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Vol. 14, Issue 8, 3230-3241, August 2003
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Departments of Cell Biology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Submitted March 6, 2003;
Revised April 9, 2003;
Accepted April 9, 2003
Monitoring Editor: Mark Solomon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The preferred carbon source for yeast, as for most cells, is glucose.
Introduction of glucose to growth medium leads to the rapid repression of
genes that are nonessential for its utilization and the induction of genes
that facilitate its uptake and metabolism. Among the many genes induced by
glucose is a family of hexose transporters encoded by the HXT genes
(Gancedo, 1998
;
Özcan and Johnston, 1999;
Van Belle and André,
2001). The HXT family consists of 17 genes encoding
proteins that are closely related but subject to distinct patterns of
regulation by glucose. The best-characterized members of the family include
HXT1, which is induced in high but not low glucose; HXT2 and
HXT4, which are induced in low but not high glucose; and
HXT3, which is induced in both low and high glucose. These patterns
of expression correlate roughly with the affinity of the specific transporter
for glucose (Özcan and Johnston,
1999).
Glucose regulation of HXT gene expression is mediated via signals
emanating from the low- and high-affinity glucose receptors Snf3 and Rgt2,
respectively, both of which are closely related to members of the hexose
transporter family but have extended carboxyterminal cytoplasmic domains that
are required for signal transduction
(Özcan et al.,
1996a; Özcan et
al., 1998). Although relatively little is known about the
arrangement of downstream elements of that pathway, several elements required
for signaling have been characterized sufficiently to predict their
function.
First, repression of HXT gene expresssion in the absence of
glucose is known to require RGT1, which encodes a DNA binding protein
that recognizes elements in the HXT promoters
(Özcan et al.,
1996b). RGT1 is required for transcriptional repression
of HXT1-HXT4 in the absence of glucose
(Vallier et al.,
1994; Özcan et
al., 1996b). In contrast, the transcriptional repression of
HXT2 and HXT4 observed in high glucose is apparently
mediated via a separate mechanism involving MIG1
(Gancedo, 1998). In addition to
its capacity to act as a transcriptional repressor, there is evidence that
RGT1 can act as a transcriptional activator
(Özcan and Johnston,
1995; Özcan et
al., 1996b). Whether those effects are all mediated at the
level of the HXT promoters is not known.
Derepression of HXT gene expression in the presence of glucose
requires the F-box protein Grr1
(Özcan et al.,
1993; Vallier et al.,
1994; Özcan and Johnston,
1995). Inactivation of RGT1 bypasses the requirement for
GRR1 to induce HXT gene expression, thereby placing it
upstream of RGT1 in the glucose-signaling pathway. Because Grr1 is an
established component of a Skp1/Cullin/F-box protein (SCF) E3 ubiquitin ligase
complex and mediates the ubiquitination of proteins destined for proteolysis
via the proteasome (Skowyra et
al., 1997; Patton et
al., 1998), it has been proposed that Grr1 antagonizes Rgt1
by targeting it for degradation
(Özcan and Johnston,
1999). Consistent with that hypothesis, two other components of
that complex, Skp1 and Cdc53, have been shown to be required for
transcriptional activation in response to glucose
(Li and Johnston, 1997).
However, Cdc34, the E2 ubiquitin-conjugating enzyme required for the
SCF-mediated ubiquitination of established SCFGrr1 targets, seems
to be dispensable for HXT gene induction
(Li and Johnston, 1997).
Surprisingly, the protein motifs of Grr1 required for recognition of
established ubiquitination targets seem to be distinct from those required for
regulation of HXT gene expression, suggesting that the properties of
the targets involved in those two processes are distinct
(Hsiung et al.,
2001).
Two other genes, MTH1 and STD1, have been shown to be
important for maintenance of HXT gene repression
(Schmidt et al.,
1999; Schulte et al.,
2000). Whereas inactivation of either gene alone results in
limited defects in regulation of HXT gene expression, inactivation of
both genes results in derepression in the absence of glucose, suggesting a
partial functional overlap. This is consistent with the high degree of
sequence homology between the encoded proteins (Std1 and Mth1 are 61%
identical) (Hubbard et al.,
1994). Although reports are conflicting, Mth1 has been reported to
interact with the cytoplasmic domains of either one or both of the
membrane-bound glucose sensors, Snf3 and Rgt2
(Schmidt et al.,
1999; Lafuente et
al., 2000). The inference that MTH1 can interact
with HXT promoters has been derived from the analysis of mutant
alleles (Özcan et al.,
1993). These interactions are consistent with its reported
localization to both the cytoplasmic membrane and the nucleus
(Schmidt et al.,
1999), although it should be noted that the retention of those
proteins at the plasma membrane does not require Rgt2 or Snf3. Std1 has been
reported to interact biochemically with Rgt1
(Tomas-Cobos and Sanz, 2002)
as well as with Snf1, a protein kinase involved in global regulation of gene
expression in response to glucose (Hubbard
et al., 1994;
Tomas-Cobos and Sanz, 2002;
Kuchin et al., 2003).
