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Molecular Microbiology (2000) 38(2), 348±358
Glucose-induced cAMP signalling in yeast requiresboth a G-protein coupled receptor system forextracellular glucose detection and a separablehexose kinase-dependent sensing process
Filip Rolland,1 Johannes H. de Winde,1
Katleen Lemaire,1,2 Eckhard Boles,3
Johan M. Thevelein1 and Joris Winderickx1*1Laboratorium voor Moleculaire Celbiologie, Katholieke
Universiteit Leuven, Kardinaal Mercierlaan 92,
B-3001 Leuven-Heverlee, Flanders, Belgium.2Flanders Interuniversity Institute for Biotechnology, VIB,
Kardinaal Mercierlaan 93, B-3001 Leuven-Heverlee,
Flanders, Belgium.3Institut fuÈr Mikrobiologie, Heinrich-Heine-UniversitaÈ t,
UniversitaÈ tsstr. 1, Geb. 26.12.01, D-40225 DuÈsseldorf,
Germany.
Summary
In Saccharomyces cerevisiae, glucose activation of
cAMP synthesis requires both the presence of the G-
protein-coupled receptor (GPCR) system, Gpr1-Gpa2,
and uptake and phosphorylation of the sugar. In a
hxt-null strain that lacks all physiologically important
glucose carriers, glucose transport as well as glu-
cose-induced cAMP signalling can be restored by
constitutive expression of the galactose permease.
Hence, the glucose transporters do not seem to have
a regulatory function but are only required for
glucose uptake. We established a system in which
the GPCR-dependent glucose-sensing process is
separated from the glucose phosphorylation process.
It is based on the specific transport and hydrolysis
of maltose providing intracellular glucose in the
absence of glucose transport. Preaddition of a low
concentration (0.7 mM) of maltose to derepressed
hxt-null cells and subsequent addition of glucose
restored the glucose-induced cAMP signalling,
although there was no glucose uptake. Addition of a
low concentration of maltose itself does not increase
the cAMP level but enhances Glu6P and apparently
fulfils the intracellular glucose phosphorylation
requirement for activation of the cAMP pathway
by extracellular glucose. This system enabled us to
analyse the affinity and specificity of the GPCR
system for fermentable sugars. Gpr1 displayed a
very low affinity for glucose (apparent Ka � 75 mM)
and responded specifically to extracellular a and b
D-glucose and sucrose, but not to fructose, mannose
or any glucose analogues tested. The presence of the
constitutively active Gpa2val132 allele in a wild-type
strain bypassed the requirement for Gpr1 and
increased the low cAMP signal induced by fructose
and by low glucose up to the same intensity as the
high glucose signal. Therefore, the low cAMP
increases observed with fructose and low glucose
in wild-type cells result only from the low sensitivity
of the Gpr1-Gpa2 system and not from the intracel-
lular sugar kinase-dependent process. In conclusion,
we have shown that the two essential requirements
for glucose-induced activation of cAMP synthesis
can be fulfilled separately: an extracellular glucose
detection process dependent on Gpr1 and an intra-
cellular sugar-sensing process requiring the hexose
kinases.
Introduction
In the yeast Saccharomyces cerevisiae, the hexoses
glucose, fructose and mannose not only serve as a
carbon and energy source, they also have a strong
regulatory effect on a range of physiological properties.
Addition of these rapidly fermented sugars to yeast cells
grown on other carbon sources triggers a wide variety of
processes directed towards the sole and optimal utiliza-
tion of these sugars and affects other seemingly unrelated
properties such as stress resistance, the level of storage
carbohydrates, sensitivity of the cell wall to lyticase, etc.
The glucose-induced transition is a multistep process
involving different signalling pathways. Although these
pathways have been studied for many years, resulting in
the characterization of many intermediate components,
only recently significant progress has been made in
elucidating the upstream sugar sensing mechanisms that
control activation of these pathways (Thevelein, 1994;
Ronne, 1995; OÈ zcan et al., 1996; 1998; de Winde et al.,
1996; 1997; Colombo et al., 1998).
Q 2000 Blackwell Science Ltd
Accepted 26 July, 2000. *For correspondence. E-mail joris.winderickx@bio.kuleuven.ac.be; Tel. (132) 16 321516 or 321500; Fax (132) 16321979.
