From the Department of Molecular & Cellular Biochemistry, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536
Received for publication, December 16, 2002
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ABSTRACT |
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Glucose, the most abundant carbon and energy
source, regulates the expression of genes required for its own
efficient metabolism. In the yeast Saccharomyces
cerevisiae, glucose induces the expression of the hexose
transporter (HXT) genes by modulating the activity of the
transcription factor Rgt1 that functions as a repressor when glucose is
absent. However, in the presence of high concentrations of glucose,
Rgt1 is converted from a repressor to an activator and is required for
maximal induction of HXT1 gene expression. We report that
Rgt1 binds to the HXT1 promoter only in the absence of
glucose, suggesting that Rgt1 increases HXT1 gene
expression at high levels of glucose by an indirect mechanism. It is
likely that Rgt1 stimulates the expression of an activator of the
HXT1 gene at high concentrations of glucose. In addition,
we demonstrate that Rgt1 becomes hyperphosphorylated in response to
high glucose levels and that this phosphorylation event is required for
Rgt1 to activate transcription. Furthermore, Rgt1 lacks the
glucose-mediated phosphorylation in the snf3 rgt2 and
grr1 mutants, which are defective in glucose induction of
HXT gene expression. In these mutants, Rgt1 behaves as a
constitutive repressor independent of the carbon source. We conclude
that phosphorylation of Rgt1 in response to glucose is required to
abolish the Rgt1-mediated repression of the HXT genes and
to convert Rgt1 from a transcriptional repressor to an activator.
The yeast Saccharomyes cerevisiae uses glucose as its
preferred carbon and energy source. Glucose not only represses the
expression of genes that are required for the metabolism of alternate
sugars but also induces the transcription of genes that are essential for its own efficient utilization (1-3). Among the genes that are
induced by glucose are the members of the HXT gene
family, which encode glucose transporters. Glucose induces the
expression of the HXT1-HXT4 genes by
10-300-fold (4, 5).
Several components of the glucose induction pathway required for
HXT gene expression have been identified, including the
glucose sensors Snf3 and Rgt2, that are responsible for sensing
extracellular glucose and generating the intracellular signal (6, 7). A
strain mutated for both sensors (snf3 rgt2 double mutant) is completely defective in glucose induction of the HXT gene
expression (7). Another component that is absolutely essential for
glucose induction of the HXT gene expression is the
ubiquitin ligase Grr1 (5, 8). Two homologous proteins, Std1 and Mth1,
have been shown to negatively regulate HXT gene expression
and to interact with the carboxyl-terminal tails of the Snf3 and Rgt2
sensors (9-11). Repression of HXT gene expression in the
absence of glucose is abolished in a std1 mth1 double mutant
(9, 11).
The target of the glucose induction signal is the Cys6
DNA-binding protein Rgt1, which belongs to the family of the Gal4
transcription factors (12). In the absence of glucose, Rgt1 represses
HXT gene expression, whereas at high concentrations of
glucose Rgt1 is required for maximal activation of HXT1 gene
expression. Repression of transcription by Rgt1 in the absence of
glucose requires the general repressor complex, Ssn6 and Tup1.
Activation of transcription by Rgt1 in response to high concentrations
of glucose requires the glucose sensors Snf3 and Rgt2 and the ubiquitin
ligase Grr1 (12).
It was previously shown that Rgt1 is required for both repression and
activation of HXT1 gene expression and binds to the HXT1 promoter in vitro by gel shift assays (12).
We report that the requirement of Rgt1 for activation of
HXT1 gene expression in response to high concentrations of
glucose is mediated by an indirect mechanism, because Rgt1 is unable to
bind to the HXT1 gene promoter in vivo when high
levels of glucose are present. Previous data indicated that the
transcription of Rgt1 is not regulated by glucose, suggesting the idea
that the transcriptional activity of Rgt1 is regulated
post-translationally (12). We demonstrate that Rgt1 is
hyperphosphorylated in response to high concentrations of glucose
and that the lack of Rgt1 phosphorylation abolishes its ability to
activate transcription.