However, despite the relatively extensive analysis of Mth1 and Std1, neither
their cellular functions nor their role in this pathway is understood.
To elucidate the mechanism of glucose regulation of HXT gene
expression, we investigated the role of Grr1 in that process. This study
confirms a recent report (Mosley et
al., 2003) that Rgt1 binds to HXT1-HXT4 promoters in
vivo under repressing conditions but dissociates from those promoters in the
presence of glucose. Dissociation of Rgt1 from these promoters is associated
with its hyperphosphorylation. Grr1 is required for both the
hyperphosphorylation of Rgt1 and its dissociation from promoters. However, we
show that Rgt1 is not a direct target for ubiquitination by
SCFGrr1. Instead, Grr1 is required to inactivate Mth1 and Std1 in
response to glucose. Mth1 inactivation seems to occur at the level of
degradation. Based upon these data, we conclude that glucose acts via Grr1 to
regulate the abundance of Mth1. Inactivation of Mth1 leads to
hyperphosphorylation of Rgt1 and dissociation from HXT promoters.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Deletion mutants were constructed using polymerase chain reaction
(PCR)-based methods as described previously
(Wach et al., 1994;
Longtine et al.,
1998; Goldstein and McCusker,
1999). Deletion mutants were confirmed by PCR. To generate
chromosomally carboxy-terminal epitope-tagged proteins, the 3' end was
amplified by PCR and cloned into the pKAN vector or one of its derivates
(Haase, Wolff, and Reed, unpublished data) for targeted integration or by the
PCR-based method as described previously
(Longtine et al.,
1998). DNA fragments were sequenced to ensure fidelity. Sequences
of all oligonucleotides used for these manipulations are available upon
request.
Protein and RNA Analysis
If not stated otherwise, cells were grown in 2% galactose to early
log-phase, half of the cells were then shifted to 4% glucose for 120 min,
after which cells were harvested by filtration. Cells were then washed in
ice-cold water and pellets were stored at [–80°C].
For reverse transcription (RT) analysis, RNA isolation, cDNA synthesis, and
PCR were performed as described previously
(Hsiung et al.,
2001). Sequences of primers are available upon request.
For Western blot analysis, protein extracts were prepared either by lyses in urea-buffer or a trichloroacetic acid (TCA)/urea extraction method. Urea-buffer extracts were prepared in urea-buffer (8 M urea, 200 mM NaCl, 10 mM Tris pH 7.5, 0.2% SDS, protease inhibitors [10 mM phenylmethylsulfonyl fluoride (PMSF), 2 mg/ml aprotenin, leupetin, and pepstatin A], phosphatase inhibitors [10 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, and 0.1 mM orthovanadate]). Cells were broken with glass beads 4x20 s in a FastPrep FP120 (BIO 101, Vista, CA/Savant Instruments, Holbrook, NY). Samples were diluted to 4 M urea before loading onto SDS-gels. For TCA-urea extraction cells were broken in 5–10 volumes of 20% TCA with glass beads 4 times 40 s in a FastPrep FP120. The TCA pellet was washed twice in acetone and then resuspended in extraction buffer (8 M urea, 4% SDS, protease inhibitors [10 mM PMSF, 2g/ml aprotenin, leupetin, and pepstatin A], phosphatase inhibitors [10 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, and 0.1 mM orthovanadate]). Samples were diluted to 4 M urea and 2% SDS before loading onto SDS-gels.
Chromatin Immunoprecipitation Assay
Cells were grown as described above. Chromatin immunoprecipitation assays
were performed by a protocol based on Tanaka et al.