One important signalling route that has been studied
in great detail is the Ras±adenylate cyclase pathway
(Broach and Deschenes, 1990; Thevelein, 1991; 1992;
Tatchell, 1993; Thevelein and de Winde, 1999). This
pathway regulates the production of cAMP, which is
synthesized by adenylate cyclase (Matsumoto et al.,
1984; Kataoka et al., 1985). Its activity is dependent on
the Ras proteins, which have been thought for many
years to replace the heterotrimeric Gs proteins that
control adenylate cyclase activity in mammalian cells
(Toda et al., 1985). In turn, cAMP causes activation of
cAMP-dependent protein kinase (cAPK) through binding
to the regulatory subunits resulting in dissociation and
concomitant activation of the catalytic subunits (Toda
et al., 1987a,b). cAPK is involved in the post-translational
regulation of a variety of proteins, for instance key
enzymes of gluconeogenesis and glycolysis. The gluco-
neogenic enzyme, fructose-1,6-bisphosphatase, is rapidly
inactivated after glucose-induced activation of cAPK,
whereas phosphofructokinase 2, an important regulator
of the major glycolytic enzyme phosphofructokinase 1, is
rapidly activated. Also, neutral trehalase and glycogen
phosphorylase are activated upon glucose addition,
resulting in mobilization of trehalose and glycogen. In
addition, cAPK exerts control at the transcriptional level,
for instance by repressing STRE-controlled genes and
inducing ribosomal protein genes (Thevelein, 1992; 1994;
Neuman-Silberberg et al., 1995; Ruis and Schuller, 1995).
Hence, the transient cAMP signal has been proposed to
trigger a fast resetting from gluconeogenic to fermentative
metabolism (Thevelein, 1991; Boy-Marcotte et al., 1996;
Jiang et al., 1998).
Only two stimuli are known to activate the Ras-
adenylate cyclase pathway in vivo. Addition of a rapidly
fermented sugar to cells growing on a non-fermentable
carbon source or stationary-phase cells (glucose-
derepressed cells) triggers a rapid, transient increase
in the cAMP level (van der Plaat, 1974; Purwin et al.,
1982; Tortora et al., 1982; Thevelein, 1984; Thevelein
et al., 1987a). Intracellular acidification, triggered for
instance by addition of the protonophore 2,4-dinitrophenol
(DNP) at low extracellular pH, triggers a more lasting
and higher cAMP increase (Caspani et al., 1985; Purwin
et al., 1986; Thevelein et al., 1987b). We have provided
evidence that fermentable sugars and intracellular acid-
ification affect adenylate cyclase activity through different
G proteins (Colombo et al., 1998). Intracellular acidifi-
cation, but not glucose, increases the GTP content on
the Ras proteins possibly through inhibition of the Ras-
GTPase activating proteins Ira1 and Ira2. The hetero-
trimeric Ga protein, Gpa2, is required for glucose-induced,
but not for intracellular acidification-induced activation of
cAMP synthesis. Recently, it has been shown that a
membrane-bound protein, Gpr1, interacts with Gpa2
(Yun et al., 1997; Xue et al., 1998; Kraakman et al., 1999).
Gpr1 displays structural and functional homology to
mammalian G-protein-coupled receptors indicating that it
may function as a receptor. Initially, it was suggested that
the GPR1-GPA2 encoded GPCR system would be
involved in nitrogen sensing (Xue et al., 1998), but
recently it was shown that this system is required for
activation of cAMP synthesis by glucose (Kraakman et al.,
1999).
In addition to Gpr1 and Gpa2, glucose activation of
cAMP synthesis also requires uptake and phosphorylation
of the sugar. The affinity of the glucose-sensing system is
relatively low (apparent Ka � 20 mM), in spite of the low
Km of glucose transport in derepressed cells (1±2 mM)
(Beullens et al., 1988). This might suggest that certain
low-affinity transporters play a regulatory role. Glucose
uptake in the yeast S. cerevisiae is generally considered
to occur via facilitated diffusion (Bisson et al., 1993). It is
catalysed by a series of glucose carriers encoded by the
HXT genes. Although more than 20 HXT genes have
been identified (AndreÂ, 1995; Kruckeberg, 1996; Boles
and Hollenberg, 1997), a strain deleted only for HXT1
to HXT7 does not show detectable glucose transport.
Therefore, this so-called hxt-null strain is unable to grow
on glucose (Reifenberger and Ciriacy, 1994; Reifenberger
et al., 1995). Expression of only one of the seven
transporters deleted in the hxt-null strain or derepression
of the galactose transporter Gal2 restores growth on
glucose of the hxt-null strain (Reifenberger et al., 1997).
All carriers also transport fructose albeit with a lower
affinity compared with glucose. Up to now, no evidence
has been found that one of the active glucose carriers
would have a regulatory role in the activation of glucose-
induced pathways such as for instance the main glucose
repression pathway (Reifenberger et al., 1997). On the
other hand, two of the HXT homologues for which no
active glucose transport could be demonstrated, Snf3 and
Rgt2, have been proposed to play a role, respectively, as
a low and high glucose sensor for glucose-induced
expression of some of the HXT genes (OÈ zcan et al.,
1996; 1998). Glucose phosphorylation can be carried out
by any one of the three known glucose kinases,
hexokinase PI, PII or glucokinase, and further metabolism
of the sugar beyond glucose phosphorylation is not
required for glucose-induced activation of cAMP synthesis
(Beullens et al., 1988; Pernambuco et al., 1996).