Construction of Strains and Plasmids--
The yeast S. cerevisiae strains used in this study are the wild type strain
YM4127, rgt1 (YM4509), snf3 rgt2 double mutant (YM6107), and the grr1 mutant (YM4576), which have been
described previously (5, 7, 12). Yeast cells were grown on YNB (0.67% yeast nitrogen base (Difco) plus 0.5% ammonium sulfate) medium lacking
the appropriate amino acids. The bacterial strain DH5 Immunofluorescence Microscopy--
The fixation and indirect
immunostaining of yeast cells was carried out as described previously
(16). The Rgt1-HA fusion protein was visualized using monoclonal HA
antibodies and Alexa532-conjugated goat anti-mouse IgG antibodies
(Molecular Probes) as the secondary antibodies. For nuclear staining,
anti-acetyl histone H3 antibodies (Upstate Biotechnology Inc.) in
combination with Alexa488-conjugated goat anti-rabbit IgG antibodies
(Molecular Probes) were used. Immunofluorescence microscopy was carried
out using a laser scanning confocal microscope (Leica).
Chromatin Immunoprecipitation (ChIP) Assay and Semi-quantitative
PCR--
Chromatin isolation was performed as previously published
(17). 100-ml cultures of yeast cells grown to
A600 of 0.6-0.8 were cross-linked with
formaldehyde (final concentration, 1%). After lysis of the cells, the
nuclear extracts were sonicated with glass beads (0.1 g) for five 10-s
pulses at 60% power using a Tekmar Sonic Disruptor. The samples were
precleared with 20 µl of blocked Pansorbin
Staphylococcus A cells (Calbiochem). After 4-fold
dilution of the samples in IP buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl) and incubation with 2 µg of HA antibodies or
mouse IgG (Sigma) overnight at 4 °C, the immunocomplexes were
recovered by incubation with blocked Staphylococcus A cells.
After rinsing the samples twice in wash buffer (2 mM EDTA,
50 mM Tris-Cl, pH 8.0, and 0.2% Sarkosyl) and four times
with IP wash buffer (1% Nonidet P-40, 100 mM Tris-HCl, pH
8.0, 500 mM LiCl, and 1% deoxycholic acid), the
immunocomplexes were eluted twice from the Staphylococcus A
cells (with 150 µl of 1% SDS in 50 mM
NaHCO3). The cross-links were reversed by adding 20 µl of
5 M NaCl and 1 µl of 10 mg/ml RNase A and by incubating at 65 °C for 8 h. After treating with 1.5 µl of proteinase K
(10µ g/µ l), the samples were extracted with phenol/chloroform and subsequently ethanol-precipitated using 20 µg of glycogen as a carrier.
PCR Analysis of Immunoprecipitated DNA--
All of the PCR
reactions were performed on a Robocycler Gradient 96 (Stratagene) in a
20-µl reaction volume containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl, 200 µM of dNTPs, and 2 µl of primers (2.5 pmol/µl). The
linear range for each primer pair was determined empirically, using
different amounts of yeast genomic DNA. The PCR products obtained with
the immunoprecipitated DNA were normalized to the products obtained
with the total input DNA. The primers used for PCR are listed in Table
I. A detailed PCR protocol is available upon request.
Western Blotting--
Yeast cell extracts from 2-10-ml cultures
were obtained using glass beads in lysis buffer (10 mM
Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 2%
Triton X-100, 1% SDS) containing protease and phosphatase inhibitors.
Approximately 50-100 µg of protein was separated by SDS-PAGE
and transferred on nitrocellulose. Monoclonal lexA (Santa Cruz) or HA
antibodies were used as primary antibodies. ECL (Amersham Biosciences)
was used for immunodetection. For the phosphatase treatment, the cell
extracts were prepared in the absence of phosphatase inhibitors and
incubated with 400 units of The Subcellular Localization of Rgt1 Is Not Regulated by
Glucose--
Several transcription factors have been shown to shuttle
between the cytoplasm and the nucleus in response to various stimuli. For example the transcription factor Mig1 that inhibits gene expression in response to glucose is in the nucleus only if high concentrations of
glucose are present. In the absence of glucose Mig1 is phosphorylated and trapped in the cytoplasm (19, 20). It has been previously demonstrated that the transcription factor Rgt1 represses
HXT gene expression in the absence of glucose and is
required for maximal activation of HXT1 gene expression when
high concentrations of glucose are present (12). To test whether
glucose regulates the subcellular localization of Rgt1, we have
transformed the rgt1 deletion strain with the
RGT1-HA construct expressed from a multicopy vector from its
own promoter. This construct was functional and complemented the
rgt1 mutant by restoring the repression of HXT
genes in the absence of glucose (data not shown). Cells grown in
absence or presence of low and high concentrations of glucose were
stained for Rgt1-HA and acetyl-histone H3 and visualized by indirect
immunofluorescence using confocal microscopy. The cells were stained
for Rgt1-HA using the HA antibody in combination with Alexa532-coupled
secondary antibodies and for acetylated histones (nuclear marker) using
anti-acetyl histone H3 and Alexa488-coupled secondary antibodies (Fig.