(1997). Briefly, DNA-protein
cross-links were induced in vivo by incubation of cells with formaldehyde
(final concentration 1%) for 20 min at room temperature, followed by the
addition of glycine to a final concentration of 125 mM for 5 min at room
temperature. Cells were washed three times with ice-cold Tris-buffered saline
and cell pellets were frozen. Frozen cell pellets were resuspended to 1.5
x 109 cells/ml in lysis buffer (50 mM HEPES pH 7.5, 140 mM
NaCl, 1% Triton X-100, 0.1% Na deoxycholate, 50 µM PMSF, 2 µg/ml
aprotinin, leupeptin, and pepstatin A). Cells were broken with glass beads 4
x 20 s at setting 4.5 in a FastPrep FP120 (BIO 101/Savant). After a
15-min centrifugation the supernatant was discarded and the pellet (chromatin
fraction) was resuspended in the initial volume of lysis buffer. The DNA was
fragmented to 
500–1000 base pairs by sonication at half maximum
(low setting) with a Braun Sonic 2000. After clarification, Rgt1–3HA was
immunoprecipitated from an equivalent of 6.75 x 108 cells
with the monoclonal anti-HA antibody 12CA5 (ascites fluid) (a generous gift
from Ian Wilson, The Scripps Research Institute, La Jolla, CA) and protein A
beads for 4 h at 4°C. Immune complexes were washed twice with 1 ml of
lysis buffer, twice with 1 ml of lysis buffer with 250 mM NaCl, twice with 1
ml of wash buffer (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na
deoxycholate, 1 mM EDTA), and twice with 1 ml of Tris-EDTA. Protein–DNA
complexes were eluted with 50 µl of elution buffer (50 mM Tris pH 8.0, 10
mM EDTA, 1% SDS) and DNA-protein cross-linking was reversed in 1%
SDS/Tris-EDTA at 65°C overnight. DNA was purified on QIAquick PCR columns
(QIAGEN, Valencia, CA) according to the manufacturer's instructions. PCR
reactions (15 min 94°C, 27 times [50 s 94°C, 1 min 30 s 50°C, 2
min 72°C], 10 min 72°C) were performed using HotStartTaq Master Mix
kit (QIAGEN) on 1/6000 of the input (pellet fraction) and 1/60 of the
immunoprecipitation. Sequences of the primers are available upon request. PCR
fragments were separated on a 2.5% agarose gel and visualized by ethidium
bromide.
Analysis of Rgt1 Phosphorylation
To analyze the phosphorylation status of Rgt1 protein extracts were
prepared in radioimmunoprecipitation assay (RIPA) buffer (1% deoxycholic acid,
1% Triton X-100, 0.1% SDS, 250 mM NaCl, 50 mM Tris-HCl pH.7.5, 10 mM sodium
pyrophosphate, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 0.1 mM orthovanadate, 1 mM
PMSF, 2 µg/ml aprotinin, leupeptin, and pepstatin A), and Rgt-3HA was
immunoprecipitated from 2.5 mg of protein with 12CA5 ascites fluid. Immune
complexes were washed twice with 1 ml of RIPA buffer, twice with 1 ml of RIPA
buffer without phosphatase inhibitors, and once in 1 ml of 50 mM Tris-HCl pH
7.5, 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin. Immunopurified Rgt1
was split into three equal parts. One part was incubated in 100 µl of
phosphatase reaction mix (50 mM Tris-HCl pH 7.5, 5 mM dithiothreitol, 0.1 mM
EDTA, 0.01% Brij35, 2 mM MnCl2) without phosphatase. The two other
parts were incubated with phosphatase reaction mix and 1200 U of
lambda-phosphatase (New England Biolabs, Beverly, MA), but to one of them a
phosphatase inhibitor cocktail (final concentration: 10 mM sodium
pyrophosphate, 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 0.1 mM orthovanadate) was
added. The reactions were incubated at 30°C for 60 min, the immuncomplexes
washed with 300 µl of RIPA buffer and analyzed by Western blotting with
monoclonal antibodies directed against the hemagglutinin (HA)-epitope (12CA5
or 16B12) or the myc-epitope (9E10).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). HXT1 is maximally induced at high glucose and only
modestly induced in low glucose, whereas HXT3 is induced efficiently
at both low glucose concentrations. HXT4 induced in low glucose but
repressed in high glucose (Figure
1A).
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Association of Rgt1-HA with chromosomal DNA was analyzed in vivo by
chromatin immunoprecipitation. Cells were either grown in 2% galactose or
shifted from galactose to low glucose (0.2%) or high glucose (4%). Consistent
with a role as a transcriptional repressor, Rgt1-HA was found to be associated
with all three promoters in galactose-grown cells
(Figure 1B). No association
with any of the three HXT promoters was observed in cells growing on
4% glucose. Although, HXT4 is repressed under those conditions, that
repression is known to be independent of RGT1
(Özcan and Johnston,
1995; Özcan,
2002). Finally, Rgt1 was associated with both the HXT1
and HXT3 promoters in 0.2% glucose, which partially induces their
expression, but was undetectable at the HXT4 promoter in 0.2% glucose
where expression is fully induced. This suggests that Rgt1 is differentially
regulated at these promoters. Although it is possible that Rgt1 remains
associated with the promoters in a form that is undetectable by chromatin
immunoprecipitation, the simplest interpretation of these results is that Rgt1
dissociates from the HXT promoters under conditions where
transcription is induced. These data are most consistent with Rgt1 acting as a
transcriptional repressor at HXT promoters in the absence of glucose
and induction of HXT gene expression by glucose occurring as a
consequence of dissociation of Rgt1 from those promoters. Furthermore, the
finding that Rgt1 is absent from these promoters under inducing conditions
seems inconsistent with the proposal, based upon studies with HXT
promoter-reporter fusions and one hybrid analysis
(Özcan et al.,
1996b), that Rgt1 acts as a transcriptional activator at
HXT promoters (see DISCUSSION). Additional support for this
contention comes from our observation that HXT gene expression is
unaffected by inactivation of Rgt1 (Figure
2A).