The connection between the requirements for uptake
and phosphorylation and the functioning of the G-protein
dependent receptor system is unclear. In addition, it is
also not known whether glucose uptake is only required
to sustain glucose phosphorylation, whether specific
glucose carriers play a role in the activation of cAMP
synthesis process and whether the apparent Ka for
activation by glucose is related to the affinity of the
Dual glucose-sensing system for cAMP induction 349
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 348±358
carriers expressed in derepressed cells. Therefore, we
have studied in more detail the connection between the
requirement of sugar uptake and phosphorylation and the
requirement of Gpa2 and Gpr1 for glucose- and fructose-
induced activation of the Ras±cAMP pathway. We show
that constitutive expression of the galactose permease in
an hxt-null strain restores glucose- and fructose-induced
cAMP synthesis making it unlikely that additional regula-
tory functions besides delivering the sugar are associated
with the hexose transporters. In addition, we developed a
system in which intracellular glucose phosphorylation
could occur in the absence of extracellular glucose by
provision of low amounts of maltose to derepressed hxt-
null cells. This system allowed us to demonstrate that the
glucose phosphorylation requirement for cAMP signalling
can be separated from the requirement for activation of
the GPCR system by glucose. We also demonstrate that
the GPCR system specifically responds to D-glucose and
sucrose but not to fructose or any other monosaccharide
tested and that it is responsible for the apparent low
affinity of glucose activation of cAMP synthesis. The much
weaker activation of the cAMP pathway by other sugars
like fructose seems only to be triggered by the intracellular
sugar phosphorylation dependent process.
Results
cAMP signalling is dependent on hexose transport but not
on a specific hexose transporter
To investigate the involvement of glucose transport and
phosphorylation in sugar-induced cAMP signalling, we
have monitored changes in cAMP and sugar phosphates
after addition of either glucose or fructose to derepressed
cells of a wild-type strain and two hxt-null strains. As
shown in Fig. 1A, addition of 100 mM glucose to wild-type
cells resulted in a sharp but transient increase in cAMP.
Addition of 100 mM fructose also resulted in an increase
in cAMP but the amplitude of the signal was only half
of that observed with glucose. The difference between
the glucose- and fructose-induced cAMP signal was not
specific for this genetic background but was consistently
observed in all wild-type strains tested (results not
shown). In spite of this difference in the amplitude of the
cAMP signal, both glucose and fructose addition resulted
in a comparable increase in Glu6P (Fig. 1B), Fru6P and
Fru1,6bP (results not shown). In both hxt-null strains,
neither addition of 100 mM glucose nor addition of
100 mM fructose triggered significant changes in cAMP
as shown in Fig. 1A. With glucose addition, a very slow
accumulation of Glu6P (Fig. 1B), Fru6P and Fru1,6bP
(results not shown) was observed indicating that the hxt-
null strains are still capable of taking up glucose albeit at a
rate which is too low to support growth or to trigger
activation of the Ras±cAMP pathway. Upon addition of
100 mM fructose to the same strains, the sugar phos-
phate concentrations did not change significantly (Fig. 1B
and results not shown).
We then tested whether constitutive expression of the
galactose permease, encoded by GAL2, could restore
glucose- and fructose-induced cAMP synthesis in the hxt-
null strains. It has been reported previously that GaI2 is
able to transport glucose (Liang and Gaber, 1996) and
fructose with high-affinity kinetics (Reifenberger et al.,
1997). Because GaI2 requires galactose induction and is
subject to glucose repression (Johnston and Carlson,
1992; Johnston et al., 1994), the permease is not involved
in glucose uptake during exponential growth on glucose in
wild-type cells. Also under our conditions it was appar-
ently largely absent as shown by the very slow increase in
the Glu6P level upon glucose addition to derepressed
hxt1-7D cells (Fig. 1B). We transformed the hxt-null
strains with a plasmid containing an ADH-GAL2 construct.
As opposed to the hxt-null strains, the transformants were
able to grow on glucose- or fructose-containing medium
(results not shown) and a clear accumulation of Glu6P
was observed upon addition of 100 mM glucose or
fructose to derepressed cells of the transformants.
Glucose addition also induced a marked cAMP increase
while fructose addition resulted only in a moderate cAMP
increase (Fig. 1A). These results demonstrate that the
galactose transporter can sustain the glucose-induced
cAMP signal. Hence, besides the transport of the sugar
no additional, regulatory function for cAMP induction
seems to be associated with the canonical hexose
transporters. The role of the hexose transporters appears
to be confined to maintaining a critical level of intracellular
sugar for the phosphorylation requirement.
The fructose- and low glucose-induced cAMP synthesis
reflects the sugar phosphorylation requirement and does
not require Gpr1 or Gpa2
As described above, addition of 100 mM fructose to wild-
type cells triggers a cAMP signal the amplitude of which is
about half of the cAMP signal obtained with 100 mM
glucose. In this way, fructose addition results in a cAMP
signal comparable to that obtained with 5 mM glucose as
shown in Fig. 2. We reported previously (Colombo et al.,
1998; Kraakman et al., 1999) that the cAMP increase after
addition of 5 mM glucose is not affected by deletion of
GPR1 or GPA2. They encode, respectively, the receptor-
like protein and the G-protein of the GPCR system that is
required for full activation of cAMP synthesis with high
(100 mM) glucose concentrations. This has now been
confirmed (Fig. 2A and C). In addition, Fig. 2B shows that
the cAMP increases obtained by addition of 100 mM
fructose to a wild-type strain, a gpr1D deletion strain or a
350 F. Rolland et al.
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 348±358
gpa2D deletion strain are comparable indicating also that
fructose sensing is independent of the GPCR system.