1). Under all carbon source conditions, Rgt1 was localized to the nucleus (Fig. 1, left panels),
indicating that Rgt1 is always in the nucleus independent of the
presence of glucose.
Rgt1 Binds to the HXT Gene Promoters Only in the Absence of
Glucose--
We have previously shown that Rgt1 is able to bind to the
HXT2 and HXT3 gene promoters in vitro
and is required for repression of these genes when glucose is absent
(5, 12, 21). Although Rgt1 is converted from a repressor to an
activator in response to high levels of glucose, we have so far no
evidence that Rgt1 is required for activation of the HXT2
and HXT3 genes when glucose levels rise. To test whether the
ability of Rgt1 to bind to the HXT gene promoters is
regulated by the carbon source, we have utilized the ChIP assay. The
rgt1 mutant strain containing the RGT1-HA
construct was grown in the absence or presence of high concentrations
of glucose and subjected to the ChIP assay using monoclonal HA
antibodies. The immunoprecipitated and the total input DNA
were used as templates for PCR analysis with primers against
HXT1, HXT2, and
HXT3 gene promoters (Figs. 2
and 3). The regions of HXT2
and HXT3 gene promoters amplified by PCR have been shown
before to bind Rgt1. As expected, Rgt1 was associated with the
HXT2 and HXT3 promoters only when glucose was
absent (Fig. 2).
Previous data indicate that Rgt1 is required for both repression and
activation of HXT1 gene expression. Deletion of
RGT1 causes increased expression of the HXT1 gene
in the absence of glucose consistent with a role for Rgt1 as a
transcriptional repressor of this gene. However, induction of
HXT1 gene expression at high levels of glucose is about
5-6-fold decreased in an rgt1 mutant, indicating that
activation by Rgt1 is required for maximal HXT1 gene
expression. These findings suggested that Rgt1 binds to the HXT1 promoter both in the absence and in the presence of
glucose to repress and activate HXT1 gene expression
directly. To test the idea that Rgt1 is bound to the HXT1
promoter in the absence and presence of glucose, we analyzed Rgt1
binding using the ChIP assay. Surprisingly, Rgt1 binds to the
HXT1 gene promoter only in the absence of glucose (Fig. 3).
A 1.2-kb region of the HXT1 promoter that has been
previously shown to be sufficient for glucose induction of this gene
was scanned for Rgt1 binding using different primers for PCR
amplification (Fig. 3). Rgt1 binding was detected for the region
spanning the sequences from Rgt1 Is Modified by Phosphorylation in Response to High
Concentrations of Glucose--
Because the expression level of
Rgt1 in cells grown on glycerol or glucose appears to be similar (12),
we investigated whether Rgt1 undergoes a post-translational
modification in response to glucose. For this purpose, we prepared
extracts from the rgt1 deletion strain containing the
LexA-RGT1 construct grown in the presence or absence of
glucose. The obtained extracts were analyzed by Western blot using a
monoclonal lexA antibody. As shown in Fig.
4A, Rgt1 from glucose-grown
cells displays a slight shift in molecular weight, resulting in
decreased mobility, when compared with Rgt1 from glycerol-grown cells.
This indicates that Rgt1 is modified in the presence glucose. Time
course analysis of Rgt1 modification indicates that the observed
modification is rapid and complete within 5 min of shifting from
glycerol to 4% glucose medium (Fig. 4B). Furthermore, we
did not see any significant changes in Rgt1 protein levels in cells
grown on glycerol versus glucose, indicating that the
stability of Rgt1 is not changed in response to glucose.