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Grr1 Is Required for Phosphorylation of Rgt1 and Dissociation
from HXT Gene Promoters
Induction of HXT gene expression requires the F-box protein Grr1.
The defect in induction of HXT gene expression resulting from
inactivation of GRR1 is suppressed by inactivation of RGT1,
suggesting that Grr1 antagonizes the activity of Rgt1 as a repressor at
HXT gene promoters
(Marshall-Carlson et al.,
1991; Özcan and Johnston,
1995) (Figure 2A).
To evaluate whether Grr1 is required for the dissociation of Rgt1 from
HXT promoters in response to glucose, we performed chromatin
immunoprecipitation of Rgt1 in a grr1
mutant by using the
protocol outlined above. Consistent with previously published observations,
cells deficient in GRR1 failed to induce HXT1, HXT3, or
HXT4 in response to glucose
(Figure 2A). Chromatin
immunoprecipitation of Rgt1 from strains growing in galactose or shifted to 4%
glucose revealed that Rgt1 remains associated with promoter DNA of all three
HXT genes in the grr1 mutant cells regardless of the
presence or absence of glucose (Figure
2B). Glucose induces the dissociation of Rgt1 from those promoters
in wild-type cells. Based upon these observations, we conclude that Grr1 is
required for dissociation of Rgt1 from HXT promoters.
Grr1 has an established role in ubiquitin-mediated proteolysis and is also required for the glucose-induced loss of Rgt1 from HXT gene promoters. Therefore, we evaluated the status of the Rgt1 protein in cells growing on galactose and after induction with 0.2 or 4% glucose. Rgt1-HA is detected as a polypeptide with apparent molecular mass of 160 kDa based upon its migration of SDS-polyacrylamide gels, significantly greater than its predicted molecular mass of 132 kDa (Figure 3A). Furthermore, the Rgt1 polypeptide migrates with a progressively lower mobility when cells are grown on increasing concentrations of glucose, suggesting that Rgt1 becomes covalently modified upon exposure to glucose. Further analysis of Rgt1 provided no evidence that the protein was ubiquitinated (our unpublished data). In contrast, Rgt1 was phosphorylated in response to glucose. When Rgt1-HA immunoprecipitated from glucose-grown cells was treated with lambda phosphatase a substantial increase in mobility was observed. The mobility of the phosphatase-treated protein was similar to untreated Rgt1-HA from galactose-grown cells (Figure 3B). An increase in mobility was not observed if phosphatase inhibitors were added along with the phosphatase. Phosphatase treatment of Rgt1 from galactose-grown cells resulted in a small, but reproducible, increase in mobility. We conclude that Rgt1 is phosphorylated under both inducing and noninducing conditions. However, the phosphorylation observed in the presence of glucose is more extensive. Thus, hyperphosphorylation of Rgt1 is associated with induction of HXT gene expression and dissociation of Rgt1 from HXT gene promoters.
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Grr1 has no known role in protein phosphorylation. Analysis of Rgt1
phosphorylation in grr1 mutants revealed that GRR1 is
required for induction of hyperphosphorylation of Rgt1 in the presence of
glucose (Figure 3C) and for
dissociation from HXT gene promoters. Although it is not clear
whether Rgt1-HA from grr1 mutants is phosphorylated, its
mobility is equivalent to phosphatase-treated Rgt1-HA. Thus, it seems that
Grr1 is required not only for the hyperphosphorylation of Rgt1 associated with
its dissociation from HXT promoters but also for the phosphorylation
observed under noninducing conditions.
Grr1 Regulation of Rgt1 Phosphorylation and Promoter Binding and
of HXT Gene Induction Is Mediated via Mth1 and Std1
The role of Grr1 in the regulation of HXT gene expression seems to
be mediated via phosphorylation of Rgt1 and dissociation from HXT
promoters. In the interest of identifying the direct target of Grr1 in this
process, we have evaluated the regulation of Rgt1 phosphorylation in cells
carrying deficiencies in protein kinases with known roles in the specific
regulation of HXT gene expression or with general roles in the
regulation of carbohydrate metabolism. These included, but were not limited to
SKS1 (Yang and Bisson,
1996; Vagnoli and Bisson,
1998), its close homolog VHS1 (YDR247W), SNF1
(Hardie et al., 1998),
and TPK1, TPK2 and TPK3
(Toda and Sass, 1988). None of
the kinases analyzed exhibited a substantial defect in the glucose-induced
phosphorylation of Rgt1 (our unpublished data). Inactivation of Reg1, a
regulatory subunit of the Glc7 protein phosphatase implicated in the
regulation of HXT gene expression
(Özcan and Johnston,
1995), resulted in a modest effect on Rgt1 phosphorylation.
