Glucose-mediated Phosphorylation Converts the Transcription Factor Rgt1 from a Repressor to an Activator*

Amber L. MosleyDagger, Jaganathan LakshmananDagger, Bishwa K. Aryal, and Sabire Özcan§

From the Department of Molecular & Cellular Biochemistry, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536

Received for publication, December 16, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 DH5alpha 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 Delta 1 (pSO421) and Delta 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 Delta 1 and OS1102 and OS668 for Delta 2 and are listed in Table I.


                              
View this table:
[in this window]
[in a new window]
 
Table I
List of the sequences of oligonucleotide primers used in this study

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 lambda  phosphatase (New England Biolabs) for 1 h at 37 °C.

beta -Galactosidase Assays-- beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (16K):
[in this window]
[in a new window]
 
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.

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).


View larger version (38K):
[in this window]
[in a new window]
 
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.


View larger version (40K):
[in this window]
[in a new window]
 
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.

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 -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.

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.


View larger version (18K):
[in this window]
[in a new window]
 
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 lambda  phosphatase for 1 h at 37 °C (C).

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 lambda  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 lambda  phosphatase (Fig. 4C). This indicates that Rgt1 undergoes hyperphosphorylation in response to glucose.

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 beta -galactosidase activity. As reported before, full-length Rgt1 fused to lexA causes a 6-fold repression of beta -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, Delta 1, line 3). Deletion of the first 395 amino acids of Rgt1 (Delta 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.


View larger version (24K):
[in this window]
[in a new window]
 
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 beta -galactosidase activity as described previously (12) (A). B, Western blot analysis of the Rgt1 deletion constructs for modification, using monoclonal lexA antibodies.

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 Delta 1 construct lacking the first 75 amino acids still displays the shift in molecular weight; however, Delta 2, which lacks the first 395 amino acids, does not change its molecular weight in response to glucose. The same construct (Delta 2) that is defective in both repression and activation by Rgt1 also lacks the modification. Both constructs Delta 1 and Delta 2 were localized in the nucleus (data not shown).

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.


View larger version (14K):
[in this window]
[in a new window]
 
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (23K):
[in this window]
[in a new window]
 
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 lambda  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).

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: HA, hemagglutinin; IP, immunoprecipitation; ChIP, chromatin immunoprecipitation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Gancedo, J. M. (1998) Microbiol. Mol. Biol. Rev. 62, 334-361[Abstract/Free Full Text]
2. Carlson, M. (1998) Curr. Opin. Genet. Dev. 8, 560-564[CrossRef][Medline] [Order article via Infotrieve]
3. Johnston, M. (1999) Trends Genet. 15, 29-33[CrossRef][Medline] [Order article via Infotrieve]
4. Özcan, S., and Johnston, M. (1999) Microbiol. Mol. Biol. Rev. 63, 554-569[Abstract/Free Full Text]
5. Özcan, S., and Johnston, M. (1995) Mol. Cell. Biol. 15, 1564-1572[Abstract]
6. Özcan, S., Dover, J., Rosenwald, A. G., Wölfl, S., and Johnston, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12428-12432[Abstract/Free Full Text]
7. Özcan, S., Dover, J., and Johnston, M. (1998) EMBO J. 17, 2566-2573[Abstract/Free Full Text]
8. Li, F. N., and Johnston, M. (1997) EMBO J. 16, 5629-5638[Abstract/Free Full Text]
9. Schmidt, M. C., McCartney, R. R., Zhang, X., Tillman, T. S., Solimeo, H., Wolfl, S., Almonte, C., and Watkins, S. C. (1999) Mol. Cell. Biol. 19, 4561-4571[Abstract/Free Full Text]
10. Schulte, F., Wieczorke, R., Hollenberg, C. P., and Boles, E. (2000) J. Bacteriol. 182, 540-542[Abstract/Free Full Text]
11. Lafuente, M., Gancedo, J. C., Jauniaux, J.-C., and Gancedo, J. M. (2000) Mol. Microbiol. 35, 161-172[CrossRef][Medline] [Order article via Infotrieve]
12. Özcan, S., Leong, T., and Johnston, M. (1996) Mol. Cell. Biol. 16, 6419-6426[Abstract]
13. Delley, P. A., and Hall, M. N. (1999) J. Cell Biol. 147, 163-174[Abstract/Free Full Text]
14. Brent, R., and Ptashne, M. (1985) Cell 43, 729-736[Medline] [Order article via Infotrieve]
15. Ma, J., and Ptashne, M. (1987) Cell 51, 113-119[Medline] [Order article via Infotrieve]
16. Beck, T., Schmidt, A., and Hall, M. N. (1999) J. Cell Biol. 146, 1227-1238[Abstract/Free Full Text]
17. Wells, J., and Farnham, P. J. (2002) Methods 26, 48-56[CrossRef][Medline] [Order article via Infotrieve]
18. Yocum, R. R., Hanley, S., West, R., Jr., and Ptashne, M. (1984) Mol. Cell. Biol. 4, 1985-1998[Medline] [Order article via Infotrieve]
19. DeVit, M. J. D., Waddle, J. A., and Johnston, M. (1997) Mol. Biol. Cell 8, 1603-1618[Abstract]
20. Treitel, M. A., Kuchin, S., and Carlson, M. (1998) Mol. Cell. Biol. 18, 6273-6280[Abstract/Free Full Text]
21. Özcan, S., and Johnston, M. (1996) Mol. Cell. Biol. 16, 5536-5545[Abstract]
22. Mandel, S., Robzyk, K., and Kassir, Y. (1994) Dev. Genet. 15, 139-147[Medline] [Order article via Infotrieve]
23. Komeili, A., and O'Shea, E. K. (1999) Science 284, 977-980[Abstract/Free Full Text]
24. Holmberg, C. I., Tran, S. E. F., Eriksson, J. E., and Sistonen, L. (2002) Trends Biochem. Sci. 27, 619-627[CrossRef][Medline] [Order article via Infotrieve]
25. Hepworth, S. R., Friesen, H., and Segall, J. (1998) Mol. Cell. Biol. 18, 5750-5761[Abstract/Free Full Text]
26. Bowdish, K. S., Yuan, H. E., and Mitchell, A. P. (1995) Mol. Cell. Biol. 15, 2955-2961[Abstract]
27. Rubin-Bejerano, I., Mandel, S., Robzyk, K., and Kassir, Y. (1996) Mol. Cell. Biol. 16, 2518-2526[Abstract]
28. Steber, C. M., and Esposito, R. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12490-12494[Abstract]
29. Washburn, B. K., and Esposito, R. E. (2001) Mol. Cell. Biol. 21, 2057-2069291[Abstract/Free Full Text]
30. Vidal, M., and Gaber, R. F. (1991) Mol. Cell. Biol. 11, 6317-6327[Medline] [Order article via Infotrieve]
31. Vidal, M., Strich, R., Esposito, R. E., and Gaber, R. F. (1991) Mol. Cell. Biol. 11, 6306-6316[Medline] [Order article via Infotrieve]
32. Kasten, M. M., Dorland, S., and Stillman, D. J. (1997) Mol. Cell. Biol. 17, 4852-4858[Abstract]
33. Xiao, Y., and Mitchell, A. P. (2000) Mol. Cell. Biol. 20, 5447-5453[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.