To test whether the observed shift in Rgt1 mobility in response to
glucose is due to phosphorylation, extracts from glucose-grown cells
containing lexA-Rgt1 were incubated with or without The Rgt1 Region Spanning Amino Acids 75-395 Is Required for
Transcriptional Activity and for Modification by Phosphorylation in
Response to Glucose--
To identify the domain(s) in Rgt1 that
regulates its transcriptional activity in response to glucose, we
generated a series of amino- and carboxyl-terminal deletions. These
deletions were fused in frame to the lexA DNA-binding domain and
transformed into the rgt1 deletion strain together with a
CYC1-lacZ reporter that contains the UAS of CYC1
and four lexA operator sites upstream of it. Because most of the
deletion constructs did not display any transcriptional activity, only
two of the eleven deletion constructs tested are shown in Fig.
5A. The ability of these
deletion proteins to repress or activate transcription was assayed by
measuring
To address the question of whether the region of Rgt1 spanning amino
acids 75-395, important for Rgt1 transcriptional activity, is also
required for Rgt1 modification, we analyzed the modification of these
constructs by Western blots with lexA antibodies. As shown in Fig.
5B, the Rgt1 Lacks the Glucose-induced Modification in grr1 and snf3 rgt2
Mutants--
We have previously shown that GRR1 encoding an
E3-ubiquitin ligase is required for the activator function of Rgt1
(12). In a grr1 mutant, Rgt1 always functions a repressor
independent of glucose. The Snf3 and Rgt2 glucose sensors are also
required for the conversion of Rgt1 from a repressor to an activator
(12).2 To analyze the modification of Rgt1 in response to
glucose in the grr1 and snf3 rgt2 double mutants,
the corresponding mutants were transformed with the
lexA-RGT1 construct, and the obtained transformants were
grown in the absence or presence of high concentrations of glucose. The
modification of the lexA-Rgt1 fusion protein in these mutants was
analyzed by Western blotting using the lexA antibody. Interestingly,
both the grr1 and the snf3 rgt2 double mutant
lack the modification that is normally observed with Rgt1 from wild
type cells grown on glucose (Fig. 6).
This indicates that the observed modification of Rgt1 is required to
release repression by Rgt1 in response to the presence of high
concentrations of glucose.
It has been previously shown that Rgt1 is the target of the
glucose induction pathway that leads to up-regulation of HXT
gene expression (5). Furthermore, it was demonstrated that Rgt1 is a
bifunctional transcription factor that is converted from a
transcriptional repressor to an activator in response to high concentrations of glucose (12). In this publication, we have addressed
the question of whether glucose regulates the transcriptional activity
of Rgt1 by mediating changes in Rgt1 subcellular localization, DNA
binding, or post-translational modification. Several transcription factors have been shown to change their subcellular localization in
response to external stimuli in yeast, including Mig1 and Pho4 (19,
23). Mig1 is phosphorylated by the Snf1 kinase in the absence of
glucose or presence of low levels of glucose and is trapped in the
cytoplasm. High concentrations of glucose cause dephosphorylation of
Mig1 and translocation into the nucleus (19, 20). The subcellular
localization of Pho4 is regulated in response to phosphate. At high
concentrations of phosphate, Pho4 is phosphorylated and exported into
the cytoplasm, whereas starvation for phosphate causes
dephosphorylation of Pho4 and translocation into the nucleus (23). Our
data obtained using indirect immunofluorescence microscopy suggest that
the subcellular localization of Rgt1 does not change in response to glucose.
We also tested the possibility that glucose may regulate the DNA
binding affinity of Rgt1 utilizing the ChIP assay. As expected we did
not see any significant binding of Rgt1 in vivo to the HXT2 and HXT3 promoters in cells grown in
presence of high concentrations of glucose. Rgt1 binding to the
HXT2 and HXT3 gene promoters was observed only in
the absence of glucose, consistent with its role as a transcriptional
repressor of the HXT2 and HXT3 genes when glucose
is absent.