However, the magnitude of its effect on phosphorylation or HXT gene
expression seemed insufficient to explain the defect observed in
grr1 mutants (our unpublished data). Thus, it is unlikely that
Reg1 is the primary target of Grr1 for this regulation. However, it is
possible that Grr1 simultaneously targets more than one of these proteins for
inactivation in response to glucose.
A number of observations implicate two closely related proteins, Mth1 and
Std1, in the glucose-dependent regulation of HXT gene expression.
First, in agreement with previous studies with HXT
promoter-lacZ reporter fusions
(Schmidt et al.,
1999), we find that inactivation of either MTH1 or
STD1 alone causes defects in repression of HXT gene
expression on nonglucose carbon sources
(Figure 4A). This is most
noticeable in the analysis of HXT3, which is fully derepressed in
mth1 mutants and partially derepressed in std1
mutants. HXT4 is modestly affected by mth1 and
HXT1 is largely unaffected by either of the single mutations.
However, inactivation of both MTH1 and STD1 results in a
dramatic derepression of all three genes under the same conditions
(Schmidt et al.,
1999; Figure 4A).
Next, the Mth1 protein is absent from cells growing on glucose. Finally, both
Mth1 and Std1 can interact with Rgt2/Snf3
(Schmidt et al.,
1999) and Std1 can bind to Rgt1
(Tomas-Cobos and Sanz, 2002).
Together, these observations led us to evaluate the role of Mth1 and Std1 in
regulation of Rgt1 phosphorylation and promoter binding and as potential
targets of Grr1.
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To determine whether Mth1 and Std1 play a role in the regulation of
HXT gene expression at the level of Rgt1, we evaluated Rgt1-HA
binding to the HXT promoters in both single mth1 and
std1 mutants and in cells carrying disruptions of both genes
(Figure 4B). Rgt1 was found to
associate at wild-type levels with all three HXT promoters in
std1 mutants growing in galactose. In contrast,
mth1 mutants exhibited substantially reduced binding at all
three promoters. Binding to HXT3 was most dramatically affected
consistent with the substantial defect in regulation of that gene in
mth1 mutants. However, glucose was found to reduce the
association of Rgt1 with all three promoters, indicating that binding was
still subject to regulation in the absence of Mth1. Strikingly, binding of
Rgt1 to each of the three promoters was fully disrupted in the
mth1 std1 mutants. Thus, the derepression of
HXT gene expression observed in those mutants is associated with a
defect in promoter binding by Rgt1.
Analysis of the Rgt1 protein in these mutants revealed that the protein
seems hypophosphorylated on galactose and hyperphosphorylated on glucose in
both mth1 and std1 single mutants
(Figure 5C). Consistent with
that observation Rgt1 retains the capacity to interact with each of those
promoters when hypophosphorylated. However, the extent of that binding is
decreased in the mth1 consistent with the derepression of
HXT3 observed under those conditions
(Figure 4A). Furthermore, Rgt1
is lost from all of those promoters in the presence of glucose, which induces
expression of the respective genes. Finally, consistent with the induction of
expression observed in galactose-grown cells, the Rgt1 protein was found to be
hyperphosphorylated in mth1 std1 mutants under
both repressing and nonrepressing conditions
(Figure 4C). Together, these
data suggest that Std1 is more important for repressing HXT1
expression, whereas Mth1 is a more important for HXT3 and, perhaps,
HXT4, a finding in general agreement with previously reported
observations (Tomas-Cobos and Sanz,
2002).
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We reasoned that if Mth1 and Std1 were targets for inactivation by Grr1,
then inactivation of those genes should bypass the requirement for Grr1 for
induction of HXT gene expression. We therefore performed an analysis
of HXT gene expression, Rgt1 promoter binding, and Rgt1
phosphorylation status with mth1 and std1
mutant cells in which GRR1 had also been inactivated
(Figure 5). That analysis
demonstrates that the defect in glucose induction of HXT gene
expression caused by grr1 is fully suppressed in the
mth1 std1 double mutant
(Figure 5A). That suppression
is associated with the persistent hyperphosphorylation of Rgt1
(Figure 5C) and its failure to
associate with HXT promoters
(Figure 5B). Furthermore,
inactivation of Grr1 fails to restore Rgt1 binding to HXT promoters
in mth1 std1 mutants
(Figure 5B). Based upon these
observations, we conclude that Grr1 is required for inactivation of Mth1 and
perhaps Std1. It is the inactivation of those proteins that leads to the
phosphorylation of Rgt1, its dissociation from HXT promoters, and, as
a consequence, HXT gene expression.
To achieve a more precise understanding of the role of Grr1 in the
regulation of individual targets, we evaluated HXT expression and
Rgt1 in cells in which either Mth1 or Std1 was inactivated along with Grr1.
Inactivation of Grr1 in the std1 mutant enhanced Rgt1 binding
to HXT promoters, blocked phosphorylation of Rgt1, and suppressed
HXT gene expression (Figure 5,
D–F). In contrast, inactivated of Grr1 in
mth1 mutants failed to block the dissociation of Rgt1 from
HXT promoters or to suppress Rgt1 phosphorylation
(Figure 5, E and F).
