Department of Biochemistry and Molecular
Biology, Louisiana State University Health Sciences Center,
Shreveport, Louisiana,1 and Department
of Biochemistry and Molecular Biology and the Walther Oncology
Center, Indiana University School of Medicine, Indianapolis,
Indiana2
Received 28 April 2000/Returned for modification 8 June
2000/Accepted 14 August 2000
Ubiquitin-mediated degradation plays a crucial role in many
fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin
ligase complexes, called SCF complexes, found throughout eukaryotes, is
involved in a variety of biological pathways. In Saccharomyces
cerevisiae, an SCF complex contains a common set of components,
namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a
variable component called an F-box protein. The F- box is a
~40-amino-acid motif that allows the F-box protein to bind Skp1p.
Each SCF complex recognizes different substrates according to which
F-box protein is associated with the complex. In yeasts, three SCF
complexes have been demonstrated to associate with the
ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity.
F-box proteins are not abundant and are unstable. As part of the
SCFMet30p complex, the F-box protein Met30p represses
methionine biosynthetic gene expression when availability of
L-methionine is high. Here we demonstrate that in vivo
SCFMet30p complex activity can be regulated by the
abundance of Met30p. Furthermore, we provide evidence that Met30p
abundance is regulated by the availability of L-methionine.
We propose that the cellular responses mediated by an SCF complex are
directly regulated by environmental conditions through the control of
F-box protein stability.
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INTRODUCTION |
Protein degradation is an essential
regulatory mechanism used by the cell in many fundamental processes,
including response to changing environmental conditions. In eukaryotes,
a major proteolytic mechanism is the ubiquitin (Ub)-proteasome pathway
(10, 12). Ub is a member of a family of conserved
polypeptides that are covalently attached to protein substrates.
Reiteration of Ub modification creates a poly-Ub chain on the substrate
that is then targeted for degradation by the proteasome, a large
multiprotein complex protease. The transfer of Ub to a protein
substrate is a multistep process requiring at least three proteins.
Free Ub is activated at the expense of ATP by a Ub-activating enzyme,
or E1. Activated Ub is then transferred to a Ub-conjugating enzyme, or
E2. Ub-ligases, or E3s, facilitate the transfer of Ub from E2 to
protein substrate. Some E3s act as intermediary Ub carriers in the
transfer of Ub from E2 to substrate (35), while other E3s
act as adapters tethering the E2 to its substrate (6). A
multiprotein complex called the SCF (named after the original
components Skp1p, Cdc53p, and F-box protein) complex is represented in
all eukaryotic taxa and has recently emerged as a major family of Ub
ligases (6, 28, 42).
In budding yeast, SCF complexes are comprised of a common set of
components, namely, Cdc53p (or cullin), Skp1p, and Hrt1p (or Rbx1p or
Roc1p) (for reviews, see references 5, 6, 28, and
42). Each SCF complex also contains an F-box
protein, which is responsible for substrate recognition (8,
37). The F box, a ~40-amino-acid motif, is believed to link the
F-box protein with the common SCF components by binding Skp1p. Although
several F-box proteins exist in the cell, only one F-box protein is
present within any one SCF complex (29). Thus, a family of
complexes that are distinguished by the F-box protein exists. The
Ub-conjugating enzyme Cdc34p is associated with several SCF complexes
and is necessary for SCF-dependent ubiquitination (8, 17, 23, 29,
36, 37, 43).
Three yeast F-box proteins, Cdc4p, Grr1p, and Met30p, are present
within SCF complexes, referred to as SCFCdc4p,
SCFGrr1p, and SCFMet30p, respectively,
and have roles in Cdc34p-mediated protein degradation events
(6, 42). An emerging feature of these three SCF Ub ligases
is their dual role in mediating cell cycle progression and metabolism.
SCFCdc4p is required for entry into S phase by degrading
the cyclin-dependent kinase-inhibitory protein Sic1p. Additionally,
SCFCdc4p controls the biosynthesis of amino acids and
purines (11, 13) by regulating the abundance of the
transcription factor Gcn4p (25). Grr1p regulates
G1 cyclin abundance (2) in addition to playing
roles in heavy metal resistance, amino acid transport, cell growth, and
glucose repression (4, 9, 14, 15, 20). SCFMet30p
regulates methionine biosynthetic gene expression in addition to
playing a positive role in cell cycle progression (16, 29, 30,
40).