Deletion of the genes encoding the hexose kinases, i.e.
HXK1, HXK2 and GLK1, however, entirely eliminated the
cAMP signal induced by high fructose or low glucose
concentrations. Moreover, preaddition of 5 mM glucose
followed by addition of either 100 mM fructose or 100 mM
glucose eliminated the fructose-induced but not the
high glucose-induced cAMP signal (Fig. 2C). These data
indicate that activation by 5 mM glucose and 100 mM
fructose is proceeded by the same sugar phosphorylation
dependent mechanism.
The requirement for glucose activation of the Gpr1-Gpa2
GPCR system can be separated from the glucose
phosphorylation requirement
The glucose phosphorylation requirement complicates
investigation of the affinity and specificity of the Gpr1-
Gpa2 GPCR system. Therefore, we established a method
where the glucose-induced activation of cAMP synthesis
via the GPCR system can be studied independently of the
requirement for glucose phosphorylation. We reasoned
that the intracellular glucose required for glucose phos-
phorylation could also be provided by addition of maltose.
Maltose is transported by a specific maltose-induced
transport system and intracellularly converted by maltase
into glucose (Vanoni et al., 1989; Needleman, 1991). We
tested the effect of addition of low concentrations of
maltose to the wild-type strain MC996A and the hxt-null
strain. As shown in Fig. 3A, addition of 2.5 mM maltose
did not increase the Glu6P concentration in the wild-type
strain whereas a pronounced accumulation was observed
in the transporter deletion strain. This difference can
be explained on the basis that glucose transport is a
facilitated diffusion process and faster than maltose
uptake. Therefore, the intracellular glucose derived from
maltose might be exported rapidly to the medium in the
wild-type strain but not in the hxt-null strain so that in the
former the concentration of intracellular glucose available
for glucose phosphorylation would remain low. In the wild
type, addition of 2.5 mM maltose did not induce an
increase in cAMP. In the hxt-null strain, we could observe
a small increase in the level of cAMP upon addition of
2.5 mM maltose to derepressed cells which is in agree-
ment with the above described results that sugar
phosphorylation upon addition of fructose or low glucose
concentrations can give a partial cAMP signal (Fig. 3B).
Subsequently, we added 100 mM glucose 2 min after
the provision of maltose. As expected, this resulted in an
Fig. 1. Glucose- and fructose-induced accumulation of Glu6P and cAMP in vivo in wild type, two hxt-null strains and the hxt1-7D gal2DpADHGAL2 strain. The cAMP signal (A) and the Glu6P level (B) were measured as a function of time after addition of 100 mM glucose (X) or100 mM fructose (W) to derepressed cells of the wild-type strain MC996A, the hxt1-7D and hxt1-7D gal2D strains and the hxt1-7D gal2Dstrain constitutively expressing the Gal2 permease as indicated.
Dual glucose-sensing system for cAMP induction 351
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 348±358
increase in the Glu6P concentration and a concomitant
spike in cAMP in the wild-type strain. In the hxt-null strain,
glucose addition did not give a supplementary increase
in Glu6P but most interestingly, however, a significant
increase in cAMP was observed. We repeated the
experiment with the hxt-null strain but with preaddition of
0.7 mM maltose and added 100 mM glucose either 2, 4 or
6 min after the addition of maltose. No significant change
in the cAMP level was obtained with this very low maltose
concentration but each time glucose was added a
pronounced cAMP increase was observed as shown in
Fig. 4A. The intensity of the cAMP signal decreased
somewhat when the time interval between maltose and
glucose addition was longer most probably because of
gradual depletion of the low maltose level in the medium.
The hxt-null strain does not transport glucose at a
significant rate and the addition of glucose also did not
enhance the intracellular glucose or Glu6P level above
that observed with maltose alone. Therefore, the immedi-
ate and large increase in the cAMP level observed upon
addition of 100 mM glucose under these conditions could
only be caused by extracellular glucose detection via
the GPCR system. Indeed, no cAMP increases were
observed upon addition of 100 mM glucose after preaddi-
tion of maltose when GPR1 or GPA2 was deleted in the
hxt1-7D gal2D strain as shown in Fig. 3B and Fig. 4B.
We have shown previously that the constitutively active
Gpa2val132 allele is able to bypass the absence of Gpr1 for
glucose-induced activation of cAMP synthesis. To confirm
that the cAMP increase upon addition of 100 mM glucose
after maltose pretreatment is not due to residual glucose
uptake in the hxt1-7D gal2D strain, we tested whether
addition of 100 mM glucose to a hxt1-7D gal2D strain that
lacks the Gpr1 receptor but expresses Gpa2val132 allele
could trigger a cAMP increase. However, also with this
strain, no glucose-induced increase in cAMP could be
observed (data not shown). This confirms that the
extracellular glucose is not able to enter the cell at least
not at a rate able to sustain induction of the cAMP
signal.