Furthermore, grr1 had little effect on the derepression of
HXT3 and HXT4 gene expression caused by mth1
(Figure 5D). We suspect that
HXT1 is regulated by STD1 in the absence of Mth1. This leads
us to conclude that Grr1 is essential for the inactivation of Mth1 but that
Std1 can be inactivated by an independent mechanism.
Loss of Mth1 in Response to Glucose Requires Grr1
The finding that inactivation of MTH1 and STD1 bypassed
the requirement for GRR1 in the regulation of HXT gene expression
prompted us to evaluate the role of Grr1 in the regulation of those genes and
their protein products. We first determined the relative abundance of
MTH1 and STD1 mRNA and protein in cells growing in galactose
or shifted into glucose. In agreement with published observations
(Schmidt et al.,
1999), STD1 mRNA
(Figure 6A) and protein
(Figure 6B) were found to be
unaffected by carbon source. Mth1 protein, which was detectable in
galactose-grown cells, was lost when cells were analyzed after addition of
glucose (Figure 6B). However,
we found that MTH1 mRNA was only modestly affected by glucose
(Figure 6A; our unpublished
data), suggesting that the loss of the Mth1 protein is posttranslational.
MTH1 RNA was previously shown to be undetectable during continuous
growth on glucose (Schmidt et
al., 1999). Because it was possible that this difference was
a consequence of differences in the culture conditions, we analyzed the
accumulation of MTH1 RNA during continuous growth on glucose or
galactose (Figure 6A, bottom).
Our data show that although MTH1 RNA accumulation is diminished on
glucose it remains detectable.
|
Whereas inactivation of GRR1 had little effect on the abundance of
MTH1 RNA (Figure 6A)
in cells growing on either carbon source, it had a substantial effect on the
abundance of the Mth1 protein (Figure
6B). The abundance of the Mth1 protein from galactose-grown
grr1 mutants was significantly increased over that from
wild-type cells and it persisted after a shift to glucose for 2 h. Both of
these effects are consistent with a role for Grr1 in determining the stability
of the Mth1 protein.
Because the known role of Grr1 in the recognition of proteins destined for
degradation via the ubiquitin proteasome pathway, we evaluated the kinetics of
loss of Mth1 in cells shifted from galactose into glucose. Cells growing in
galactose were either maintained in that carbon source or shifted to glucose
and samples taken each 5 min over a 30-min time course
(Figure 6C). Mth1 was rapidly
lost from the glucose-, but not the galactose-grown culture. Thus, it seems
that although the MTH1 mRNA persists in the presence of glucose
(Figure 6A), the protein is
rapidly depleted. We repeated this experiment analyzing MTH1 RNA and
protein each 15 min over a time course of 2 h
(Figure 6D). Although a
decrease in MTH1 RNA was observed after 30 min in that experiment,
the Mth1 protein was undetectable within 15 min (and probably less).
Consistent with the finding that GRR1 is required for that
instability, parallel analysis of Mth1 abundance in grr1
mutants revealed that Mth1 persists even in the presence of glucose for the
duration of the experiment (Figure
6D). The behavior of the Mth1 protein is consistent with Grr1
playing a direct role in Mth1 destabilization in response to glucose by acting
as an adaptor for substrate recognition by the SCF E3 ubiquitin ligase.
In contrast to Mth1, both STD1 RNA
(Figure 6A) and protein
(Figure 6B) undergo a
noticeable decrease in abundance under both inducing and repressing conditions
in the grr1 cells. Although the relevance of this decrease is
unclear, these data suggest that, if Std1 is regulated by glucose, the
regulation occurs via a mechanism that does not involve the regulation of
protein abundance. Furthermore, the mechanism is likely to be largely
independent of Grr1.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
This study demonstrates that glucose acts via Grr1 to block the function of
Mth1 and, perhaps, Std1 as negative regulators of HXT transcription,
thereby inducing Rgt1 phosphorylation and dissociation from HXT
promoters. One of those proteins, Mth1, is regulated at the level of its
abundance via a mechanism involving Grr1.
We show herein that glucose regulates Rgt1 at the level of protein
phosphorylation and promoter binding. A similar observation was recently
reported (Mosley et al, 2003).
Dissociation of Rgt1 from HXT promoters is tightly correlated with
its hyperphosphorylation and with activation of the HXT
transcription. Furthermore, glucose induction of HXT transcripts is
unaffected in cells lacking Rgt1. This seems inconsistent with the proposal
that Rgt1 acts as a transactivator at HXT promoters
(Özcan et al.,
1996b). That model is based, in part, upon the observation that
Rgt1 can act as a transcriptional activator when tethered to a heterologous
promoter via LexA. Our data suggests instead that Rgt1 is absent from
transcriptionally active HXT promoters arguing that if Rgt1 plays a
role in the activation of HXT genes, it is indirect.
