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Nutrient Sensing through the Plasma Membrane of Eukaryotic Cells
Glucose sensing through the Hxk2-dependent
signalling pathway
F. Moreno1 , D. Ahuatzi, A. Riera, C.A. Palomino and P. Herrero
Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Oviedo, 33006 Oviedo, Spain
Abstract
In this work, we describe the hexokinase 2 (Hxk2) signalling pathway within the yeast cell. Hxk2 and Mig1
are the two major factors of glucose repression in Saccharomyces cerevisiae. The functions of both proteins
have been extensively studied but there is no information about possible interactions among them in the
repression pathway. Our results demonstrate that Hxk2 interacts directly with Mig1 in vivo and in vitro and
that the ten amino acids motif between K6 and M15 is required for their interaction. This interaction has been
detected at the DNA level both in vivo by chromatin immunoprecipitation experiments and in vitro using
purified proteins and a DNA fragment containing the MIG1 site of the SUC2 promoter. This demonstrates that
the interaction is of physiological relevance. Our findings show that the main role of Hxk2 in the glucose
signalling pathway is the interaction with Mig1 to generate a repressor complex located in the nucleus of
S. cerevisiae.
Introduction
The ability of the yeast Saccharomyces cerevisiae to grow
in different media is due to its capacity to sense and respond
to changes in the availability of nutrients. Among sugars, glucose is probably the major signalling nutrient for S. cerevisiae
as well as the carbon and energy source used preferentially.
S. cerevisiae is able to detect the glucose levels of extracellular
medium and to generate intracellular signals that result in
an adequate cellular response to variations in the glucose
medium composition. Many of these responses involve alterations in gene expression. The majority of these alterations
occur at the level of mRNA transcription by the repression
or activation of the transcription of many genes that encode
enzymes implicated in carbon metabolism [1–3].
Present knowledge concerning factors required to transmit
the glucose signal from the environment to the cell nucleus
is mainly derived from studies with mutants affected in different regulatory circuits governed by glucose [2]. In the last
few years important advances in this field have been made
[4–6]. These studies have characterized, in different detail,
several signal transduction pathways that allow the yeast to
perceive the level of glucose in the medium and to initiate the
appropriate metabolic response (Figure 1).
S. cerevisiae has membrane proteins that act as glucose receptors. Glucose binds to these receptors and generates an
intracellular signal. In the Rgt2/Snf3 pathway, these two proteins act as glucose receptors. The Rgt2 and Snf3 proteins
resemble hexose transporters in structure but have long cytoplasmic tails that are required for signal transduction [7].
Glucose binding to these transmembrane proteins initiates
Key words: glucose, glucose repression, glucose sensing, hexokinase 2, Hxk2-dependent
signalling pathway, Mig1.
Abbreviations used: Gpr1, G-protein coupled receptor 1; GFP, green fluorescent protein; HA,
haemagglutinin; Hxk2, hexokinase 2; PKA, protein kinase A.
1
To whom correspondence should be addressed (email [email protected]).
signals that activate a pathway that allows hexose transporter
gene expression by repressing Rgt1 function [8].
An additional pathway that involves transcriptional
changes in response to glucose is the stimulation of adenylyl
cyclase and the increase in intracellular cyclic AMP. This pathway includes a G-protein coupled receptor (Gpr1) and two
G proteins Gpa1 and 2, necessary for the glucose-specific increase in cAMP [9,10]. Finally, glucose activation of adenylyl
cyclase leads to activation of the cAMP-dependent protein
kinase A (PKA). Upon activation of PKA by cAMP the
Rap1 transcription factor activity is increased inducing expression of genes encoding ribosomal proteins and proteins
required for glycolysis. In addition, PKA inactivates other
transcription factors, such as Msn2 and Msn4, down-regulating the expression of STRE-controlled genes [11].