Affinity and specificity of the Gpr1-dependent extracellular
glucose-sensing process
Using the approach described above, we determined the
specificity of the GPCR system. Addition of 100 mM of the
a- or b-anomer of D-glucose to cells of the hxt1-7D gal2D
strain after preaddition of 0.7 mM maltose triggered in
both cases an increase in the cAMP level. The b-anomer
of D-glucose was somewhat more potent than the a-
anomer (Fig. 5A). Addition of 100 mM sucrose also
triggered a similar Gpr1-Gpa2 dependent cAMP increase.
This was not due to extracellular cleavage of sucrose to
glucose and fructose by periplasmic invertase as inver-
tase mutants displayed the same sucrose-induced cAMP
signal (data not shown). However, addition of 100 mM
fructose (Fig. 5B) or 100 mM each of mannose, galac-
tose, xylose, L-glucose or the glucose analogues, 3-O-
methyl glucose, 6-deoxyglucose or 2-deoxyglucose
(results not shown) did not cause any significant change
in the cAMP level. Even with 300 mM fructose no cAMP
Fig. 2. High-fructose and low-/high-glucose-induced cAMPsignalling in Gpr1, Gpa2 and glucose kinase mutants. cAMP signalwith 100 mM glucose (A) or 100 mM fructose (B) in the wild-typestrain (X), the gpr1D (O), the gpa2D (K), the hxk1D hxk2D glk1Dgpr1D (B) and the hxk1D hxk2D glk1D gpa2D (A) deletion strainsand (C) cAMP signal with 5 mM glucose followed by 100 mMglucose (X) or fructose (W) in the wild-type strain and with 5 mMglucose followed by 100 mM glucose in the gpr1D (O), and thegpa2D (K) deletion strains
352 F. Rolland et al.
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response was observed (Fig. 5B). Similar results were
obtained with derepressed wild-type cells when these
sugars and sugar analogues were added 2 min after
addition of 5 mM glucose. Addition of 100 mM maltose
with or without preaddition of 5 mM glucose did not trigger
a cAMP signal indicating that maltose cannot activate
the Gpr1-Gpa2 dependent system (results not shown).
Hence, the Gpr1-dependent extracellular-sensing system
appears to be specific for extracellular D-glucose, at least
among the monosaccharides tested, and for sucrose.
With the same experimental set-up, we also determined
the affinity of the Gpr1-dependent glucose detection system
using different glucose concentrations. As shown in Fig. 6A,
the maximum cAMP increase was observed with 400 mM
glucose. When the amplitude of the cAMP signal was
plotted vs. the concentration of glucose used, an apparent
Ka of about 75 mM was found as illustrated in Fig. 6B.
Gpa2val132 substitutes for the requirement of Gpr1-
dependent extracellular glucose sensing
To gain further support for the dual sensing system,
we compared the glucose- and fructose-induced cAMP
response of a wild-type strain with that of an isogenic
strain lacking the Gpr1 protein and expressing the
Gpa2val132 allele (Kraakman et al., 1999). In the latter,
activation of cAMP synthesis should be independent of
extracellular Gpr1-dependent sugar detection and should
only require sugar phosphorylation. As shown in Fig. 7A
and B, addition of different concentrations of glucose or
100 mM fructose all triggered an equally high cAMP
response in the gpr1D GPA2val132 strain. These results
confirm that Gpa2val132 indeed bypasses the need for
the high extracellular glucose concentrations normally
required for activation of the Gpr1-Gpa2 system and that
only the phosphorylation requirement has to be fulfilled for
cAMP signalling in a strain with a constitutively active
GPCR system. The data further support the idea that
fructose is not able to activate the Gpr1-Gpa2 system and
that the fructose-induced cAMP increase is largely, if not
exclusively, due to stimulation of cAMP synthesis by
intracellular glucose phosphorylation.
Fig. 4. Maltose-mediated fulfilment of theglucose phosphorylation requirement foractivation of cAMP synthesis by extracellularglucose.A. cAMP level after addition of 0.7 mMmaltose (X) alone or after addition of 100 mMglucose, 2 min (W), 4 min (O) and 6 min (K)after addition of 0.7 mM maltose toderepressed hxt1-7D gal2D cells.B. cAMP level after addition of 100 mMglucose, 4 min after addition of 0.7 mMmaltose to derepressed hxt1-7D gal2D (X),hxt1-7D gal2D gpr1D (W), hxt1-7D gal2Dgpa2D (O).
Fig. 3. Glu6P and cAMP level in the wild type, the hxt1-7D gal2Dand the hxt1-7D gal2D gpa2D strain after addition of maltose.Glu6P (A) and cAMP (B) levels in the wild type (X), the hxt1-7Dgal2D (W), and the and hxt1-7D gal2D gpa2D (O) after addition of2.5 mM maltose followed 2 min later by addition of 100 mM glucose.