A well-described role for SCFMet30p is its regulation of
methionine biosynthesis (29, 32). The MET genes
encode enzymes and transporters necessary for the uptake of inorganic
sulfur and its assimilation into methionine and cysteine
(39). A negative feedback loop regulates MET gene
expression: cells supplied with L-methionine repress
MET gene expression, whereas depression occurs in media
lacking L-methionine. The transcription factor Met4p positively regulates methionine biosynthesis. Recently, Met4p was
proposed to be a substrate for the SCFMet30p Ub ligase
complex (32). How SCFMet30p activity is
regulated by L-methionine is unknown. One means of regulating the activity of some SCF complexes is at the level of
substrate recognition. There are well-characterized examples of
substrates being phosphorylated prior to their recognition by an SCF
complex (42). However, Met4p modification has not been
attributed to phosphorylation, and thus, SCFMet30p complex
activity may be regulated by an alternate mechanism.
Here we show that increased Met30p abundance positively regulates
SCFMet30p activity. Additionally, we report that
Met30p abundance is regulated by the availability of
L-methionine. Therefore, SCFMet30p complex
activity is, at least partially, regulated at the level of Met30p
abundance. Finally, the F-box region of Met30p plays a critical role in
regulating Met30p stability in a methionine-dependent manner,
suggesting that this sequence may have crucial regulatory roles in
addition to linking the F-box protein with the SCF complex.
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MATERIALS AND METHODS |
Yeast strains and manipulations.
The yeast
(Saccharomyces cerevisiae) strains used in this study are
Y382 MAT
ade2 ade3 ura3 leu2 trp1 (kindly provided
by A. Bender), PY283 MATa met30-6 bar1
ura3 ade1 his2 leu2-3,112 trp1-1 (kindly provided by Steve Reed
[16]), YPH1172 MATa ura3-52
trp1-63 his3-200 leu2-1 ade2-101 skp1::TRP
skp1-3::LEU2 (kindly provided by Phil Hieter), C114
MAT ura3 leu2::MET25-lacZ::LEU2 (kindly provided by Yolande Surdin-Kerjan), NMmet30 MAT
ura3-52 trp1-1 his3-200 leu2-3,112 met30::HIS3
(this study), NMMET30/met30 MATa/
ura3-52/ura3-52 leu2/leu2 his3/his3 trp1-1/trp1-1 MET30/met30::HIS3 (this study), and
NMmet30MET25-lacZ MATa ura3-52 his3-200 trp1-1
met30::HIS3
leu2::MET25-lacZ::LEU2 (this study).
NMmet30 was created as follows. A heterozygous met30
disruption strain, NMMET30met30 (MET30
met30::HIS3), was transformed with pGST Met30-3.
Transformants were sporulated, and asci were dissected onto yeast
extract-peptone-dextrose medium. Ura+ and His+
colonies were identified, one of which we named NMmet30.
NMmet30MET25-lacZ is a meiotic product of a cross between C114 and
NMmet30. Standard rich (YPD) and defined minimal SD media were
prepared as described previously (31). Transformations were
carried out as described previously (7). For plasmid
selection, yeast cells were grown on defined minimal medium
supplemented with the appropriate amino acids. For galactose induction,
cells were first grown to early logarithmic phase in minimal medium
containing sucrose instead of dextrose and then galactose-containing
medium (2%) was added and the culture was incubated for a further
3 h. To measure protein half-lives, glutathione
S-transferase (GST)-Met30p fusions were transiently induced
from the GAL1 promoter for 3 h. Glucose (2%) and
cycloheximide (1 mg/ml) were added to repress transcription and
translation, respectively. For complementation experiments, patches
derived from single colonies were grown under permissive conditions
(23°C) and then replica plated and incubated further under
nonpermissive conditions of 37°C.
Flow cytometry.