Once inside the cell, glucose activates another pathway
involved in the repression of genes not needed during growth
on glucose. In this pathway, both the Mig1 and the hexokinase 2 (Hxk2) proteins are necessary to generate the glucose
repression signal. Thus, in hxk2 mutants repression of several
genes by glucose is no longer operative even in the presence
of the functional MIG1 gene. The mechanism by which Mig1
operates in this system is quite well known [12–14]. However, the mechanism by which Hxk2 participates in the glucose signalling pathway, is almost unknown until now, in
particular, the position of this factor in the signalling cascade
and the interactions with the other factors of the cascade. Here
we will briefly describe the Hxk2 signalling pathway in the
S. cerevisiae cell, a protein that in addition to its classical metabolic role plays an important function in glucose signalling.
Hxk2 interacts with the Mig1 protein and
can enter the nucleus
Hxk2, Mig1 and Med8 form part of the same protein complex that interacts with a regulatory element of the SUC2
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Figure 1 Overview of Snf3/Rgt2, Gpr1 and Mig1/Hxk2 signalling pathways
The diagram shows the signalling cascades that flow from the complex between glucose and receptor proteins of the cell
membrane to the target gene promoters. For details see the text.
promoter located between −451 and −416 bp [15–20]. However, an interaction between Hxk2 and Mig1 proteins has
not been demonstrated. To test this possibility, we have performed yeast two-hybrid assays, immunoprecipitation and
glutathione S-transferase (GST)-pull down experiments.
Our results demonstrate that about 15% of the total Hxk2
protein is localized in the nucleus and that this localization is
required for glucose repression of several genes [16]. A possible explanation of why in a wild-type strain a limited fraction of total Hxk2, about 15%, is located in the nucleus and
that this amount was not modified when the cell content of
Hxk2 was increased (for example, by expressing the HXK2
gene contained in a high copy number plasmid), could be that
Hxk2 is sequestered in the nucleus by direct interaction with
Mig1 and thus, the amount of nuclear Hxk2 depends on the
amount of Mig1 present in the nucleus. We have also found
that Hxk2 interacts directly with Mig1 in vivo and in vitro
and that the ten amino acid motif between K6 and M15,
previously identified as essential for Hxk2 translocation to
the nucleus, is actually required for the Hxk2–Mig1 interaction [21].
Figure 2 Both Mig1 and Hxk2 are bound to the SUC2 promoter
under high-glucose conditions as detected by chromatin
immunoprecipitation analysis
A strain containing a Mig1 haemagglutinin (HA)-tagged protein was
grown in the presence of high glucose. Immunoprecipitation was performed by using mouse anti-HA monoclonal antibodies or anti-Hxk2
polyclonal antibodies (A). PCR was performed with primers spanning a
305 bp DNA fragment of the SUC2 promoter containing a Mig1 binding
site (B).
Hxk2 binds to a SUC2–Mig1 complex both
in vivo and in vitro
To determine the biological relevance of the interaction
between Hxk2 and Mig1, we have investigated whether
these two proteins co-localize in DNA–protein complexes
in vivo by chromatin immunoprecipitation experiments
and in vitro by gel-retardation assays.
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Chromatin immunoprecipitation experiments have
shown that the Mig1 and Hxk2 proteins are actively recruited
to a DNA fragment of the SUC2 promoter (−451/−416)
containing the Mig1 binding site in response to highglucose conditions (Figure 2).
Nutrient Sensing through the Plasma Membrane of Eukaryotic Cells
Figure 3 Involvement of Mig1 in the subcellular localization of Hxk2
For details see the text.
To demonstrate that the interaction is produced directly
between both proteins without the concurrence of other
auxiliary proteins, we have performed an in vitro gel mobility
shift analysis by using purified proteins from Escherichia coli
expressed genes. We have studied the different mobility of
the complex formed with Mig1 and a DNA fragment carrying the Mig1 binding site of the SUC2 promoter and the
complex formed with Mig1, Hxk2 and the same DNA fragment. The different mobility of these complexes suggests
that Hxk2 interacts directly with Mig1. An additional
control experiment using an anti-Hxk2 antibody confirms
this finding since this antibody inhibits the formation of the
Hxk2–Mig1 complex [21].
These results allow us to conclude that the Hxk2 can bind
directly to a complex formed by the transcriptional repressor
Mig1 and the MIG1 element of the SUC2 promoter.