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Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 348±358
Discussion
The hexose carriers do not seem to play a specific
regulatory role in glucose activation of the cAMP pathway
Glucose activation of cAMP synthesis is known to require
glucose phosphorylation (Beullens et al., 1988) and the G-
protein-coupled receptor system consisting of Gpr1 and
Gpa2 (Kraakman et al., 1999). Our present result that the
glucose-induced cAMP signal is entirely absent in a yeast
strain without functional glucose carriers confirms that the
presence of the GPCR system is not enough to trigger
activation of the cAMP pathway by glucose. Transport
and phosphorylation of glucose are apparently required
for transduction of the glucose signal to adenylate
cyclase. Up to now, it had not been investigated whether
the glucose phosphorylation requirement reflected only
the need for glucose phosphorylation or whether one or
more specific glucose carriers in some way also played a
role. We have now demonstrated that the presence of
glucose-induced cAMP signalling does not require a
specific glucose carrier because the cAMP increase can
also be triggered in a hxt-null strain when the glucose is
transported solely via the galactose transporter or when
extracellular glucose is provided together with low
amounts of maltose. These data seem to indicate that it
is not important by which means the intracellular glucose
required for glucose phosphorylation is provided and that
therefore the transporters themselves do not seem to play
a regulatory role in glucose activation of cAMP synthesis.
Experiments with yeast strains expressing a single
functional glucose carrier have confirmed this conclusion
(F. Rolland et al.. unpublished results). In addition, the
glucose carrier homologues, Snf3 and Rgt2, previously
suggested to function as glucose sensors, are also not
required for glucose activation of cAMP synthesis (F.
Rolland, J.M. Thevelein and J. Winderickx, unpublished
results). The conclusion that the role of the glucose carriers is
confined to glucose uptake was also obtained for activation of
the main glucose repression pathway, which also requires
glucose phosphorylation (Reifenberger et al., 1997).
The exact nature of the signal given by the hexose
kinase dependent glucose phosphorylation process
remains to be elucidated. Data obtained in wild-type
strains and glucose carrier deletion strains clearly
indicated that there is no correlation between the Glu6P
increase and the amplitude of the glucose-induced cAMP
signal (Fig. 3, Beullens et al., 1988; F. Rolland et al.,
unpublished results). This can be interpreted as indicating
that Glu6P is not involved in the activation process or
alternatively that another factor is limiting the activation of
adenylate cyclase. Furthermore, results obtained with
glycolysis mutants and from experiments in which
Fig. 5. Specificity of the Gpr1-Gpa2dependent glucose detection process. cAMPafter addition of 100 mM a-D-glucose (X) orb-D-glucose (W) to derepressed hxt1-7Dgal2D cells 4 min after the addition of 0.7 mMmaltose (A) cAMP after addition of 100 mMglucose (X),100 mM fructose (W) and 300 mMfructose (O) to derepressed hxt1-7D gal2Dcells, 4 min after the addition of 0.7 mMmaltose (B).
Fig. 6. Affinity of the Gpr1-Gpa2 dependentglucose detection process. cAMP levelsobtained after addition of different glucoseconcentrations 4 min after addition of 0.7 mMmaltose to derepressed hxt1-7D gal2D cells.Glucose concentrations: 2 mM (X), 10 mM(W), 20 mM (O), 50 mM (K), 100 mM (B),300 mM (A), 400 mM (V), 500 mM (S),750 mM (P), 1000 mM (L) (A). Half-maximalcAMP accumulation is obtained with aglucose concentration of about 75 mM asindicated (B).
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iodoacetate was used to inhibit ATP generation in
glycolysis, showed that the glucose-induced increase in
cAMP is not dependent on an increase in the ATP level.
The cAMP signal remains independent of the ATP level
provided that enough ATP is available as substrate for
adenylate cyclase (Beullens et al., 1988; F. Rolland et al.,
unpublished results). Hence, neither Glu6P nor ATP seem
to act as simple metabolic messengers for glucose
activation of cAMP synthesis
Extracellular glucose detection by the Gpr1-Gpa2
dependent mechanism can be separated from glucose
phosphorylation in the hxt-null strain
Because glucose activation of cAMP synthesis requires
glucose phosphorylation in addition to the presence of
the Gpr1-Gpa2 G-protein coupled receptor system, the
properties of the latter system cannot be studied in a
straightforward manner. For instance, it is difficult to
determine whether the receptor responds to extracellular
or intracellular glucose. One can also not test activation of
the GPCR system by glucose analogues because they
have to be able to sustain also the glucose phosphoryla-
tion requirement. For instance, it has been unclear
whether L-glucose can activate the GPCR system
because L-glucose cannot be transported and phosphory-
lated. 2-Deoxyglucose presents another problem: it is
efficiently transported and phosphorylated but not further
metabolized and therefore causes a precipitous drop in
the ATP level. Because cAMP synthesis requires ATP,
absence of a cAMP signal with 2-deoxyglucose might be
due to an ATP depletion effect. We have now established
a system in which the requirement for activation of the
Gpr1-Gpa2 detection mechanism and the requirement
for glucose phosphorylation can be fulfilled separately
(Figs 3 and 4). It makes use of the hxt1-7D gal2D strain
which is unable to take up glucose from the medium. The
strain is given maltose in a concentration too low to trigger
by itself an increase in the cAMP level but apparently high
enough to fulfil the glucose phosphorylation requirement,
because subsequent addition of glucose caused an
immediate spike in the cAMP level. This novel system
has led to several new conclusions. First of all, this
system uncouples for the first time the requirement
for extracellular glucose from the requirement for
intracellular glucose phosphorylation. Apparently, the
glucose detected by the Gpr1-Gpa2 system does not
have to be phosphorylated itself.