Several lines of evidence indicate that Rgt1 regulates both repression
and activation of HXT1 gene expression. First, Rgt1 is able
to bind to a 280-bp promoter region of the HXT1 gene
in vitro that is sufficient for glucose induction of this
gene (12).2 Second, deletion of RGT1 causes a
10-fold increase in HXT1 expression when glucose is absent
but a 5-6-fold decrease in HXT1 gene expression at high
concentrations of glucose. Third, a lexA-Rgt1 fusion protein functions
as a repressor of transcription in the absence of glucose and as an
activator of transcription when glucose is abundant (12). These data
suggested that Rgt1 might directly regulate HXT1 gene
expression in the absence and presence of high concentrations of
glucose. However, ChIP analysis of Rgt1 binding in vivo
indicates that the positive regulatory effect of Rgt1 on
HXT1 gene expression is mediated by an indirect mechanism,
because Rgt1 is unable to bind to the HXT1 promoter in
response to glucose. It is likely that Rgt1 is required for expression
of a transcriptional activator of the HXT1 gene at high
concentrations of glucose (Fig. 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F' was used as
a host for plasmids. The Rgt1-HA plasmid (pSO913), which contains the
RGT1 promoter (900 bp) and the coding region fused in frame
to a triple HA1 epitope tag
at the carboxyl terminus, was constructed in two steps. First, the
RGT1 coding region was amplified by PCR using the primers OS669 and OS95. The amplified PCR product was subcloned into the vector pAD80 (13) as BamHI and SalI in
frame with a triple HA tag. Then the obtained plasmid was cut with
EcoRI and XbaI (which removes the first 1,063 bp
of the RGT1 coding region), and an
EcoRI/XbaI fragment containing the
RGT1 promoter and the missing coding region was inserted.
The construction of the CYC1-lacZ and the
lexA-RGT1 plasmids have been described previously (12). The
constructs
1 (pSO421) and
2 (pSO462) were created by subcloning PCR products as BamHI/SalI fragments into the
lexA vector pSH2-1 (14, 15). The oligonucleotide primers
used are OS155 and OS668 for
1 and OS1102 and OS668 for
2 and are
listed in Table I.
List of the sequences of oligonucleotide primers used in this study
phosphatase (New England Biolabs) for
1 h at 37 °C.
-Galactosidase Assays--
-Galactosidase activity assays
were performed with permeabilized yeast cells grown to mid-log phase as
described previously (18). The mean activities are given in Miller
units and are the averages of three to four assays of at least four
independent yeast transformants.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rgt1 is always localized to the nucleus
independent of carbon source. Yeast cells were grown overnight on
5% glycerol and then transferred to 5% glycerol, 0.1% glucose, and
4% glucose and incubated for 2 h. Rgt1-HA and acetylated histone
H3 were visualized by confocal microscopy, using HA and acetyl histone
H3 antibodies in combination with Alexa532- and Alexa488-coupled
secondary antibodies, respectively. Acetyl histone H3 was used as a
marker to detect nuclear localization. The rgt1 strain
without the Rgt1-HA construct was used as a negative control for
staining with HA antibodies.
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Fig. 2.
Rgt1 binds to the HXT2 and
HXT3 gene promoter regions in vivo,
only when glucose is absent. The rgt1 mutant containing
the RGT1-HA construct was grown overnight on 5% glycerol
and then transferred to 5% glycerol, 0.1% glucose, and 4% glucose
and incubated for 2 h. The cells were subjected to ChIP assay
analysis using HA antibodies. Rgt1 binding under various conditions was
analyzed by PCR using primers to amplify the HXT2 and
HXT3 promoter regions with total input or immunoprecipitated
DNA as template. The PCR products were separated on 8% nondenaturing
PAGE gels and stained with ethidium bromide. Input DNA was used as
template to determine whether equal amounts of total DNA were used in
each immunoprecipitation experiment.
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Fig. 3.
Rgt1 binds to the HXT1
promoter region from 648 to
361 only in the absence of
glucose. The ChIP assay analysis was carried out as described in
the legend to Fig. 2. The HXT1 gene promoter was scanned for
Rgt1 binding by PCR using primers covering different regions of the
promoter. A shows the regions of the HXT1
promoter scanned for Rgt1 binding. The PCR products obtained using
primers to amplify different regions of HXT1 promoter with
input and HA-immunoprecipitated DNA as template are shown in
B.
648 to
361 bp from the transcription
initiation site, and Rgt1 binds this region only in the absence of
glucose (Fig. 3). This region of the HXT1 gene promoter is
sufficient to mediate glucose-regulated expression of a heterologous
gene.2 The data
obtained with the ChIP assay indicate that Rgt1 does not bind to the
HXT1 promoter in response to glucose and that it activates
HXT1 gene expression probably by an indirect mechanism.