The nuclear localization of Hxk2 is
regulated by glucose and is
Mig1-dependent
To analyse the factors that regulate the subcellular localization
of Hxk2, green fluorescent protein (GFP) was fused to the
C-terminal end of the Hxk2 protein. The fusion protein was
expressed in several yeast strains from the HXK2 promoter.
Fluorescent microscopy experiments demonstrated that in
glucose-growing cells, Hxk2 is predominantly localized in the
cytoplasm although a small fraction is located in the nucleus
in good agreement with previous results [16,21]. In cells
grown overnight in 3% ethanol, conditions under which
repression does not occur, Hxk2 is found only in the cytoplasm, apparently excluded from the nucleus. This result sug-
gests that the cell modulates the regulatory function of Hxk2
by controlling the transport of the protein between the
nucleus and the cytoplasm.
To determine whether Mig1 participates in the regulation
of Hxk2 localization, we examined it in a mig1 mutant strain.
In glucose-grown cells, Hxk2 was found only in the cytoplasm. This suggests that either Mig1 retains Hxk2 in the
nucleus or Mig1 is necessary for the translocation of the Hxk2
protein to the nucleus. Since it is known that glucose causes
a rapid translocation of Mig1 to the nucleus and that the removal of the sugar results in cytoplasm localization [22], it
could be thought that the amount of Hxk2 located in the
nucleus under glucose-grown conditions could be dependent
on the Mig1 level. To check this, we increased the amount of
Mig1 in the cell by introducing a high copy number plasmid
containing the MIG1 gene under control of its own promoter.
Under this condition we examined the Hxk2 localization
during glucose growth. Interestingly, Hxk2 was intensely
enriched in the nucleus [21].
These results suggest that the cell modulates the Hxk2
regulatory function by controlling the amount of the protein
localized in the nucleus and that Hxk2 is sequestered in
the nucleus through interaction with Mig1. However, these
experiments do not clarify if the Mig1–Hxk2 complex is a
carrier necessary for Mig1 translocation to the nucleus.
The subcellular localization of Mig1 is
regulated by Snf1 and is not
Hxk2-dependent
In order to investigate this possibility GFP was fused to
the C-terminal end of the Mig1 protein and the subcellular
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localization of Mig1 was determined under different metabolic conditions and in several mutant strains. As has been
reported previously, Mig1 has a nuclear location in cells
growing in glucose medium and a cytosolic localization in
cells growing in ethanol medium [19]. However, in an hxk2
mutant cell, Mig1 always has a cytosolic localization. This
result could mean that the formation of the heterodimeric
complex Hxk2–Mig1 is necessary to carry on the Mig1 translocation to the nucleus.
However, on the basis of other previously reported results
[23–25], it is necessary to take into account that the activity of
the Snf1 kinase complex is regulated by the Rgt1–Glc7 phosphatase complex and by Hxk2. In the absence of any one of
these proteins a conformational change is produced in the
Snf1 complex that activates its protein kinase function. Thus,
when Snf1 is activated by mutations in the HXK2 gene, it
was expected that the Mig1 protein would be phosphorylated
and translocated to the cytosol. Interestingly, in a snf1/hxk2
double mutant, the Mig1 protein is always located in the
nucleus, indicating that the presence of the Hxk2 protein is
not necessary for a nuclear localization of Mig1.
Our results are in agreement with the model shown in
Figure 3, explaining the involvement of Hxk2 in the glucose
signalling pathway. At high glucose concentrations, Mig1
is dephosphorylated by the Glc7 phosphatase complex and
imported to the nucleus. At the same time Hxk2 enters
the nucleus and there it is sequestered by interacting with
Mig1. The amount of Hxk2 present in the nucleus is limited
by the Mig1 levels of the cell. Once access to the nucleus has
been gained the heterodimeric Hxk2–Mig1 complex binds to
the Mig1 target gene promoters and recruits the general corepressor complex Cyc8–Tup1 to repress the transcription of
genes not required for growth in glucose.
Upon glucose depletion, Snf1 protein kinase is activated
and phosphorylates Mig1. Phosphorylation induces Mig1
nuclear export, sequestering the protein in the cytoplasm
together with Hxk2. Genes needed for growth on other
carbon sources different from glucose are thus derepressed.
