinchon & Gancedo, 2007b) and, while

long-term repression of SUC2 requires Hxk2, SUC2 mRNA

levels show a strong, transient, decrease on addition of

glucose to an hxk1hxk2 strain (Sanz et al., 1996). A further

element modulating repression by glucose is the protein

kinase Pho85, because in its absence genes involved in

gluconeogenesis and the glyoxylate cycle can be expressed

at low concentrations of glucose (Nishizawa et al., 2004).

Genes induced by glucose

A genome-wide screening has shown that, for most of the

genes induced by high glucose, the increase in transcript

Table 1. Elements affecting different glucose repressed genes

Element(s) Stimulus Strain used Strong response

Weak

response

Response very weak

or absent

cAMP Galactose� GAL10-RAS2Val19

gal1

FBP1, GDH2, HXK1, COQ3,

CYB2, TPS2, FOX2

COX6, ADH2,

MLS1

ICL1, PCK1, CYC1,

SUC2

cAMP External cAMPw

(no glucose present)

pde2 FBP1 PCK1, ICL1,

GDH2

SUC2

cAMP-dependent

protein kinase

2% Glucose� tpkw bcy1 FBP1, ICL1, PCK1, FOX2 TPS1, HXK1,

GLK1, TKL2

TPS2, GPH1, GPD1,

COQ3, YJR036

Snf3, Rgt2, Gpr1 2% Glucosez snf3 rgt2 gpr1 FBP1, GDH2, SUC2 – –

Hxk2 2% Glucosez,‰ hxk2 FBP1, ICL1, ADH2, FOX1 GDH2, GLK1,

CYC1, CYB2

SUC2, HXK1, GAL1

Hxk1, Hxk2 2% Glucosez,‰ hxk1 hxk2 FBP1, ICL1, PCK1 GDH2 GLK1

Hxk1, Hxk2, Glk1 2% Glucosez hxk1 hxk2 glk1 – – FBP1, ICL1, GDH2

To establish the role of different elements related with glucose signalling on transcriptional repression, repression of selected genes has been measured

in different mutant strains (see text for details). Strong response is equivalent to the response of a wild-type strain to 2% glucose.�Wang et al. (2004).wZaragoza et al. (1999).zBelinchon & Gancedo (2007b).‰See text for other references.


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levels triggered by glucose is modest, between 1.5- and

4-fold. This increase is usually rapid, o 20 min, but it is

sometimes transient, mRNA levels decreasing markedly at

60 min. There are exceptions, as for some genes the degree of

induction is higher, about fivefold for RPA12 or RPC53, 20-

fold for HXT3, and even 100-fold for HXT1, while for genes

such as TRP3, ARG3 or MET14, the kinetics of induction are

slower (Wang et al., 2004). The role that different sensing

elements play in the induction of different genes is examined

in the next paragraphs.

Binding of glucose to the plasma membrane sensors Snf3/

Rgt2 triggers the induction of a limited number of genes

through a cascade of reactions discussed previously. Most of

these genes encode glucose transporters (Ozcan & Jonhston,

1999), but a few others have been identified (Kaniak et al.,

2004), and among them MIG2 is of special interest, as

increased levels of the DNA-binding protein Mig2 contri-

bute to glucose repression (Lutfiyya et al., 1998). The other

plasma membrane sensor, Gpr1 (see the corresponding

section), is often required for a maximal response to glucose;

in its absence, the degree of induction decreases by a factor

of two in many cases (Wang et al., 2004). Gpr1 is not

needed, however, for the complete induction of HXT1 or

pyruvate decarboxylase (Belinchon & Gancedo, 2007a)

(Table 2).

Artificial activation of Ras2, in the absence of glucose (see

section on ‘Signalling by intracellular metabolites’), can

cause an increase in mRNA levels for many genes; for some

genes the extent of induction is similar to that triggered by

glucose, while for others induction is low, or even absent for

genes such as HXK2 or ENO2 (Table 2). Ras2 activation

experiments performed in a strain with a weak PKA

insensitive to cAMP (tpkw bcy1 background) have led to the

conclusion that Ras signalling takes place exclusively

through a cAMP-regulated PKA (Wang et al., 2004). It

should be noted, however, that for HXT1 and HXT3 there

was at least a 10-fold induction by activated Ras2 in this

background (supplementary information in Wang et al.,

2004). Although these results have not yet been confirmed

by northern analysis or quantitative reverse transcriptase

(RT)-PCR, it would be interesting to follow this lead and

investigate a potential alternative pathway for Ras signalling.

To investigate whether a cAMP-regulated PKA is not only

sufficient, but also necessary, for glucose induction of

transcription, glucose was added to a tpkw bcy1 mutant

strain growing on glycerol (Wang et al., 2004), because in

such a strain PKA is no longer sensitive to the changes in

cAMP intracellular concentration triggered by glucose. In a

large number of cases there was a strong induction of

transcription by glucose (at least 10-fold), thus showing that

metabolism of glucose can provide for most genes an

induction mechanism independent of an increase in PKA

activity. It may be observed that for many genes, mRNA

levels were much lower (5–20-fold) in the mutant strain

than in the wild type, during growth in glycerol. This

suggests that in a wild-type yeast grown in glycerol, the

PKA activity is greater than in the mutant and sufficient to

cause a partial induction of the corresponding genes.

Surprisingly, while galactose does not act as an inducer in

the wild-type strain, for some genes it can cause large

increases in mRNA levels in the tpkw bcy1 mutant, although

the strain lacks Gal1 and is therefore unable to metabolize

galactose (Wang et al., 2004). This is a result that remains

difficult to interpret.

The role of the glucose phosphorylating enzymes in the

induction of transcription by high glucose is not the same

for different genes (Table 2). In mutant strains lacking Hxk2,

there is a strong decrease in the induction of HXT1 and

HXK2, but HXT3 and pyruvate decarboxylase are fully

induced (Ozcan & Johnston, 1995; Rodrıguez et al., 2001;

Table 2. Elements affecting different genes induced by glucose

Element(s) Stimulus Strain used Strong response Weak response

Response very weak

or absent

cAMP Galactose GAL10-RAS2Val19 gal1 THR4, RPC53, RPO31 ARG3, MET14, HXT3 HXT1, HXK2, ENO2

cAMP-dependent

protein kinase

Glucose� tpkw bcy1 ENO2, ARG3, THR4,

GCN3, RPA43, RPC53

HXT1, HXT3, RPC82 MET14, RPO31

Snf3, Rgt2 Glucose� snf3 rgt2 PDC1 SUC2w HXT1, HXT3

Gpr1 Glucose� gpr1 PDC1, HXT1 SUC2w

Hxk2 Glucose� hxk2 PDC1, HXT3 HXT1, HXT2w, HXT3w –

Hxk1, Hxk2 Glucose� hxk1 hxk2 SUC2w, PDC1w PDC1 –

Hxk1, Hxk2, Glk1 Glucose� hxk1 hxk2 glk1 SUC2 HXT1 PDC1

Hxk1, Hxk2, Glk1, Gpr1 Glucose� hxk1 hxk2 glk1 gpr1 – HXT1 SUC2

To establish the role of different elements related with glucose signalling on transcriptional induction, induction of selected genes has been measured in

different mutant strains (see text for details). Strong response is equivalent to the response of a wild-type strain to the same concentration of glucose.�Induction by 2–4% glucose, unless otherwise indicated.wInduction by 0.05% glucose.