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Fig. 4.
Rgt1 is rapidly phosphorylated in response to
glucose. Cells transformed with the lexA-Rgt1 construct were
precultured on 5% glycerol overnight and transferred to 5% glycerol
and 4% glucose for 12 to 16 h. After cell lysis, the protein
extracts (100 µg) were separated on 8% SDS-PAGE gel and transferred
to nitrocellulose membrane. The lexA-Rgt1 fusion protein was detected
using monoclonal lexA antibodies (A). For the time course
shown in B, the cells were pregrown on 5% glycerol and
shifted to 4% glucose at time 0. The samples were taken at 1-, 5-, and
10-min intervals after incubation with glucose. The phosphatase
treatment of cell extracts from glucose-grown cells was carried out
using 400 units of phosphatase for 1 h at 37 °C
(C).
phosphatase. Western blot analysis of these samples with the lexA antibody demonstrate that the observed shift in Rgt1 mobility in response to
glucose is abolished after treatment with
phosphatase (Fig. 4C). This indicates that Rgt1 undergoes hyperphosphorylation
in response to glucose.
-galactosidase activity. As reported before, full-length
Rgt1 fused to lexA causes a 6-fold repression of
-galactosidase
expression when glucose is absent and a 8-fold activation in the
presence of glucose, compared with the control (lexA alone) (Fig.
5A, compare lines 1 and 2) (12). As
demonstrated before, deletion of the DNA-binding domain of Rgt1 (the
first 75 amino acids) has no effect on its transcriptional activity,
indicating that the DNA-binding domain of Rgt1 is not required for
regulation of its transcriptional activity in response to glucose (Fig.
5A,
1, line 3). Deletion of the
first 395 amino acids of Rgt1 (
2) abolishes both the repressor and
activator functions of Rgt1 (Fig. 5A, line 4).
This indicates that the amino acids between 75 and 395 are essential
for Rgt1 repressor as well as activator function.
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Fig. 5.
The region spanning amino acids 75-395 is
required for modification and regulation of Rgt1 transcriptional
activity. The transcriptional activity of the Rgt1 deletion
constructs was analyzed and quantified by measuring -galactosidase
activity as described previously (12) (A). B,
Western blot analysis of the Rgt1 deletion constructs for modification,
using monoclonal lexA antibodies.
1 construct lacking the first 75 amino acids
still displays the shift in molecular weight; however,
2, which
lacks the first 395 amino acids, does not change its molecular weight
in response to glucose. The same construct (
2) that is defective in
both repression and activation by Rgt1 also lacks the modification.
Both constructs
1 and
2 were localized in the nucleus (data not shown).
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Fig. 6.
The grr1 and the snf3
rgt2 double mutants are defective in Rgt1 modification in
response to glucose. Western blot analysis of Rgt1 modification in
wild type (WT), grr1, and snf3 rgt2
double mutants was carried as described for Fig. 4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A model explaining the regulation and
modification of Rgt1 in response to glucose. In the absence of
glucose, Rgt1 associates with a repressor complex containing Ssn6,
Tup1, Mth1, and Std1 and represses the transcription of the
HXT1 gene by direct binding to its promoter region. When
glucose is abundant, Rgt1 becomes hyperphosphorylated and dissociates
from the repressor complex. Phosphorylation of Rgt1 in response to
glucose converts it to an activator, which then may stimulate the
expression of a transcriptional activator (Act) that is
required for maximal expression of the HXT1 gene.
PM, plasma membrane; CYT, cytosol;
NUC, nucleus.
We have consistently observed a change in Rgt1 molecular weight in
response to glucose, suggesting that glucose regulates the
transcriptional activity of Rgt1 by a post-translational modification event. Treatment of extracts of glucose-grown cells with phosphatase indicated that Rgt1 is phosphorylated in response to
glucose. Because the molecular mass of the lexA-Rgt1 fusion
construct is about 140 kDa, the observed shift in Rgt1 mobility at high
concentrations of glucose is likely due to hyperphosphorylation or
phosphorylation at multiple sites. The activity of several eukaryotic
transcription factors is regulated by reversible phosphorylation in
response to changes in the cellular environment. Multisite
phosphorylation in particular has been shown to provide a dynamic and
precise tuning of the transactivating potential of transcription
factors rather than being a static on/off switch. Multisite
phosphorylation of transcription factors such as heat shock factor 1 and p53 enables the integration of several different signals (24).