An HXK2 mutation also activates Snf1 complex, and therefore Mig1 is phosphorylated and translocated to the cytosol,
signalling derepression of Mig1 target genes. Thus in the
absence of Hxk2, the Snf1 protein kinase complex is active
even in the presence of high levels of glucose in the medium.
Our results suggest that one major function of Hxk2 may
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be to inhibit Snf1 protein kinase function by blocking Mig1
phosphorylation at the nuclear level.
We are very grateful to J.M. Gancedo and C. Gancedo for fruitful
discussions. This work was supported by Grant PB97-1213-C0202
from the Dirección General de Enseñanza Superior e Investigación
Cientı́fica (DGESIC, Spain).
References
1 Johnston, M. (1987) Microbiol. Rev. 51, 458–476
2 Ozcan, S., Vallier, L.G., Flick, J.S., Carlson, M. and Johnston, M. (1997)
Yeast 13, 127–137
3 Gancedo, J.M. (1998) Microbiol. Mol. Biol. Rev. 62, 334–361
4 Özcan, S., Dover, J., Rosenwald, A.G., Woelft, S. and Johnston, M. (1996)
Proc. Natl. Acad. Sci. U.S.A. 93, 12428–12432
5 Thevelein, J.M. and de Winde, J.H. (1999) Mol. Microbiol. 32, 904–918
6 Moreno, F. and Herrero, P. (2002) FEMS Microbiol. Rev. 26, 83–90
7 Özcan, S. and Johnston, M. (1999) Microbiol. Mol. Biol. Rev. 63,
554–569
8 Özcan, S., Leong, T. and Johnston, M. (1996) Mol. Cell. Biol. 16,
6419–6426
9 Xue, Y., Batlle, M. and Hirsch, J.P. (1998) EMBO J. 17, 1996–2007
10 Kraakman, L., Lemaire, K., Ma, P., Teunissen, A.W., Donaton, M.C.,
Van Dijck, P., Winderickx, J., de Winde, J.H. and Thevelein, J.M. (1999)
Mol. Microbiol. 32, 1002–1012
11 Rolland, F., Winderickx, J. and Thevelein, J.M. (2001) Trends Biochem. Sci.
26, 310–317
12 Nehlin, J.O. and Ronne, H. (1990) EMBO J. 9, 2891–2898
13 Lundin, M., Nehlin, J.O. and Ronne, H. (1994) Mol. Cell. Biol. 14,
1979–1985
14 Treitel, M.A. and Carlson, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,
3132–3136
15 Bu, Y. and Schmidt, M.C. (1998) Nucleic Acids Res. 26, 1002–1009
16 Herrero, P., Martı́nez-Campa, C. and Moreno, F. (1998) FEBS Lett. 434,
71–76
17 Moreno-Herrero, F., Herrero, P., Colchero, J., Baro, A.M. and Moreno, F.
(1999) FEBS Lett. 459, 427–432
18 Meijer, M.M., Boonstra, J., Verkleij, A.J. and Verrips, C.T. (1998)
J. Biol. Chem. 273, 24102–24107
19 Mayordomo, I. and Sanz, P. (2001) Yeast 18, 923–930
20 Sanz, P., Nieto, A. and Prieto, J.A. (1996) J. Bacteriol. 178, 4721–4723
21 Ahuatzi, D., Herrero, P., de la Cera, T. and Moreno, F. (2004)
J. Biol. Chem. 279, 14440–14446
22 DeVit, M.J., Waddle, J.A. and Johnston, M. (1997) Mol. Biol. Cell 8,
1603–1618
23 Alms, G.R., Sanz, P., Carlson, M. and Haystead, T.A.J. (1999) EMBO J. 18,
4157–4168
24 Sanz, P., Alms, G.R., Haystead, T.A.J. and Carlson, M. (2000)
Mol. Cell. Biol. 20, 1321–1328
25 Treitel, M.A., Kuchin, S. and Carlson, M. (1998) Mol. Cell. Biol. 18,
6273–6280
Received 17 September 2004