It was anticipated, based upon the role of Grr1 in the ubiquitin-proteasome
pathway, that Rgt1 would be a target for ubiquitin-mediated degradation.
Surprisingly, inactivation of Grr1 does not substantially affect the abundance
of Rgt1 nor is Rgt1 ubiquitinated in response to glucose. Instead, we find
that Rgt1 is phosphorylated in response to glucose in Grr1-dependent manner
consistent with the recent report by Özcan and colleagues
(Mosley et al.,
2003). Furthermore, our genetic and biochemical analysis places
Mth1 and Std1 upstream of Rgt1 and downstream of Grr1. Consistent with its
role as a negative regulator of HXT gene expression, we find that in
the absence of glucose Mth1 and, to a lesser extent, Std1 are required to
maintain Rgt1 in the hypophosphorylated state, and, consequently, for its
association with the HXT promoters. Finally, a role for Grr1 in that
process is supported by the finding that inactivation of these genes relieves
the requirement for Grr1 for HXT gene expression.
The rapid Grr1-dependent loss of Mth1 suggests it as a likely target for
ubiquitin-dependent degradation. Yet, there remains significant ambiguity both
in the literature and in our own analysis regarding a role for ubiquitination
in that context. Although Skp1 and Cdc53 are clearly important for
derepression induced by glucose (Li and
Johnston, 1997), studies of thermosensitive mutants of Cdc34, the
E2 ubiquitin-conjugating enzyme responsible for all know ubiquitination
involving SCFGrr1, suggest that it is not required
(Li and Johnston, 1997; our
unpublished data). Another E2 enzyme, Ubc8, has been implicated in regulation
of fructose-1,6-bisphosphatase (Fbp1) stability by glucose
(Schule et al.,
2000). Like HXT gene expression, Fbp1 proteolysis depends
upon Grr1 (Horak et al.,
2002). However, the two processes are distinguishable based upon
the strong dependence of Fbp1 proteolysis upon Reg1 and its lack of dependence
upon Rgt2 and Snf3. Although no role for Ubc8 in SCF-dependent ubiquitination
has been described, it remains a potential collaborator with
SCFGrr1 in regulation of HXT gene expression and Mth1
stability. However, it remains to be established that the role of Grr1 in
regulation of Mth1 abundance involves protein ubiquitination.
Std1 is both structurally and functionally related to Mth1
(Hubbard et al.,
1994). Both proteins have been shown to associate with the
cytoplasmic domains of one or both hexose receptors, Rgt2 or Snf3, as well as
with Rgt1 (Schmidt et al.,
1999; Lafuente et
al., 2000). Although inactivation of Mth1 is sufficient to
derepress HXT2, HXT3, and HXT4, both Mth1 and Std1 must be
inactivated for full induction of Rgt1 hyperphoshorylation and HXT
gene expression in the absence of glucose (Figures
4 and
5;
Schmidt et al.,
1999). Furthermore, both genes must be eliminated to bypass the
requirement for Grr1 in those processes. Consequently, we can conclude that
Grr1 is somehow involved in the regulation of Std1. However, whereas the
behavior of Mth1 in response to glucose and in grr1 mutants
argues in favor of a role for Grr1 in Mth1 turnover, Std1 is not similarly
regulated. Rather, the level of the Std1 protein seems to be unaffected by
glucose (Figure 6;
Schmidt et al.,
1999). Unlike Mth1, both STD1 RNA and protein seem to
decrease in grr1 mutants. Finally, although inactivation of Grr1 has
little or no effect on Rgt1 modification or HXT gene expression in
cells lacking Mth1, it has a dramatic effect on those phenotypes in cells
lacking Std1, suggesting that Grr1 is primarily involved in the regulation of
Mth1. Together, these results argue that Mth1 is the primary target of Grr1
for regulation of HXT gene expression by glucose.
Std1 associates with Snf1, a protein kinase involved in global regulation
by glucose via a pathway independent of Snf3 and Rgt2
(Hubbard et al.,
1994; Tomas-Cobos and Sanz,
2002; Kuchin et al.,
2003). Furthermore, Std1 seems to act as a positive regulator of
Snf1 (Kuchin et al.,
2003). However, it is not clear whether this association is
relevant to the regulation of HXT gene expression by glucose.
Although Snf1 may be responsible for a portion of the regulation of Rgt1, it
is not essential for glucose-inducible phosphorylation of Rgt1 (our
unpublished results) nor for induction of HXT gene expression by
glucose (Özcan and Johnston,
1995). Consequently, it seems likely that there are multiple
signal transduction pathways involved in glucose regulation of the
HXT genes that are, at least in part, independently regulated. The
distinction between these pathways may account, in part, for the differences
in the behavior of the HXT genes in response to different levels of
glucose.