Multisite phosphorylation of Rgt1 could have several functions such as
the disruption of Rgt1 interaction with a repressor complex in the absence of glucose (Fig. 7) and conversion of Rgt1 from a
transcriptional repressor to an activator via unmasking of a specific
activation domain.
Based on several lines of evidence, we propose that Rgt1 functions as a transcriptional repressor of the HXT genes in the absence of glucose, in a complex with the Ssn6-Tup1 and Mth1-Std1 repressor proteins (Fig. 7) (12).3 When glucose is abundant, Rgt1 becomes hyperphosphorylated and dissociates from the repressor complex. Hyperphosphorylation also converts Rgt1 from a transcriptional repressor to an activator (Fig. 7). Consistent with this idea, deletion of the region from amino acids 75 to 395 abolishes the ability of Rgt1 to both activate transcription and become hyperphosphorylated. Furthermore, the grr1 and snf3 rgt2 mutant strains, where Rgt1 functions always as a transcriptional repressor independent of glucose, lack the glucose-induced modification of Rgt1. The presented data also suggest that Rgt1 is not directly involved in the activation of the HXT1 gene expression but rather stimulates the expression of a transcription factor that is required for maximal expression of the HXT1 gene when glucose is abundant (Fig. 7).
The region of Rgt1 required for its glucose-mediated phosphorylation (amino acids 75-395) has several potential phosphorylation sites. Our preliminary data indicate that nuclear localization of Rgt1 is required for the glucose-induced modification, suggesting the idea that the kinase(s) that phosphorylate Rgt1 in response to glucose is nuclear. We are currently testing whether any of the known nuclear kinases are involved in phosphorylation of Rgt1.
The transcription factor Ume6 belongs to the same family of DNA-binding proteins as Rgt1 and is also a repressor and an activator of gene expression (26-28). Ume6 in a complex with Sin3 and Rpd3 functions as a repressor; however, its interaction with Ime1 during meiosis converts Ume6 to an activator of early genes (29). The co-repressor Sin3 interacts with the deacetylase Rpd3 and causes repression by deacetylation of histones (30-32). Interaction of Ume6 with Ime1 is required for the phosphorylation of Ume6 at the amino terminus by the Rim11 and Mck1 kinases (33). Because Ime1 functions as a transcriptional activator when fused to a DNA-binding domain, it has been proposed that Ime1 provides the activation domain for Ume6 (22).
Like with Ume6, phosphorylation of Rgt1 in response to high
concentrations of glucose is important for Rgt1 to function as a
transcriptional activator. However, it is not known whether phosphorylation facilitates Rgt1 to interact with an activator like
Ime1. It is likely that repression by Rgt1 also involves a co-repressor
complex like the Sin3 and Rpd3 that mediates deacetylation of histones.
Indeed we have preliminary data indicating that glucose induction of
HXT genes involves changes in histone acetylation levels.2 Experiments are under way to test whether the
observed changes in histone acetylation at the HXT gene
promoters are mediated by Rgt1. In addition, being an activator itself,
Ume6 also induces the expression of another transcription factor,
Ndt80, that activates middle genes (25). Similar to Ume6, Rgt1 is also
likely to stimulate the expression of a transcriptional activator that
is required for maximal expression of the HXT1 gene in
response to high concentrations of glucose (Fig. 7).
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ACKNOWLEDGEMENTS |
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We thank Mark Johnston and the members of his laboratory for sharing data prior to publication and Bob Dickson and Bob Lester for useful suggestions. We thank Dr. Wally Whiteheart for providing us with HA antibodies and Courtney Reynolds for excellent technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These two authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky, College of Medicine, 800 Rose St., MN 608, Lexington, KY 40536. Tel.: 859-257-4821; Fax: 859-323-1037; E-mail: sozcan@uky.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212802200
2 S. Özcan, unpublished data.
3 J. Lakshmanan, A. L. Mosley, and S. Özcan, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: HA, hemagglutinin; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation.
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