See text for references.



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Belinchon & Gancedo, 2007a). While induction of pyruvate

decarboxylase is decreased by 50% in a double mutant hxk1

hxk2 and blocked in the triple hxk1 hxk2 glk1 mutant, in this

last mutant there is a partial induction of HXT1 and SUC2 is

highly induced. The need for glucose phosphorylation to

induce pyruvate decarboxylase is in agreement with the

observation that this induction is dependent on products

of glucose metabolism such as glucose-6P and three-carbon

metabolites (Boles & Zimmermann, 1993). It should be

noted that, in the absence of glucose phosphorylation, Gpr1

is absolutely required for the induction of SUC2 but is

dispensable for that of HXT1 (Belinchon & Gancedo,

2007a).

A low concentration of glucose, o 0.1%, is able to induce

a number of genes, and in this case also, there are differ-

ent signalling pathways controlling the induction process

(Ozcan & Johnston, 1995; Belinchon & Gancedo, 2007a).

The induction of HXT2, HXT3 and HXT4, encoding glucose

transporters, requires the glucose sensor Snf3 and the

expression of these genes decreases two- to fivefold in the

absence of Hxk2 (Ozcan & Johnston, 1995). The induction

of SUC2 has only a partial requirement for Snf3/Rgt2 and

does not depend on Hxk2, or Hxk1, but it is slightly

impaired in the absence of Gpr1 (Belinchon & Gancedo,

2007a) and considerably decreased in mutants lacking PKA

(J.M. Gancedo, unpublished results). Induction of pyruvate

decarboxylase by low glucose is little affected by the lack of

both Hxk1 and Hxk2 (Belinchon & Gancedo, 2007a).

Proteins activated by glucose

When glucose becomes available to glucose-starved yeast,

the activity of a number of enzymes increases. Prominent

among them, because it initiates an important signalling

pathway, is adenylate cyclase. Activation of adenylate cyclase

requires the Ras1/Ras2 proteins, located in the plasma

membrane and able to interact directly with the adenylate

cyclase complex (Shima et al., 2000). The exact mechanism

for activation of the Ras proteins is not known, but it has

been established that glucose causes a modest increase in

Ras1/2 GTP loading and that this increase requires glucose

phosphorylation; the increase is impaired in a strain lacking

Cdc25, the GTP/GDP exchange factor for the Ras proteins.

Inhibition of the Ira1/Ira2 proteins, which increase the

GTPase activity of Ras1/2, contributes to the activation of

the Ras proteins triggered by glucose (Colombo et al., 2004).

The increased GTP loading of the Ras proteins does not

depend on the glucose receptor Gpr1 or on its G-binding

protein Gpa2 (Colombo et al., 2004); however, the Gpr1/

Gpa2 system is required for full activation of adenylate

cyclase (Rolland et al., 2000). Only the GTP-bound, active

form of Gpa2 is able to bind adenylate cyclase (Peeters et al.,

2006), but this binding is not enough to trigger a full

response, because in strains unable to phosphorylate glucose

the activation of adenylate cyclase observed, which is

dependent on Gpr1 and on a high concentration of glucose,

is very weak (Kraakman et al., 1999a). The activation of

adenylate cyclase causes an increase in the intracellular

concentration of cAMP, which in turn activates PKA.

Activated PKA phosphorylates different enzymes such as

the neutral trehalase Nth1 (Ortiz et al., 1983; Uno et al.,

1983; Francois & Parrou, 2001) or the 6-phosphofructo-2-

kinase Pfk26 (Francois et al., 1984; Dihazi et al., 2003) and

thereby increases their activity.

The activation by glucose of the plasma membrane

H1-ATPase is also related to phosphorylation of the protein

at different positions, but PKA is not involved in the process,

at least directly (Portillo et al., 1991). While phosphoryla-

tion of Thr912 from the H1-ATPase appears to mediate an

increase in the Vmax of the enzyme (Portillo et al., 1991),

phosphorylation of Ser899 would cause a decrease in the Km

for ATP (Eraso & Portillo, 1994). Phosphorylation of Ser899

is performed by the protein kinase Ptk2 (Eraso et al., 2006),

but the protein kinase responsible for Thr912 phosphoryla-

tion has not been identified, in spite of a systematic search

using protein kinase mutants (Goossens et al., 2000). The

activity of Ptk2 is the same in membranes isolated from cells

incubated in the presence or in the absence of glucose and is

not affected by glucose itself (Eraso et al., 2006), but it could

be modulated by some glucose-derived metabolite as in-

dicated by the fact that activation of the H1-ATPase is

strongly reduced in an hxk1 hxk2 mutant and extremely low

in a mutant unable to phosphorylate glucose (Belinchon &

Gancedo, 2007b). On the other hand, the activation of the

H1-ATPase is independent of the glucose sensors Snf3/Rgt2

and Gpr1 (Belinchon & Gancedo, 2007b).

Proteins inactivated by glucose

Glucose also triggers the inactivation of different proteins, a

phenomenon called catabolite inactivation (Holzer, 1976).

Phosphorylation of FbPase by PKA causes a partial inactiva-

tion of the enzyme (Gancedo et al., 1983; Rittenhouse et al.,

1987); this process is impaired in a mutant unable to

phosphorylate glucose and the impairment is stronger when

both the glucose phosphorylating enzymes and the glucose

sensor Gpr1 are absent (Belinchon & Gancedo, 2007b). This

is consistent with the observation that phosphorylation of

FbPase by PKA is strongly activated by fructose-2,6-bispho-

sphate, a regulatory metabolite that is formed when glucose

is metabolized (Gancedo et al., 1983). While partial inacti-

vation of isocitrate lyase by glucose is also dependent on

phosphorylation by PKA (Ordiz et al., 1996), there is no

information on the protein kinase(s) performing the phos-

phorylation of the malate dehydrogenase isoenzyme Mdh2


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that takes place on glucose addition and causes inactivation

of the enzyme (Minard & McAlister-Henn, 1994).

Glucose not only causes the inactivation of metabolic

enzymes, but also that of the protein kinase Snf1, which

plays a major role in allowing the transcription of glucose-

repressed genes. In this case inactivation is due to depho-

sphorylation of Snf1 by the Glc7-Reg1 complex (Sanz et al.,

2000). Glucose may shift the equilibrium between the

phosphorylated and nonphosphorylated forms of Snf1,

by activating Glc7-Reg1 and/or by inhibiting the protein

kinases Sak1, Tos3 and Elm1 that phosphorylate Snf1 (Hong

et al., 2003; Nath et al., 2003; Sutherland et al., 2003).

However, the mechanism by which glucose modifies Snf1

activity has not yet been unravelled (Hedbacker & Carlson,

2008; Rubenstein et al., 2008).