Y382 transformants containing pGSTMET30-3
were grown to a density of 5 × 106 cells/ml in
sucrose-containing medium that lacked methionine. Galactose was added,
and the culture was incubated further until cells had become arrested,
which occurred within two divisions. Cells were harvested, sonicated,
fixed in 70% ethanol, and stored at 4°C. To prepare for flow
cytometry, cells were washed once in 10 mM Tris (pH 7.4)-15 mM NaCl
and resuspended in the same buffer containing 0.1 mg of RNase A (Roche
Molecular Biochemicals) per ml for 1.5 h. The cells were then
harvested and resuspended in phosphate-buffered saline at
106 cells/ml, and propidium iodide was added to a final
concentration of 40 µg/ml. Flow cytometry was performed using
FACSvantage (Becton Dickinson, Mountain View, Calif.) with an
excitation wavelength of 488 nm and monitoring of emission in the f12
channel. Data were collected in the four-parameter list mode of 20,000 cells/run.
Plasmid constructions.
Escherichia coli DH5
was used to propagate plasmids. Plasmid manipulations used standard
protocols (34). The vector pEG(KG) was used for the
expression of GST fusion proteins (26). Expression of the
GST fusion proteins was from the GAL1 promoter. All
GST-MET30 fusion constructs were created by cloning
PCR-generated DNA fragments using plasmid-borne MET30 DNA as
template (pP58, kindly provided by Steve Reed) as described previously
(21). Full-length MET30 was generated using
primers 5'-TTTTCTAGACATGAGGAGAGAGAGGCAAAGG-3' and
5'-CCCGTCGACCTAATCATTGAGATCGAATTTG-3'. The primer annealing to the 3' end of MET30 incorporated an XbaI
restriction site, and the primer annealing to the 5' end of
MET30 incorporated a SalI restriction site. The
PCR product was restricted with XbaI and SalI and
ligated into pEG(KG) that had been restricted with the same enzyme to
yield plasmid pGSTMet30-3. To generate MET30(185-277), a PCR
fragment was amplified from primers
5'-CCCTCTAGACATCTACAGAGAACGGTTCAAAG-3' and
5'-CCCGTCGACCTAATCATTGAGATCGAATTTG-3'. This fragment
contained XbaI and SalI restriction enzyme sites
and was ligated into pEG(KT) digested with the same enzymes. This
yielded a plasmid, pGSTMET30-6, which contained a DNA fragment encoding
Met30p residues 277 to 640. A second PCR fragment was amplified using
primers 5'-AAAAACCCGGGAATGAGGAGAGAGAGGCAAAGG-3' and
5'-GGGGTCTAGAATGCTGATGAAGTCGATCTTGATC-3' which incorporated SmaI and XbaI and was ligated into pGSTMET30-6
restricted with the same enzymes yielding plasmid
pGSTMET30(185-277). MT839 encoding hemagglutinin (HA)-tagged
CDC53 was provided by Mike Tyers.
Protein preparation and Western immunoblotting techniques.
Yeast lysate preparations and Western immunoblotting were carried out
as described previously (22). Data from Western blots detected with ECL or ECL+ were exposed to film or imaged with a Storm
Phosphorimager (Amersham Pharmacia Biotech, Piscataway, N.J.) in the
blue fluorescence mode. Quantification of Western blot data used Image
Quant image analysis software according to the manufacturer's
instructions. Antibodies raised against GST were purchased from Sigma,
and antibodies raised against the HA epitope were purchased from Roche
Molecular Biochemicals. Antibodies raised against Cdc34p have been
previously described (22).
Assay for MET25-lacZ enzyme activity.
-Galactosidase activity was measured as described previously
(45). For repressing conditions, cultures were grown in the presence of 2 mM L-methionine, whereas for nonrepressing
conditions, cultures were grown in the absence of
L-methionine. Cultures were grown to 106 to
107 cells/ml in sucrose, filtered, and resuspended in
medium containing galactose to induce production of GST or GST-Met30p
fusion proteins. Values reported here are the averages from at least
two independent assays. -Galactosidase activities were expressed as
nanomoles of
o-nitrophenyl--D-galactopyranoside hydrolyzed
per minute per milligram of protein.
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RESULTS |
Characterization of GST-Met30p fusion.