Based upon our analysis of the role of Grr1 in the regulation of
HXT gene expression and observations of others, we propose the model
presented in Figure 7. We
suggest that repression of HXT gene expression occurs via Rgt1
binding to HXT gene promoters. However, maintenance of repression
depends upon Mth1 and Std1 perhaps via a direct interaction, consistent with
the observed interaction between Std1 and Rgt1
(Tomas-Cobos and Sanz, 2002).
The extent to which these regulators affect expression varies between the
different HXT genes, Std1 having its predominant effect on
HXT1 and Mth1 having a more pronounced effect on HXT3 and
HXT4 (and probably HXT2). On exposure to glucose multiple
signaling pathways are activated, a primary pathway for HXT
regulation involving the glucose receptors, Rgt2 and Snf3, and a second, as
yet undefined, pathway. Our data suggest that Rgt2/Snf3 signaling occurs
primarily by elimination of the Mth1 protein via a Grr1-dependent mechanism.
Unlike Mth1, inactivation of Std1 seems to be largely Grr1-independent. That
pathway may involve Snf1, a global regulator of glucose repression, consistent
with the capacity of those proteins to form a complex
(Hubbard et al.,
1994; Tomas-Cobos and Sanz,
2002; Kuchin et al.,
2003). Inactivation of Mth1 and Std1 may occur as a consequence of
their retention in the cytoplasm by the Rgt2/Snf3 receptor proteins. Both
proteins have been reported to interact with the cytoplasmic tails of these
transmembrane receptors via two-hybrid analysis
(Schmidt et al.,
1999; Lafuente et
al., 2000), although the specific conditions under which
those interactions occur is not known. This would require cycling of these
proteins between the nucleus and the cytoplasm. Finally, we propose that the
inactivation of Mth1 and Std1 leads to the phosphorylation of Rgt1 by an as
yet unidentified protein kinase. Phosphorylation leads to dissociation of Rgt1
from HXT promoters, thereby activating HXT gene expression.
Clearly, many of the details of this model remain to be established.
|
Grr1 plays an important, but as yet undescribed role in a number of
nutrient-regulated transcription systems. The targets of the SPS signaling
system required for the response of cells to extracellular amino acids
(Forsberg and Ljungdahl, 2001)
and the Rgt1/Snf3 sensor system for regulation of hexose permeases are among
the best studied. There are similarities between these signal transduction
systems in addition to the involvement of Grr1. Notably, both use members of a
family of membrane-bound sensors related to the permeases that they regulate
(Van Belle and André,
2001) and both exert transcriptional repression via the Ssn6/Tup1
transcriptional corepressor (Özcan
and Johnston, 1999;
Andrèasson and Ljungdahl,
2002). Like HXT gene regulation, activation of the
targets of SPS (Ssy1-Ptr3-Ssy5) signaling
seems to involve SCF components (Iraqui
et al., 1999) but is unaffected by mutations that affect
binding of targets for phosphorylation-dependent ubiquitination
(Hsiung et al.,
2001). However, beyond Grr1 the analogy between elements of the
signal transduction systems conveying signals to the nucleus from the cell
membrane remains unclear. Strikingly, the transcriptional activation in the
SPS system involves proteolytic processing of two transcriptional regulators,
Stp1 and Stp2 (Andrèasson and
Ljungdahl, 2002). However, processing of those proteins does not
seem to involve Grr1 nor have regulators analogous to Mth1 and Std1 been
identified in that system (Özcan and
Johnston, 1999). Consequently, it is difficult, based upon analogy
to the HXT system, to predict the role of Grr1 in SPS signaling.
However, it remains possible that the mechanism by which Grr1 regulates Mth1
is conserved between these pathways or that a single process mediated by Grr1
leads to the inactivation of Mth1 along with elements of those other
pathways.
The diverse roles of F-box proteins in cellular regulation are only
beginning to be fully appreciated. Activation of the transcriptional regulator
nuclear factor-
B, which has been known to involve ubiquitination target
for many years, is now known to occur via SCF-mediated
phosphorylation-dependent ubiquitination
(Brivanlou and Darnell, 2002).
Over the past several years, a number of other ubiquitin-dependent mechanisms
for gene-specific, as well as general, transcriptional regulation have begun
to be elucidated (Hoppe et al.,
2001; Brivanlou and Darnell,
2002). More sophisticated understanding of these regulatory
networks will undoubtedly reveal a tightly regulated and highly integrated
system of ubiquitination-dependent events. Grr1, as an F-box protein involved
in a broad range of cellular processes including cell cycle regulation,
morphogenesis, and transcriptional control provides an excellent subject for
such studies.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
* Present address: Department of Biological Chemistry, University of
California, Irvine, Irvine, CA 92697 
Present address: Department of Radiology, The Ohio State University,
Columbus, OH 43210
Present address: Department of Ophthalmology, University of Minnesota,
Minneapolis, MN 55455.
Corresponding author. E-mail address:
curtw{at}scripps.edu.
|
REFERENCES |
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