Proteins degraded by glucose

Glucose also triggers the degradation of a number of

enzymes, a process that has been most thoroughly studied

for FbPase and Mdh2. Two conflicting views on the mechan-

ism of FbPase degradation have been offered. One of them

contemplates a regulated transfer of FbPase to the vacuole and

its degradation by vacuolar proteases (Chiang & Schekman,

1991; Shieh et al., 2001); the other one proposes that FbPase

is first subject to ubiquitination (Schork et al., 1995) and then

degraded by the proteasome (Schork et al., 1994). These views

may be reconciled by the observation that, depending on the

physiological conditions of the yeast cells, degradation of

FbPase, and also of Mdh2, can take place in the vacuole or in

the proteasome (Hung et al., 2004). Degradation in the

vacuole requires the phosphorylation of glucose but may

proceed in the absence of Hxk1 and Hxk2 (Hung et al., 2004;

Belinchon & Gancedo, 2007b), while degradation by the

proteasome is strictly dependent on Hxk2 (Horak et al.,

2002). Reg1 and Grr1 play a role in the degradation of FbPase

in the proteasome (Horak et al., 2002), perhaps related to their

possible involvement in the ubiquitination process. Reg1 is

also required for the transport of FbPase from intermediate

vesicles to the vacuoles (Cui et al., 2004). On the other hand,

the cAMP-signalling pathway is required for the vacuolar-

dependent degradation of FbPase (Hung et al., 2004;

Belinchon & Gancedo, 2007b) but not for degradation by the

proteasome (Horak et al., 2002; Hung et al., 2004). The

transcriptional repressor Rgt1 is required for FbPase degrada-

tion in some conditions (Belinchon & Gancedo, 2007b) but

not in others (Horak et al., 2002); its specific role, however,

has not been elucidated. Degradation of phosphoenolpyruvate

carboxykinase appears to proceed by a mechanism similar to

that described for FbPase and Mdh2 (Muller et al., 1981;

Hammerle et al., 1998).

Mth1, a regulatory protein involved in the transcription

control of the HXT genes, is also degraded on glucose

addition as described in the section on Snf3 and Rgt2. This

degradation is considerably reduced in a reg1 mutant, and

this seems to be related to the fact that the Glc7-Reg1

complex is required for glucose activation of the kinases

Yck1/2 (Gadura et al., 2006), as activation of Yck1/2 is an

early step in the pathway leading to Mth1 ubiquitination

and degradation (Moriya & Johnston, 2004). This role of

Reg1 could explain the observation that HXT1 induction is

strongly impaired in a reg1 mutant but appears in contra-

diction with the fact that the HXT2, HXT3 or HXT4 genes

are normally induced in this mutant (Ozcan & Johnston,

1995).

On glucose addition different kinds of transporters are

internalized and degraded, among them the maltose/H1

symporter Mal61 (Riballo et al., 1995), the galactose per-

mease Gal2 (Horak & Wolf, 1997), the monocarboxylate/H1

symporter Jen1 (Paiva et al., 2002) and the glycerol/H1

symporter Stl1 (Ferreira et al., 2005). Because the first step

in the internalization of the transporters is their ubiquitina-

tion (Horak & Wolf, 1997; Lucero & Lagunas, 1997; Paiva

et al., 2002), the corresponding signal is likely to be the

binding of glucose to Snf3/Rgt2 and the subsequent activa-

tion of Yck1/2 as occurs for Mth1 (Gadura et al., 2006). The

fact that Gal2 inactivation is not prevented in an snf3 or in

an rgt2 mutant (Horak et al., 2002) may be due to the

partial functional redundancy of Snf3 and Rgt2. Degrada-

tion of the ubiquitinated transporters is independent of the

proteasome and takes place within the vacuole. Although

Grr1, the F-box protein of one of the SCF ubiquitin ligase

complexes, is required for the degradation of the Gal2

transporter, it does not seem to be required for the ubiqui-

tination of Gal2 itself (Horak & Wolf, 2005). Degradation

of the maltose permease Mal61 by glucose requires Rgt2

and Grr1 but it is not impaired in an rgt1 mutant (Jiang

et al., 1997).

Other processes controlled by glucose

In addition to the processes already considered, the presence

of glucose affects yeast physiology in many other ways.

Glucose increases the turnover of a number of mRNA

species (Lombardo et al., 1992; Mercado et al., 1994), while

it causes a transient stabilization of ribosomal protein

mRNAs (Yin et al., 2003). The increased turnover of SDH2

mRNA, encoding the iron–sulfur protein subunit of succi-

nate dehydrogenase, takes place only at high levels of

glucose; in contrast, the degradation rates of the FBP1

and PCK1 mRNAs, encoding gluconeogenic enzymes, are

already increased by the addition of 0.02% glucose (Yin

et al., 2000). Glucose phosphorylation is required to en-

hance the turnover of SDH2, FBP1, PCK1 or SUC2 mRNAs,

but Hxk2 is not specifically needed, except for SDH2

(Cereghino & Scheffler, 1996; Yin et al., 2000). In all cases



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the effect of glucose is blocked in an reg1 mutant, where the

activity of the protein kinase Snf1 is less sensitive to glucose

(Sanz, 2007). Although external cAMP (in a pde2 back-

ground) can trigger degradation of PCK1 and SDH2

mRNAs, the increased degradation of PCK1 mRNA ob-

served in the presence of 0.02% glucose also occurs in the

absence of a Ras-cAMP pathway (Yin et al., 2000). It appears

therefore that glucose controls mRNA turnover through

alternative, partially redundant signalling pathways. The

same applies to the stabilization by glucose of different

ribosomal protein encoding mRNAs (Yin et al., 2003).

Specifically, the effect of glucose on RPL3 mRNA stability,

but not on that of RPS6 mRNA, is retained in the absence of

glucose phosphorylation, while the Ras-cAMP pathway is

required for stabilization of RPL3 and RPL24 mRNAs but

not for that of RPS6 mRNA.

Glucose also affects the translation rate of mRNA. On

transfer of glucose-growing yeast to a medium lacking

glucose, there is a very rapid (1–2 min) inhibition of

translation (Ashe et al., 2000). This effect of carbon source

removal is specific for glucose (or fructose), as it does not

occur in yeast grown in sucrose, maltose or galactose.

Inhibition of translation upon glucose withdrawal does not

involve the pathways implicated in the inhibition of transla-

tion caused by amino acid starvation (Ashe et al., 2000).

Translation is restored rapidly (5 min) by addition of

glucose (Ashe et al., 2000) and slowly (hours) by incubation

of the yeast cells under aerobic conditions in the absence of a

carbon source (Uesono et al., 2004). Although several

proteins such as Pat1, Ddh1 or Sbp1 have been identified as

destabilizing translation initiation complexes upon glucose

removal (Coller & Parker, 2005; Segal et al., 2006), there is

no information on the mechanism(s) through which glu-

cose may control their activity. Inhibition of translation after

glucose removal is prevented if yeast cells are in a metabolic

condition that mimicks to some extent the absence of

glucose. This occurs in mutants with a very low PKA activity

or in reg1 or hxk2 mutants, provided Snf1 is present (Ashe

et al., 2000). Translation is also strongly inhibited when yeast

cells are transferred from a medium with glucose to a

medium with ethanol, and this inhibition requires the Gat1

protein, involved in signalling the quality of the carbon

source through the Tor pathway (Kuruvilla et al., 2001).