We tagged Met30p with
GST, expressed from the GAL1 promoter (see Materials and
Methods), which permits low-level transcription when cells are grown in
the presence of dextrose and strongly induces transcription in
galactose-grown cells. Therefore, we could analyze GST-Met30p activity
when it was either poorly or highly produced. The plasmid pGSTMet30-3
complements the growth defect of a met30-6
temperature-sensitive mutant (Fig. 1a)
and of a met30 null strain (data not shown; see Materials
and Methods). Thus, the GST tag does not affect the essential function
of Met30p. Since complementation of met30
temperature-sensitive and null mutations by pGSTMet30-3 was achieved on
medium containing dextrose as the sole carbon source, low levels of
GST-Met30p are sufficient for its function. To test whether low-level
production of GST-Met30p correctly regulated MET gene
expression, we examined the regulation of the MET25
promoter, whose activity is repressed by L-methionine through the SCFMet30p complex (29). In cells
containing either wild-type MET30 or GST-MET30, expression of a MET25-lacZ fusion was
repressed in the presence of 2 mM L-methionine and
derepressed in the absence of L-methionine (Fig. 1b). Thus,
the GST-Met30p fusion appears to be functional for MET30
essential function and regulation of MET gene expression.
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FIG. 1.
Characterization of the GST-Met30p fusion. (a) Low-level
production of GST-Met30p is sufficient for complementation of a
met30-6 temperature-sensitive allele. PY283 cells containing
the met30-6 temperature-sensitive mutation were transformed
with either pEG(KG) or pGSTMET30-3, which allowed the production of the
indicated protein. Patches were made on SD medium, and cells were
incubated at the indicated temperature for 3 days. (b)
NMYmet30MET25-lacZ cells containing pGSTMET30-3 or a
wild-type MET30 congenic strain were grown in medium in the
presence (+) of a repressing concentration of L-methionine
or in the absence ( ) of L-methioine. The reported values
represent averages of two independent assays and were expressed as
nanomoles of substrate transformed per minute per milligram of protein.
The individual measurements deviated from the average values shown here
by 20% or less. (c) Induction time course of GST-Met30p and GST. We
performed an anti-GST Western immunoblot analysis of lysate prepared
from cells, grown to mid-logarithmic growth phase, containing either
pEG(KG) or pGST-MET30, which produced the indicated protein that had
been induced for 0, 1, 2, and 3 h by galactose. A cross-reacting
band is used as a loading control. (d) Met30p is an unstable protein.
We performed time course experiments to measure the stability of
GST-Met30p fusion (see Materials and Methods for details). A
cross-reacting band is used as a loading control.
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Met30p is an unstable protein.
To confirm GST-Met30p
production, Western immunoblot analysis was performed. Whereas GST was
readily detected after the first time point, 60 min, GST-Met30p was
detectable only after prolonged incubation of the cells in the presence
of galactose (Fig. 1c), suggesting that Met30p may be an unstable
protein. Indeed, a promoter shutoff experiment showed that GST-Met30p
is very unstable (Fig. 1d), like the yeast F-box proteins Cdc4p,
Ctf13p, and Grr1p (24, 33, 44).
Met30p abundance regulates SCFMet30p activity.
Since Met30p acts through the SCFMet30p complex to repress
MET gene expression (29, 32), we tested the
hypothesis that limiting Met30p abundance is a means of regulating
activity of the SCFMet30p complex. We assessed the ability
of methionine prototrophs to grow in the absence of
L-methionine while overproducing either GST or GST-Met30p.
Cells overproducing GST were able to grow in the absence of
L-methionine, illustrating the fact that the host strain
was indeed a methionine prototroph (Fig.
2a). The same host strain overproducing
GST-Met30p, however, was unable to grow in the absence of
L-methionine (Fig. 2a). Therefore, overproduction of
GST-Met30p induces methionine auxotrophy in cells otherwise wild type
for methionine biosynthesis.
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FIG. 2.
Effect of Met30p overproduction. (a) Methionine
prototrophic Y382 cells were transformed with either pEG(KG) or
pGSTMET30-3, which allowed the production of the indicated protein.
Patches were made on the indicated medium, and cells were incubated for
3 to 4 days. (b) Flow cytometry analysis of Y382 cells, transformed
with pGSTMET30-3, grown in the absence of methionine, before production
of GST-Met30p (upper panel) and after production of GST-Met30p (lower
panel). (c) C114 cells, MET25-lacZ, transformed with a
plasmid that allowed the production of the indicated protein, were
grown in medium containing (+) a repressing concentration of
L-methionine or in the absence () of
L-methionine. The reported values represent averages of
three independent assays and were expressed as nanomoles of substrate
transformed per minute per milligram of protein. The individual
measurements deviated from the average values shown here by 20% or
less.