Secondly, this system clearly shows that Gpr1 is
required for the detection of extracellular glucose.
Although our data do not provide evidence for a direct
interaction between Gpr1 and glucose, such an interac-
tion offers by far the simplest explanation for the results
described in this paper. In the control experiment with the
hxt-null strain expressing the Gpa2val132 allele in the
absence of Gpr1, glucose addition was unable to trigger
any increase in cAMP. This indicates that any residual
glucose transport or any change in intracellular meta-
bolites or composition that might be caused by the
extracellular glucose in the hxt-null strain is unable to
affect the cAMP level. Whatever such residual changes
are, they are clearly not able to sustain enough glucose
phosphorylation to trigger the cAMP signal.
Thirdly, this system allows for the first time to study the
specificity of the GPCR system for the extracellular sugar
independent of the metabolism of this sugar. In this way
we have shown that Gpr1-dependent extracellular sugar
sensing is specific for D-glucose with a preference for
the b-anomer. None of the other hexoses or glucose
analogues tested was able to activate cAMP synthesis
under the same conditions as glucose. This explains why
deletion of Gpr1 or Gpa2 has no effect on the cAMP
increase observed after addition of 100 mM fructose
(Fig. 3). This increase is apparently only triggered by the
intracellular phosphorylation dependent process. When
the increase in cAMP was measured with different
extracellular glucose concentrations, an apparent Ka
of about 75 mM for the extracellular glucose detection
system was deduced. This apparent Ka is somewhat
higher than that previously obtained for glucose-induced
activation of cAMP synthesis in wild-type cells, which was
Fig. 7. Activation of cAMP synthesis in a gpr1D strain expressingthe constitutively active Gpa2val132 allele. cAMP after addition of5 mM (X), 25 mM (W), 50 mM (O) or 100 mM (K) glucose toderepressed W303-1A wild type and gpr1D Gpa2val132 cells (A).cAMP after addition of 100 mM glucose (X) or 100 mM fructose (W)to derepressed W303-1A wild type and gpr1D Gpa2val132 cells (B).
Dual glucose-sensing system for cAMP induction 355
Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 348±358
around 25 mM (Beullens et al., 1988). However, it can be
considered in the same range in view of the different
strain and experimental set-up. Previously, we have
suggested that the low-affinity glucose uptake system
might be involved in glucose-activation of cAMP synthesis
because its Km of 20±25 mM is similar to the apparent Ka
of glucose activation of cAMP synthesis in wild-type cells.
Our present results show that the low-affinity system is
the Gpr1-Gpa2 system (or at least the Gpr1-dependent
glucose-sensing system) and not the low-affinity glucose
uptake system. The natural environment of yeast cells
(e.g. grape juice) can contain very high glucose concen-
trations which are fermented rather than respired. The
very low affinity of the GPCR system for its ligand might
therefore be related to its function in stimulating through
the cAMP pathway the switch from respirative/gluconeo-
genic growth to fermentative growth which only fully
occurs at glucose concentrations of around 20 mM
(Thevelein, 1991; Johnston and Carlson, 1992).
Gpa2val132 can fully substitute for the requirement of high
extracellular glucose
We have previously reported that for glucose-induced
cAMP signalling Gpr1 deletion can be bypassed by the
constitutively active Gpa2val132 allele (Kraakman et al.,
1999). In this paper, we have confirmed these results and
in addition we have shown that Gpa2val132 can substitute
for the requirement of high extracellular glucose. Indeed,
we demonstrated that in a gpr1D GPA2val132 strain cAMP
responses of similar magnitude are obtained with glucose
and fructose. This confirms that the fructose-induced
cAMP increase is only triggered by the sugar phospho-
rylation dependent process while the high glucose
induced increase is supported in addition by the Gpr1-
Gpa2 dependent process. We have further demonstrated
that in the gpr1D GPA2val132 strain low glucose concen-
trations trigger similar cAMP spikes as the high glucose
concentration. This further supports that the low glucose
sensitivity of activation of cAMP synthesis in wild-type
strains is due to the low sensitivity for glucose of the Gpr1-
Gpa2-dependent process. The glucose kinases have Km
values below 1.5 mM for both glucose and fructose (Lobo
and Maitra, 1977; Maitra and Lobo, 1983), which is much
less than the apparent Ka of the Gpr1-Gpa2 system for
glucose. Hence, in a wild-type strain, the latter system acts
as limiting factor for glucose activation of cAMP synthesis.