The yeast vacuolar H1ATPase (V-ATPase) is a multi-

subunit complex responsible for the acidification of the

organelle. The assembly of this V-ATPase is regulated by

glucose, 0.1% glucose increasing the level of assembly from

10 to 25%, while in the presence of 2% glucose, 60% of the

subunits are assembled in complexes (Parra & Kane, 1998).

Glucose metabolism beyond glucose-6-phosphate is re-

quired for triggering V-ATPase assembly but a cAMP signal

is neither sufficient nor necessary for the process to take

place (Parra & Kane, 1998). Because aldolase is specifically

needed for assembly, it has been suggested that aldolase may

act in this process as a sensor for the presence of glucose (Lu

et al., 2004). V-ATPase assembly is also dependent on the

RAVE complex, formed by the Rav1, Rav2 and Skp1 proteins

(Seol et al., 2001), but it is not known whether glucose

directly controls RAVE.

The growth rate of S. cerevisiae and its cell size depend on

the carbon source in the medium and both are highest in

glucose-grown cells (Johnston et al., 1979). The effect of

glucose on cell size is mediated by the Gpr1-Gpa2 system

(Tamaki et al., 2005; Vanoni et al., 2005) and requires

Cdc25, the Ras GTP–GDP exchange protein (Belotti et al.,

2006). Glucose increases the growth rate by inducing a

diverse set of genes: glycolytic and ribosomal protein genes,

as well as genes related to the cell division cycle. An

important signal for this process is the activation of PKA

by cAMP, which increases the transcription rate of Rap1

target genes (Klein & Struhl, 1994) and causes an increase

in the level of the cyclin Cln3 (Hall et al., 1998). The increase

in Cln3 concentration triggered by glucose is due both

to a PKA-dependent stimulation of its synthesis (Hall

et al., 1998) and to a PKA-independent activation of

CLN3 transcription, involving the transcription factor Azf1

(Newcomb et al., 2002). It has been shown that the induc-

tion by glucose of genes such as CLN3, BCK2 and CDC28,

which promote progress through the start phase of the cell

cycle, does not require signalling through Snf3/Rgt2 or

Hxk2 (Newcomb et al., 2003). On the other hand, it does

not occur in pfk1 or pfk2 mutants or in the presence of

iodoacetate, an inhibitor of glyceraldehyde-3-phosphate

dehydrogenase, thus suggesting that some product of glu-

cose metabolism plays a role in the control of growth rate

(Newcomb et al., 2003). The transcriptional activator Gcr1

also affects the response to glucose, as it is required to

stimulate cellular metabolism and transcription of CLN

genes (Willis et al., 2003; Barbara et al., 2007). Although

the effects of Gcr1 may be indirect, it appears more likely

that Gcr1 acts in concert with the transcription factor Rap1

(Santangelo, 2006).

Glucose blocks the sporulation developmental pathway,

initiated in diploid cells by the phosphorylation of the

transcriptional activator Ime1. Glucose regulates the process

by inhibiting Rim11, a homologue of glycogen synthase

kinase 3-b, able to phosphorylate a tyrosine residue, and

possibly a serine residue, in Ime1 (Rubin-Bejerano et al.,

2004). The glucose signal is transmitted through the cAMP/

PKA pathway that mediates, directly or indirectly, the

phosphorylation of Ser5, Ser8 and/or Ser12 in Rim11, and

the inhibition of its kinase activity (Rubin-Bejerano et al.,

2004). An additional element involved in sporulation is the

protein kinase Ime2, able to phosphorylate Sic1, an inhibi-

tor of meiotic DNA replication, thus allowing its degrada-

tion. Ime2 also stimulates a transcription factor, Ndt80,


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involved in the G2-M transition (Benjamin et al., 2003). In

the presence of glucose Ime2 is destabilized, a process

dependent on ubiquitination by the SCFGrr1 ubiquitin ligase

(Purnapatre et al., 2005). The mode of action of glucose has

not yet been established, but by analogy with the mechanism

of regulation of Mth1 (Moriya & Johnston, 2004) it could

involve the binding of glucose to the Snf3/Rgt2 receptors.

In some strains of S. cerevisiae, a further developmental

pathway may take place, pseudohyphal growth caused by

nitrogen starvation (Gimeno et al., 1992; Gancedo, 2001).

Under such conditions, glucose (or sucrose) and the Gpr1-

Gpa2 system are required to trigger the formation of

pseudohyphae (Lorenz et al., 2000). It has been reported

recently that pseudohyphal growth can be triggered by

maltose or maltotriose through a signalling pathway inde-

pendent of Gpr1 (Van de Velde & Thevelein, 2008).

In other yeasts

In other yeasts, there is only scattered information on the

elements controlling repression by glucose. In S. pombe,

repression of fbp1, encoding FbPase, is dependent on the

phosphorylation of the transcription factor Rst2 by a cAMP-

dependent protein kinase (Higuchi et al., 2002). In mutants

lacking this protein kinase, or unable to activate it, fbp1 is no

longer sensitive to repression by glucose (Byrne & Hoffman,

1993; Hoffman, 2005b). In contrast, glucose repression of

inv1, encoding invertase, is maintained in the absence of the

cAMP-signalling pathway (Tanaka et al., 1998). While the

lack of Scr1, a repressor protein orthologous of Mig1,

relieves repression of inv1, it has no effect on the repression

of fbp1 (Tanaka et al., 1998). Nevertheless, in the presence of

glucose Scr1 binds to the UAS2 site of the fbp1 promoter,

and when glucose is removed Scr1 is exported to the

cytoplasm, in a process not regulated by the cAMP pathway,

and replaced by the activator Rst2 (Hirota et al., 2006). In

K. lactis, glucose repression of different genes is impaired in

strains with a decreased rate of glucose transport (Weirich

et al., 1997; Milkowski et al., 2001). The fact that, in K. lactis,

the strong repression of invertase by glucose is independent

of Mig1 (Georis et al., 1999) may be related to the observa-

tion that expression of invertase in this yeast does not

require Fog2, the orthologue of Snf1 (Goffrini et al., 1996).

In P. angusta, glucose fails to repress peroxisomal enzymes in

a mutant lacking the orthologue of Snf3, Gcr1 (Stasyk et al.,

2004), while repression of alcohol oxidase is only partially

relieved in a mig1 mig2 double mutant (Stasyk et al., 2007).

Regarding the signals controlling induction of transcrip-

tion by glucose in other yeasts, there are few data available.

In K. lactis, induction of pyruvate decarboxylase by glu-

cose is abolished in a mutant where the genes KHT1 and

KHT2, encoding for glucose transporters, have been de-

leted (Milkowski et al., 2001). Induction of RAG1, a gene

encoding a glucose transporter, requires the RAG5 gene,

encoding hexokinase; although the mode of action of Rag5

is not known, it should be noted that induction of RAG1 by

galactose is also dependent on Rag5, suggesting that this

protein plays a regulatory role independent of its metabolic

function (Prior et al., 1993). Induction by glucose of

RAG1 and of several glycolytic genes is only partial in the

absence of the transcriptional activator Sck1 (Lemaire et al.,

2002). Because expression of both RAG1 and SCK1 is

blocked by the repression factor Rgt1 (Rolland et al., 2006;

Neil et al., 2007), the Rag4/Rag8 pathway that inactivates

Rgt1 (Rolland et al., 2006)[cf. section above on Snf3/Rgt2] is

essential for the induction by glucose of RAG1 and of

other glycolytic genes. In C. albicans, induction by glucose

of genes encoding hexose transporters requires Hgt4, an

orthologue of the Snf3 glucose sensor (Brown et al., 2006).