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Methionine auxotrophs arrest growth in the G1 phase of the
cell division cycle (41). The majority of the cells
overproducing GST-Met30p in the absence of methionine were unbudded
(data not shown), suggesting a G1 arrest. To confirm a
G1 arrest, we performed flow cytometry. Y382 cells
containing pGSTMET30-3 were grown in sucrose-containing medium that
lacked methionine. GST-Met30p production was induced by the addition of
galactose. In order to determine DNA content, samples were analyzed by
flow cytometry. Induction of GST-Met30p caused cells to be uniformly
arrested with 1N DNA content, consistent with our previous observations
that methionine auxotrophy had been induced (Fig. 2b).
To test whether the induced methionine auxotrophy caused by
overproduction of GST-Met30p was the result of repression of
MET gene expression, we examined the regulation of the
MET25 promoter. In cells overproducing GST, expression of a
MET25-lacZ gene fusion was repressed in the presence of 2 mM
L-methionine (Fig. 2c). In cells overproducing GST-Met30p,
the MET25 promoter was repressed independently of the
availability of L-methionine (Fig. 2c); however, the levels
of repression were not equivalent. When cells were overproducing
GST-Met30p, MET25 promoter activity repression was approximately fivefold greater in the presence of
L-methionine than in the absence of
L-methionine. Thus, overproducing GST-Met30p enhanced the
repressing effect of medium containing 2 mM L-methionine. Taken together, these data suggest that the abundance of Met30p regulates methionine biosynthesis.
Met30p abundance is regulated by L-methionine.
We
tested whether methionine availability regulates Met30p abundance. Y382
cells were grown in medium containing 2% galactose in either the
presence or absence of L-methionine. The steady-state abundance of GST-Met30p was elevated 4.5-fold after the addition of
L-methionine (Fig. 3a to c).
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FIG. 3.
GST-Met30p steady-state abundance and stability are
dependent on methionine availability. (a) Anti-GST and anti-Cdc34p
immunoblot analysis of soluble protein extracts from Y382 cells
containing either pEG(KG) or pGSTMET30-3, whose expression was induced
for 3 h by the addition of galactose. Cdc34p, whose abundance is
unaffected by methionine (Fig. 4), is used as a loading control, and
its position is indicated (*). (b) A fivefold-longer exposure time of
panel a. (c) Quantitation of GST and GST-Met30p immunoblot signals of
which panel a is an example. Values represent averages derived from
five independent experiments. (d) Time course experiments to measure
the stability of GST-Met30p fusions after promoter shutoff in medium
containing or lacking methionine. Y382 cells, containing pGSTMET30-3,
were grown either in the presence or in the absence of 2 mM methionine,
and GST-Met30p synthesis was induced by the addition of galactose for
3 h. After the addition of dextrose and cycloheximide, samples
were taken at the indicated time points. (e) Quantification of data in
panel d by Storm Phosphorimager analysis. The amount of GST-Met30p
protein detected at the indicated time points is represented as a
percentage of GST-Met30p protein detected at time point zero.
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Because both GST and GST-Met30p were expressed from the same promoter,
it is unlikely that GAL1 promoter activity is affected by
L-methionine availability. Therefore, in order to observe a difference in the steady-state abundance of GST-Met30p, we postulated that GST-Met30p stability is altered. To test whether the degradation rate of GST-Met30p is altered by the presence of
L-methionine, we measured the half-lives of GST-Met30p in
the presence and absence of 2 mM L-methionine. Although we
observed that GST-Met30p was slightly unstable in the presence of 2 mM
L-methionine, it had a longer half-life than in the absence
of L-methionine (Fig. 3d and e). Thus, these data indicate
that GST-Met30p stability is dependent upon the presence of
L-methionine.
To test whether additional SCF components or F-box proteins are
affected by L-methionine availability, we measured the
steady-state abundances of Cdc34p, GST-Skp1p, and HA-Cdc53p by
following the same regimen as described above. The steady-state
abundance of Met30p, but of none of the other SCF components tested, is
dependent on the availability of L-methionine (Fig.
4).
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FIG. 4.