Conclusions
We have demonstrated that glucose activation of cAMP
synthesis consists of two processes: an extracellular
glucose-sensing process that is dependent on the G-
protein coupled receptor system consisting of Gpr1 and
Gpa2, and an intracellular glucose sensing process that is
dependent on glucose phosphorylation by the glucose
kinases. These two processes can be separated using a
strain in which all physiologically relevant glucose carriers
are deleted and in which the glucose phosphorylation
requirement is fulfilled through maltose transport and
hydrolysis. This shows that extracellular glucose detection
and glucose phosphorylation are independent processes
and that the extracellularly detected glucose molecules do
not need to be phosphorylated. We have also shown that
the requirement for the Gpr1-Gpa2 dependent extracel-
lular glucose sensing process can be fully substituted by
the constitutively active Gpa2val132 allele. This allows
ligands that are phosphorylated but not detected by the
Gpr1-Gpa2 system, such as fructose and low glucose, to
cause full activation of cAMP accumulation.
Experimental procedures
Strains and plasmids
The wild-type strain (MC996A) and the hxt-null strain hxt1-7D(RE700A) have been described by Reifenberger et al. (1995).The hxt1-7D gal2D (RE800A) strain has been described byReifenberger et al. (1997). We transformed the wild-typestrain (MC996A), the hxt1-7D (RE700A) and the hxt1-7Dgal2D (RE800A) strains with an episomal plasmid (pHL125)expressing GAL2 under the control of the ADH2 promoter, aconstruct kindly provided by Liang and Gaber (1996). Thestrains hxt1-7D gal2D gpa2D (FR801) and hxt1-7D gal2Dgpr1D (FR802) were obtained by deletion of, respectively,GPA2 and GPR1 in strain RE800A using a constructdescribed previously (Colombo et al., 1998; Kraakman et al.,1999). The strain hxt1-7D gal2D gpr1D GPA2val132 wasobtained by transforming strain FR802 with a centromericplasmid bearing the constitutive GPA2val132 allele. The wild-type strain W303-1A, the congenic triple hexose kinasedeletion strain and the gpr1D GPA2val132 have beendescribed before (de Winde et al., 1996; Kraakman et al.,1999). GPR1 and GPA2 were deleted in the triple hexosekinase deletion strain by replacing the coding region with theURA3 gene
Growth media
Yeast cells were grown at 308C in 1% yeast extract, 2%Bacto-peptone (YP) supplemented with either 2% maltose(YPM), 2% glucose (YPD), 2% fructose (YPF), 2% galactose(YPGal) or 3% glycerol (YPG) as indicated. To avoid thepossibility of generating suppressors, we tested the hxt1-7D ,the hxt1-7D gal2D and their derived deletion strains forgrowth on YPD medium before and after each experiment.For the strains carrying the pADHGAL2 plasmid, cells weregrown on nutrient supplementation media lacking uracil (SC-ura) that contained 0.67% Difco yeast nitrogen basesupplemented with 10 mM (NH4)2SO4 the required aminoacids and either 2% of the appropriate sugar (SCM, SCD,SCF, SCGal) or 3% glycerol (SCG).
356 F. Rolland et al.
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Biochemical analyses
For the determination of the glycolytic metabolites and cAMP,cells were grown on YPG or SCG media and harvested bycentrifugation. The pellet was washed once, resuspendedand then incubated in 25 mM MES buffer pH 6 at 308C.Before and after the addition of sugars to this cell suspensionat a concentration of 100 mM unless otherwise mentioned,samples were taken at the time intervals indicated andimmediately quenched in 60% methanol at 2408C. Forglycolytic metabolite determination, cells were extractedusing chloroform at 2408C at neutral pH, essentially asdescribed by de Koning and van Dam (1992). Using the totalamount of protein in the sample as determined according toLowry et al. (1951), and the assumption of a yeast cytosolicvolume of 12 ml mg21 protein, cytosolic concentrations in mMwere calculated. For determination of the cAMP levels, cellswere extracted using perchloric acid. After neutralizationcAMP levels were determined using the cyclic AMP [3H]assay system from Amersham as described previously(Thevelein et al., 1987b). All experiments were performedat least three times and representative results are shown.
Acknowledgements
We are grateful to J. Rosseels and W. Verheyden forexcellent technical assistance. This work was supported byfellowships from the Institute for Scientific and TechnologicalResearch (IWT) to F.R. and the Fund for Scientific Research± Flanders to J.W. and by grants from the Fund for ScientificResearch ± Flanders and the Research Fund of theKatholieke Universiteit Leuven (Concerted Research Actions)to J.W. and J.M.T., the European Commission (Biotechnol-ogy BIO4-CT98-0562 to E.B. and J.M.T.), and InteruniversityAttraction Poles Network P4/30 to J.M.T. and the DeutscheForschungsgemeinschaft (grant BO 1517/1-1) to E.B.
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