In yeast genera different from Saccharomyces, there is little

evidence for activation or inactivation of proteins by glu-

cose. An exception is the activation of adenylate cyclase by

glucose in S. pombe (Hoffman, 2005a, b). As discussed in the

section on Gpr1, it depends on the binding of glucose to

Git3, a plasma membrane protein that plays the same role as

ScGpr1, and on the trimeric G-protein formed by the Gpa2,

Git5 and Git11 subunits. In contrast with the situation in S.

cerevisiae, the SpRas1 protein from S. pombe plays no role in

glucose/cAMP signalling (Fukui et al., 1986) but the direct

activation of adenylate cyclase by Gpa2 requires the Git1

protein (Kao et al., 2006). Git1 lacks sequence homologues

in other fungi and its mechanism of action remains to be

determined. Regarding catabolite inactivation, only a few

systems have been studied and it was found that it does not

affect FbPase in S. pombe (Vassarotti et al., 1982) or

isocitrate lyase in K. lactis (Lopez et al., 2004). In methylo-

trophic yeasts, peroxisomal enzymes can be degraded after

peroxisomes have been engulfed within the vacuole (Dunn

et al., 2005), but this process of pexophagy is induced both

by ethanol and by glucose and is therefore not equivalent to

the classical catabolite inactivation. There is, nevertheless,

a case where degradation is specifically triggered by glu-

cose; this is the glucose-induced microautophagy in Pichia

pastoris, which requires the a-subunit of phosphofructoki-

nase (Yuan et al., 1997). The role of phosphofructokinase

has not yet been established, but a catalytically inactive

phosphofructokinase is still functional for this process.

Interestingly, the glucose-induced degradation of peroxiso-

mal enzymes in P. pastoris does not require the Gpr1/Gpa2

proteins, while in S. cerevisiae lack of Gpr1 or Gpa2

suppresses the degradation of the peroxisomal thiolase

induced by glucose (Nazarko et al., 2007, 2008).

As observed in S. cerevisiae for pseudohyphal growth,

hyphae formation in C. albicans, in a glucose-containing

medium, is also dependent on both Gpr1 and Gpa2 (Miwa

et al., 2004).



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Signalling pathways for other nutrients

Amino acids

Saccharomyces cerevisiae may use a large number of com-

pounds as sources of nitrogen, but it does not grow at the

same rate on the different nitrogen sources. A regulatory

mechanism, called nitrogen catabolite repression, allows the

yeast to use preferentially the nitrogen sources that support

the faster growth (Cooper, 2002). Superimposed to this

control system, other regulatory pathways respond to the

presence of amino acids in the growth medium.

An important sensor of external amino acids is the

plasma membrane protein Ssy1, with strong structural

homology with amino acid permeases (Didion et al., 1998;

Poulsen et al., 2005). The targets of this signalling pathway

are the transcription factors Stp1 and Stp2, able to activate

the expression of a number of genes encoding amino acid

permeases (Andreasson & Ljungdahl, 2002). As shown in

Fig. 4, Ssy1 forms a complex with the Prt3 protein and the

endoprotease Ssy5 (Forsberg & Ljungdahl, 2001b) and,

upon binding an amino acid, activates Ssy5 (Abdel-Sater

et al., 2004; Andreasson et al., 2006). Branched amino acids,

in particular leucine, are most effective, while charged

amino acids have a smaller effect and proline is ineffective

(Iraqui et al., 1999; Gaber et al., 2003). Activated Ssy5, in

turn, causes the endoproteolytic processing of the transcrip-

tion factors Stp1 and Stp2 (Andreasson & Ljungdahl, 2002).

In the absence of amino acids, Stp1 and Stp2, that are not

integral membrane proteins, are associated with the plasma

membrane through their N-terminal domains. After the

limited endoproteolysis brought about by Ssy5, the released

C-terminal domains from Stp1/Stp2 enter the nucleus and

activate the expression of different genes encoding amino

acid permeases. Although the unprocessed forms of Stp1/

Stp2 are not totally excluded from the nucleus, they are

prevented to act as inducers of transcription by three inner

nuclear membrane proteins, Asi1, Asi2 and Asi3, acting in

concert (Zargari et al., 2007).

The processing of Stp1/Stp2 requires the SCFGrr1 ubiqui-

tin–ligase complex (Bernard & Andre, 2001) and the casein

kinases Yck1/2 (Abdel-Sater et al., 2004). Yck1/2 phosphor-

ylate Stp1, and possibly Stp2, but this phosphorylation is

not dependent on the presence of external amino acids

(Abdel-Sater et al., 2004). The substrate of the ubiquitin–

ligase complex has not yet been identified, but it has been

established that Grr1 acts upstream of the protease Ssy5

(Abdel-Sater et al., 2004), that the protein Ptr3 is modified

in the presence of amino acids (Forsberg & Ljungdahl,

2001b) and that the modification of the Ptr3 migration

profile is Grr1-dependent (Abdel-Sater et al., 2004). It is

therefore tempting to propose that Ptr3 is ubiquitinated

by SCFGrr1, and that this modification is required for Ssy5

activation (Fig. 4). It is interesting to note that, while the

SCFGrr1 complex is required for amino acid signalling,

the activity of the proteasome is not necessary (Andreasson

& Ljungdahl, 2002; Abdel-Sater et al., 2004), thus suggesting

that in this case ubiquitination does not tag a protein

for degradation but modifies its capacity to interact with

other proteins, a function for ubiquitination that has been

discussed previously (Schnell & Hicke, 2003).

An authenticated amino acid permease, Gap1, also plays a

role as nutrient sensor, because its interaction with amino

acids results in activation of PKA. This activation, however,

does not seem to involve an increase in the intracellular

Fig. 4. Amino acid-signalling pathway. In the absence of amino acids the transcription factors Stp1/2 are phosphorylated by the casein kinases Yck1/2

but are not further processed. Although these forms of Stp1/2 are not fully excluded from the nucleus, they are prevented from activating gene

transcription by the inner nuclear membrane proteins Asi1, Asi2, Asi3, acting together. When amino acids are present, they bind the transmembrane

protein Ssy1 and activate the protease Ssy5, in a process that requires the protein Ptr3 and the SCFGrr1 complex, that may play a role in ubiquitinating

Ptr3. Ssy5 performs a limited endoproteolysis of phosphorylated Stp1/2 and the processed Stp1/2 enter the nucleus and activate transcription. See text

for details.


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concentration of cAMP (Donaton et al., 2003), but depends

on Sch9, a protein kinase paralogous to mammalian protein

kinase B (Crauwels et al., 1997). It is interesting to note that

signals given by different nutrients are integrated to mod-

ulate the activity of PKA and control the growth rate of the

yeast cells, as well as other processes. For instance, in

glucose-grown, nitrogen-starved yeast, the addition of a

nitrogen source triggers different effects, such as the activa-

tion of trehalase or changes in the rate of transcription of a

variety of genes (Pernambuco et al., 1996). Some of these

effects depend on the presence of glucose (or fructose) but

do not require the phosphorylation of the sugar. Other

effects, such as the induction of genes encoding ribosomal

proteins, are stronger in the presence of glucose, but only

when the sugar can be phosphorylated (Pernambuco et al.,

1996).