Met30p is the only component of SCFMet30p
whose steady-state abundance is methionine dependent. Y382 cells
transformed with the appropriate epitope-tagged plasmid were grown
either in the presence (+) or in the absence () of 2 mM methionine.
GST-Met30p and GST-Skp1p were detected using anti-GST antibodies
(Sigma), Cdc34p was detected by anti-Cdc34p antibodies, and HA-Cdc53p
was detected using anti-HA antibodies (Roche Molecular Biochemicals).
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We had previously demonstrated that Cdc4p abundance was dependent on
the interaction of Cdc4p with Skp1p and proposed that Skp1p shields
Cdc4p from degradation events (24). To investigate whether
Met30p abundance was similarly affected, we measured the steady-state
level of GST-Met30p in lysate prepared from cells containing the
skp1-3 temperature-sensitive mutation that were grown at
either the permissive temperature (23°C) or the nonpermissive temperature (37°C) in the presence or absence of 2 mM
L-methionine. Figure 5 shows
that the steady-state abundance of GST-Met30p is decreased in cells
lacking Skp1p activity when cells are grown in the absence of
L-methionine. These data suggest that Skp1p may enhance
Met30p stability. However, the steady-state abundance of GST-Met30p is
unaffected in cells lacking Skp1p activity when cells are grown in the
presence of L-methionine. Possibly, the decision to degrade
Met30p may be regulated prior to its incorporation into an SCF complex.
Alternatively, Skp1p binding to Met30p may be tighter in the presence
of L-methionine.
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FIG. 5.
Met30p steady-state abundance is dependent on Skp1p
activity. skp1-3-containing cells, transformed with
pGSTMET30-3, were grown either in the presence (+) or in the absence
() or 2 mM methionine and induced to produce the GST fusion protein
for 3 h by the addition of galactose at the indicated
temperature.
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Identification of an L-methionine-responsive element in
the Met30p family of proteins.
We wished to identify the Met30p
element that responds to L-methionine availability. Our
previous investigations of Cdc4p demonstrated that the F-box region is
partly responsible for regulating Cdc4p abundance (24).
During the analysis of Cdc4p degradation, we had constructed a
GST-MET30 fusion containing Met30p residues 176 to 275, which include the Met30p F box. We had previously demonstrated that
GST-Met30(176-275)p levels are low, which suggested that we had
identified a potential Met30p degradation sequence. We, therefore,
tested whether we had identified a region on Met30p that regulated the
abundance of Met30p in a manner dependent on the presence of
L-methionine. Cells containing plasmids encoding either
GST, GST-Met30p, or GST-Met30(176-275)p were grown in the presence or
absence of 2 mM L-methionine and induced by the addition of
galactose to produce the GST fusion proteins. Western immunoblot analysis demonstrated that the steady-state abundance of both GST-Met30p and GST-Met30(176-275)p was dependent on the presence of
L-methionine in the medium whereas the steady-state
abundance of GST was unaffected (Fig. 6a). Thus, we have identified a
region of Met30p that responds to the presence of
L-methionine in the medium.
We tested whether the degradation rate of Met30(176-275)p was altered
depending upon the availability of L-methionine in the medium. Promoter shutoff experiments demonstrated that
GST-Met30(176-275)p is more stable in the presence of 2 mM
L-methionine (Fig. 6b). Therefore, Met30p residues 176 to 275 are sufficient to target Met30p
for degradation in a manner dependent on the presence of L-methionine.
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FIG. 6.
Identification of a methionine-responsive element in the
Met30p family of proteins. (a and c) Anti-GST Western immunoblot
analysis of soluble protein extracted from Y382 cells transformed with
either pEG(KG), pGSTMET30-3, pGSTMET30(176-275), or
pGSTMET30(185-277) producing the indicated GST-Met30p fusion. Cells
were grown either in the presence (+) or in the absence () of 2 mM
methionine and induced to produce the GST fusion protein for 3 h
by the addition of galactose. Either Cdc34p (*) or a cross-reacting
band was used as a loading control. (b) Quantification of the
degradation rate of GST-Met30(176-275)p in the presence or absence of
2 mM methionine by Storm Phosphorimager analysis. Y382 cells
transformed with pGSTMET30(176-275) were grown in the presence or
absence of methionine and induced to produce GST-Met(176-275)p by the
addition of galactose for 3 h. The amount of GST-Met30(176-275)p
detected at the indicated time points is represented as a percentage of
GST-Met30(176-275)p protein detected at time point zero.