Intracellular sensors for amino acids have also been

described. When the intracellular concentration of amino

acids declines, the corresponding tRNAs remain partially

uncharged and activate the protein kinase Gcn2 that phos-

phorylates the translation initiation factor eIF2; phosphory-

lated eIF2, in turn, stimulates the translation of the GCN4

gene (Hinnebusch, 1997). The subsequent increase in the

intracellular concentration of the transcription factor Gcn4

causes large modifications in the pattern of transcription of

genes involved in amino acid metabolism (Natarajan et al.,

2001). Besides Gcn4, there are other transcription factors

that respond to the presence or absence of specific amino

acids. Put3, for instance, binds proline and can then activate

the transcription of the genes PUT1 and PUT2, which

encode proteins involved in proline catabolism (Sellick &

Reece, 2005), while the activity of the transcription factor

Arg81 depends on its binding to arginine (Amar et al.,

2000). Lys14, which activates the transcription of genes

involved in lysine biosynthesis, is only operative when

bound to a-aminoadipate semialdehyde, an intermediate of

the biosynthetic pathway that accumulates in the absence of

lysine (Feller et al., 1994). The transcription factor Leu3

responds indirectly to leucine, as it is able to bind isopro-

pylmalate, a product of leucine catabolism. In the absence of

isopropylmalate Leu3 represses the transcription of a num-

ber of genes involved in amino acid metabolism, while Leu3

bound to isopropylmalate can act as a transcriptional

activator (Sze et al., 1992; Kohlhaw, 2003).

Sulfur-containing compounds

The transcription of genes encoding enzymes involved in the

assimilation of sulfur-containing compounds and in the

biosynthesis of methionine is the subject of a particularly

complex regulatory network. This may be explained by the

fact that S-adenosylmethionine (SAM) serves as a methyl

donor for a very large number of transmethylation reactions

involving not only proteins but also nucleic acids or lipids

(Menant et al., 2006a). Transcription of many genes is

repressed in the presence of methionine or SAM in the

medium (Cherest et al., 1969) but also of cysteine or

glutathione (Ono et al., 1996). It has been established that

under these conditions the expression of most MET genes is

turned off, due to degradation or inactivation of the

transcription factor Met4, mediated by the ubiquitin ligase

SCFMet30 (Kaiser et al., 2000; Rouillon et al., 2000).

Although it had been proposed that the signal for repression

of the MET genes is an increase in the intracellular concen-

tration of SAM (Thomas & Surdin-Kerjan, 1997), there is

now convincing evidence that it is the intracellular concen-

tration of cysteine that regulates the rate of transcription of

these genes (Ono et al., 1996; Hansen & Johannesen, 2000;

Srikanth et al., 2005; Menant et al., 2006b). It is not yet

known, however, whether cysteine controls directly the

activity of SCFMet30. Although sulfur amino acid metabo-

lism in S. pombe presents some particularities with respect to

S. cerevisiae, it also appears to be regulated by the intracel-

lular concentration of cysteine (Brzywczy et al., 2002).

Ammonium

In S. cerevisiae, there are three ammonium transporters,

Mep1, Mep2 and Mep3, with similar sequences (Marini

et al., 1997). It has been proposed that these proteins include

11 transmembrane domains, an extracytosolic N-terminus

and an intracellular C-terminal tail (Marini & Andre, 2000).

Mep2 is specifically required for pseudohyphal differentia-

tion in response to nitrogen starvation, and it has therefore

been suggested that it acts as an ammonium sensor (Lorenz

& Heitman, 1998). Although Mep2 is N-glycosylated in an

asparagine residue at its N-terminus, while Mep1 and Mep3

are not, the glycosylation is not necessary for pseudohyphal

growth (Marini & Andre, 2000). Mep2, and to a lesser extent

Mep1, mediate the activation of PKA triggered by ammo-

nium (Van Nuland et al., 2006). As also reported for the

activation triggered by amino acids, this process is not

associated with an increase in cAMP levels (Thevelein et al.,

2005). Because specific mutations in Mep2 did not affect

activation of the PKA pathway and pseudohyphal growth in

the same way, it has been suggested that the two processes

are not regulated through the same mechanism (Van Nu-

land et al., 2006). This contrasts with the observation that

the requirement for Mep2 to trigger pseudohyphal growth

when ammonium is limiting is relieved when the PKA

pathway is activated by dominant-active forms of Gpa2 or

Ras2 or by addition of exogenous cAMP (Lorenz & Heit-

man, 1998). In C. albicans, when ammonium is absent or

present at low concentrations, CaMep2 activates, via its

C-terminal cytoplasmic tail, both a cAMP-dependent path-

way and a MAP kinase cascade (Biswas & Morschhauser,



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2005). It would be interesting to examine whether a MAP

kinase is also a target for ScMep2.

Phosphate

Saccharomyces cerevisiae responds to phosphate starvation

by increasing the transcription rate of genes encoding

phosphate transporters and acid and alkaline phosphatases.

These genes are activated by the transcription factor Pho4,

which is regulated by the proteins Pho85, Pho80 and Pho81.

When there is enough phosphate available, the complex

Pho80-Pho85, formed by a cyclin and a cyclin-dependent

kinase, phosphorylates Pho4, which is then excluded from

the nucleus; when phosphate is limiting Pho81, the inhibitor

of the cyclin-dependent kinase, inhibits Pho85 activity and

Pho4 remains in the nucleus (reviewed in Lenburg &

O’Shea, 1996; Oshima, 1997; Persson et al., 2003). It is not

yet clear, however, how phosphate availability is sensed and

how the corresponding signal is transmitted to Pho81

(Mouillon & Persson, 2006).

As Pho84, a high-affinity phosphate transporter, has

some similarity to the glucose sensors Snf3 and Rgt2, it

could be a phosphate sensor; however, it is not essential for

sensing the external phosphate concentration (Wykoff &

O’Shea, 2001). It has been proposed that the low-affinity

transporters Pho87, Pho90 and Pho91 are the sensors that

monitor external phosphate (Pinson et al., 2004). More

recent evidence indicates that, whereas Pho87 and Pho90

are localized in the plasma membrane, Pho91 is found at the

vacuolar periphery and is likely to export phosphate from

the vacuolar lumen to the cytosol (Hurlimann et al., 2007).

While it has been shown that the concentration of intracel-

lular phosphate serves as a signal for the control of the

genes regulated by phosphate, the identity of the intracel-

lular phosphate sensor is not known, and a potential

candidate, Pho81, is not regarded as likely (Auesukaree

et al., 2004).

Addition of phosphate to phosphate-starved yeast cells

activates the protein kinase pathway, although it does not

trigger a cAMP signal. In this case the phosphate transpor-

ters Pho84 and Pho87 may act as phosphate sensors (Giots

et al., 2003). The stimulation of processes activated by PKA,

triggered by phosphate, requires the presence of glucose in

the medium, but may proceed in the absence of glucose

phosphorylation, as long as the glucose-signalling pathway

depending on Gpr1 is operative (Giots et al., 2003). While

the activation of PKA is required for downregulation of

Pho84 and its translocation to the vacuole where it is

degraded, inhibition of PKA does not affect the down-

regulation of acid phosphatase in response to high phos-

phate (Mouillon & Persson, 2005). This supports the

suggestion that the response to phosphate involves two

different pathways: there would be a rapid response to

external phosphate levels, dependent on Pho84/Pho87,

PKA and an abundant carbon source, and a long-term

response dependent on the internal concentration of phos-

phate and a phosphate sensor that remains to be identified

(Thomas & O’Shea, 2005; Mouillon & Persson, 2006). Other

proteins involved in nucleotide metabolism (adenylate ki-

nase, Adk1, and adenosine kinase, Ado1) or the synthesis of

inositol polyphosphate (phosphoinositide-specific phos-

pholipase, Plc1, inositol polyphosphate kinase, Arg82, and

inositol hexaphosphate kinase, Kcs1) function upstream of

Pho81, but it is not clear whether they play some role in

transmitting a phosphate signal (El Alami et al., 2003;

Auesukaree et al., 2005; Huang & O’Shea, 2005).

Final remarks

Gene regulation via reverse recruitment

The effects of glucose on the rate of transcription of different

yeast genes have been explained as a consequence of the

modification of transcriptional activators and repressors

that takes place in the presence of glucose. In the classical

recruitment paradigm these modifications affect the capa-

city of these factors, and also of diverse elements of the

polymerase II complex, to assemble on each gene. Recently,

it has been proposed that induction of a gene occurs when

this gene associates with a preassembled transcription com-

plex situated at the internal nuclear periphery and con-

nected with a nuclear pore; this process has been called gene

regulation via reverse recruitment (Santangelo, 2006; Sarma

et al., 2007). The mechanism of reverse recruitment is an

intriguing possibility, but it is not clear how glucose would

act to modify the capacity of the genes to associate with the

preformed transcription complex. If the association depends

on the presence or on the absence of activators or repressors

bound to the gene, the models of direct and reverse

recruitment would be equivalent with respect to the way in

which early signalling elements control transcription in

response to glucose. The difference between the models,

however, remains important to establish, from a mechanistic

point of view, how the transcription process is regulated.

Glucose signalling in mammalian systems

A comparison between glucose signalling in yeast and in

mammalian systems shows a number of common themes,

but also important variations. In yeast, glucose acts by

causing changes in the conformation of the proteins Snf3/

Rgt2 and Gpr1 in the plasma membrane, by interacting

with the intracellular protein Hxk2 or by modifying the

pattern of intracellular metabolites, a process that requires

metabolism, except for a small increase in the cAMP

concentration, which can be observed in the absence of

glucose phosphorylation.


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In mammalian cells, glucose operates through two kind of

signals, either by causing a depolarization of the plasma

membrane that affects the secretion of the hormones insulin

and glucagon, or by modifying the levels of intermediary

metabolites, which in turn control the activity of different

regulatory proteins (Towle, 2005).

Although two proteins in the plasma membrane of

mammalian cells, GLUT2 and SGLT3, act as glucose sensors,

their mode of action is unrelated to those of Snf3/Rgt2 or

Gpr1. GLUT2, a low-affinity glucose transporter, appears to

work mainly through its control of the glycolytic flux in liver

cells (Antoine et al., 1997), but not in pancreatic b cells

(Efrat et al., 1994). In hepatic cells, GLUT2 also plays a more

direct role in the regulation of transcription, through

specific binding to karyopherin a2, a protein required for

induction of glucose-responsive genes such as L-pyruvate

kinase (Guillemain et al., 2002). Glucose induces karyopher-

in a2 nuclear efflux, but the mechanism(s) regulating this

efflux and the precise role of karyopherin in the control of

transcription have not yet been elucidated (Cassany et al.,

2004). SGLT3, a protein belonging to the sodium/glucose

cotransporter family, causes in the presence of glucose a

depolarization of the plasma membrane as a result either of

Na1/glucose cotransport or of the activation of an ion

channel sensitive to glucose (Dıez-Sampedro et al., 2003).

This depolarization triggers an increase in intracellular

Ca11 that, in the case of pancreatic b cells, stimulates

insulin secretion (Tarasov et al., 2004) and, in the case of

glucose-sensing neurons, inhibits glucagon secretion from

pancreatic a cells, mediated by the autonomous nervous

system (Gromada et al., 2007).

Mammalian glucokinase works as an intracellular glucose

sensor and could be seen as the counterpart of yeast

hexokinase. However, its mode of action is again different.

The low affinity of mammalian glucokinase for glucose

makes it able to couple the rate of glucose metabolism to

the concentration of glucose available to the cells. Although

glucokinase, like yeast hexokinase, is able to shuttle between

the nucleus and the cytoplasm, at low concentrations of

glucose glucokinase binds the nuclear glucokinase regula-

tory protein GKRP, and has a nuclear localization, while at a

high glucose concentration the proteins dissociate and

glucokinase is found mostly in the cytoplasm (de la Iglesia

et al., 1999). Binding of glucokinase to 6-phosphofructo-2-

kinase/fructose-2,6-bisphosphatase facilitates the retention

of glucokinase in the cytoplasm (Payne et al., 2005). Binding

is enhanced in the presence of glucose (Baltrusch et al.,

2006) and increases 6-phosphofructo-2-kinase activity

(Smith et al., 2007), which yields fructose-2,6-bisphosphate,

an activator of phosphofructokinase (Hers, 1984). This

allows the coordination of two key steps of glucose metabo-

lism; glucose phosphorylation and phosphorylation of

fructose-6P.

A high rate of glucose metabolism increases the ATP/ADP

ratio and, in mammalian cells, the elevated ATP concentra-

tion causes the closure of an ATP-sensitive K1 channel and

membrane depolarization (Cook & Hales, 1984), with

consequences similar to those described above for glucose

binding to SGLT3. Other intracellular metabolites, whose

concentration changes in relation with the rate of glucose

metabolism and that have been shown to act as glucose

signals in mammalian cells, are cAMP, AMP, glucose-6P and

xylulose-5P, an intermediary in the pentose cycle pathway. It

is interesting to note that the relationship between cAMP

concentration and nutritional status varies depending of the

organism (Gancedo et al., 1985). While the intracellular

concentration of cAMP increases in yeast in the presence of

glucose (Eraso & Gancedo, 1984), it decreases in mamma-

lian cells due to a lowered glucagon secretion. The decrease

in the concentrations of cAMP and AMP diminishes the

activity of the cAMP-dependent protein kinase (PKA) and

of AMPK, a protein kinase activated by AMP, homologous

to the protein kinase Snf1 from yeast (Hardie et al., 1998),

while the increase in xylulose-5P activates the protein

phosphatase PP2A (Nishimura & Uyeda, 1995). This is

important for the activation of the glucose-responding

protein ChREBP that, together with Mlx, activates the

transcription of different genes, such as those encoding

acetylCoA carboxylase, fatty acid synthase or L-pyruvate

kinase (Ma et al., 2006). Because ChREBP is phosphorylated

by both PKA and AMPK (Kawaguchi et al., 2001, 2002), and

dephosphorylated by PP2A (Kabashima et al., 2003), glu-

cose facilitates the formation of the dephosphorylated form

of ChREBP, which is the active form able to accumulate in

the nucleus and to bind DNA. Finally, glucose-6P and

glucose itself are able to bind LXR, a transcription factor

found in liver cells that targets genes involved in cholesterol,

fatty acid and carbohydrate metabolism (Mitro et al., 2007).

While in yeast Snf1 is required for the induction of

gluconeogenic genes (Scholer & Schuller, 1994), in liver

active AMPK decreases the expression of genes encoding

gluconeogenic enzymes such as phosphoenolpyruvate car-

boxykinase or glucose-6-phosphatase (Lochhead et al., 2000;

Inoue & Yamauchi, 2006).

Glucose signalling vs. signalling for othernutrients

The yeast systems sensing the presence of different nutrients

share a number of features, regarding both their structure

and their regulatory elements. For instance, the first element

in a signalling pathway is often a membrane protein, acting

as a sensor for external nutrients. This protein can be a

transporter, such as Gap1 for amino acids, Mep2 for

ammonium, and Pho84, Pho87, Pho90 and Pho91 for

phosphate, or a transporter homologue, such as Snf3 and



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Rgt2 for glucose or Ssy1 for amino acids. However, only in

the case of glucose (and sucrose) has a G-protein-coupled

nutrient receptor, Gpr1, been identified. Once the cell has

taken up the nutrient, intracellular sensors may interact

directly with it, or with some metabolite whose concentra-

tion changes in the presence of the nutrient. Well-character-

ized examples of such sensors are transcription factors such

as Put3, Arg81, Lys14 or Leu3, which bind either an amino

acid (proline or arginine) or an intermediary metabolite

in a biosynthetic (lysine) or a catabolic (leucine) pathway.

A case presenting singular characteristics is the sensing of

biotin, as it is not mediated by the biotin transporter or by

free intracellular biotin (Pirner & Stolz, 2006). Sensing

requires biotin-protein ligase and it has been proposed that

biotinyl-AMP formed as intermediary in the reaction cata-

lyzed by biotin-protein ligase acts as a signal for the presence

of biotin; however, the downstream molecular target of

biotinyl-AMP remains unknown (Pirner & Stolz, 2006).

An important common target for many nutrients is PKA,

activated by fermentable carbon sources, amino acids,

ammonium or phosphate (Thevelein et al., 2005). In the

case of fermentable carbon sources such as glucose, fructose

or galactose, activation requires metabolism of the sugar,

proceeds through Ras1/Ras2 and involves an increase in

intracellular cAMP. Glucose and sucrose produce an addi-

tional activation mediated by Gpr1 and its G-coupled

protein Gpa2. In contrast, activation of PKA by other

nutrients is not accompanied by changes in intracellular

cAMP and the activation mechanism is not known (Thevelein

et al., 2005).

Other components shared by different nutrient-signalling

pathways are the casein kinases Yck1/2 and SCF ubiquitin

ligase complexes (Spielewoy et al., 2004). However, while the

casein kinases phosphorylate the transcription factor Stp1,

independently of the presence of amino acids (Abdel-Sater

et al., 2004), their phosphorylation of the regulatory pro-

teins Mth1 and Std1 is dependent on the binding of glucose

to Rgt2, or possibly to Snf3 (Moriya & Johnston, 2004). The

complex SCFGrr1 is required for both glucose and amino

acid signalling through the transporter homologues Snf3/

Rgt2 and Ssy1, respectively, but here again the precise

regulatory mechanisms differ. In the case of glucose, the

ubiquitination of Mth1 and Std1 tags these proteins for

degradation in the proteasome (Moriya & Johnston, 2004),

while the transmission of the amino acids signal, presum-

ably mediated by ubiquitination of Ptr3 (Abdel-Sater et al.,

2004), does not require the activity of the proteasome

(Andreasson & Ljungdahl, 2002; Abdel-Sater et al., 2004).

SCFGrr1 is also involved in the ubiquitination of the protein

kinase Ime2, required for sporulation, and there is evidence

that ubiquitination destabilizes Ime2, but it is not known

whether degradation takes place within the proteasome

(Purnapatre et al., 2005). A different SCF complex,

SCFMet30, plays a specific role in the control of methionine

metabolism. In the presence of high levels of methionine,

SCFMet30 ubiquitinates the transcriptional activator Met4

and reduces its activity. Although inactivation of Met4 is not

necessarily linked to subsequent proteolysis, release of poly-

ubiquitinated Met4 from SCFGrr1 results in proteasome-

dependent degradation (Chandrasekaran et al., 2006). A

number of connections between the changes induced in yeast

by different nutrients have become apparent. However, our

knowledge is still patchy and we lack a real understanding

of how the multiple signalling pathways are integrated to

produce an appropriate response (Schneper et al., 2004).

Conclusions

Some aspects of glucose signalling in yeast are now well

established. The cascade of reactions triggered by the bind-

ing of glucose to the sensors Snf3/Rgt2 in the plasma

membrane has been delineated in detail, showing the role

of the proteins Yck1/2, Mth1/Std1, Grr1 in the relief of the

inhibition by Rgt1 of the transcription of a number of genes.

While Hxk2 is required for glucose to affect the transcrip-

tion of some genes, it is dispensable for the effects of glucose

on many other genes. An important consequence of the

availability of glucose is an increase in the activity of PKA,

mediated both by Gpr1 and by a signalling pathway depen-

dent on metabolism. It has been shown that activation of

PKA is sufficient to modify the pattern of gene transcription,

with some genes being activated and others repressed. There

is also clear evidence that there are no single systems able to

cause either repression or induction of transcription by

glucose, but that the rate of transcription of different genes

responds to different combinations of signals. The effects of

glucose are not limited to changes in transcription rates but

include modifications in the stability of mRNAs and pro-

teins and in the activity of enzymes, as well as in the

concentration of intracellular metabolites.

Transporters have been recruited to act as sensors for

many nutrients and in some cases (Snf3/Rgt2, Ssy1) the

sensors have lost the capacity to take up the corresponding

ligand. Among the intermediary elements in the corre-

sponding signalling pathways, some are shared by different

nutrients (Yck1/2, Grr1, PKA), but the role they play may

not be the same in all cases.

Although glucose-signalling pathways in yeasts and in

mammalian cells share a number of similar elements (Hxk2/

Glucokinase, Snf1/AMPK), these elements are regulated

differently and have different functions in the two kinds of

cell.

An important aspect of yeast glucose signalling remains

to be elucidated: the identification of intracellular metabo-

lites able to play a regulatory role and the determination of

their targets.


692 J.M. Gancedo

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Noteadded in proof

It has recently been reported that it is hyperphosphorylation

of Ptr3 that mediates activation of SPS signalling, and it has

been suggested that Grrl is required to allow the phosphor-

ylation of Ptr3 by the kinases Yck1/2 (Liu et al., 2008).

Acknowledgements

I acknowledge the work performed by my students in the

field of glucose signalling, with special thanks to M.M.

Belinchon. I am grateful to C. Gancedo for useful insights

during the writing of this review and for carefully reading

the manuscript. This work has been supported by grant

BFU2004-02855-C02-01 from the Direccion General de

Investigacion Cientıfica y Tecnica (DGICYT).

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The early steps of glucose signalling in yeast

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