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We next wanted to test whether mutations of Met30p residues 176 to 275 rendered Met30p insensitive to the availability of L-methionine in the medium. We therefore made a deletion of
Met30p lacking residues 185 to 277 and measured the ability of the
mutant protein to sense L-methionine availability in the
medium (Fig. 6c). The steady-state abundance of the mutant protein is
not affected by the availability of L-methionine in the
medium (Fig. 6c). Thus, it appears that we have identified a region
that is necessary for the abundance of Met30p to be modulated in a
manner dependent on the presence of L-methionine. Database
searches revealed that this region is highly conserved in the Met30p
family of proteins (Fig. 7).
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FIG. 7.
Sequence alignment of the region that regulates Met30p
in a methionine-dependent manner in the Met30p family of proteins.
Residues 177 to 252 in Met30p from S. cerevisiae are shown
aligned with the same region of SCONB from Emericella
nidulans, open reading frame YDJ5 from S. pombe, and
scon2 from N. crassa. Identical residues in at least three
species are highlighted. The location of the F box is indicated.
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DISCUSSION |
In this paper, we demonstrate that SCFMet30p activity
can be modulated by changes in the abundance of Met30p. Furthermore,
repressing levels of L-methionine increases Met30p
stability. Finally, we have identified a region on Met30p that is
degraded in a methionine-dependent manner.
Why the cell limits F-box protein abundance has been unclear.
Potentially, maintaining limiting pools of F-box proteins prevents inter-F-box protein competition for common SCF components. Indeed, inter-F-box protein competition has been invoked to explain genetic interactions between mutants encoding SCF components (20, 22, 24,
29). The cell may also control F-box protein abundance as a means
of regulating SCF complex formation and activity. F-box proteins appear
to play an essential role in SCF complex activity, by acting as
adapters bridging the Ub-conjugating machinery with the protein
substrate that is to be ubiquitinated. Regulating the abundance of a
particular F-box protein would be a means of regulating the activity of
a particular SCF complex. In this paper, we present data that support
the notion that a specific SCF activity is regulated by modulating the
abundance of the respective F-box protein.
MET30 negatively regulates MET gene expression in
the presence of 2 mM L-methionine. We demonstrate that
Met30p abundance is limiting for SCFMet30p activity. First,
ectopic expression of GST-MET30 from the GAL1 promoter results in methionine auxotrophy in a strain wild type for
methionine biosynthesis (Fig. 2a). Additionally, GST-Met30p overproduction causes a cell cycle arrest when cells are grown in the
absence of methionine (Fig. 2b), an additional hallmark of methionine
auxotrophs (41). Finally, by measuring the activity of the
MET25 promoter we demonstrate that increased Met30p
abundance represses the transcriptional induction of the MET
genes (Fig. 2c). Indeed, overproducing GST-Met30p in the presence of
repressing concentrations of L-methionine enhances the
repression of the same reporter (Fig. 2c). Together, these data
demonstrate that the regulation of Met30p abundance plays a key role in
regulating SCFMet30p activity. In vitro and in vivo data
support the hypothesis that some SCF complexes recognize phosphorylated
substrates and do not recognize nonphosphorylated substrates. However,
modification of Met4p has not been attributed to phosphorylation
(32). We demonstrate here that an additional means of
regulating SCFMet30p complex activity is at the level of
F-box protein abundance.
Because SCFMet30p complex activity is required to repress
MET gene expression in the presence of
L-methionine (29, 32), we investigated whether
Met30p abundance is regulated by the availability of
L-methionine. We demonstrate that the steady-state
abundance and stability of Met30p are dependent on the availability of
L-methionine in the growth medium (Fig. 3). In the absence
of methionine, GST-Met30p has a low abundance and is unstable, whereas
in the presence of 2 mM L-methionine, Met30p is more
abundant and more stable. Thus, we propose that the availability
of L-methionine regulates SCFMet30p
activity by regulating Met30p stability (Fig.
8). These data appear to be at odds with
those of Rouillon et al. (32), who did not detect changes in
the stability of Met30p according to the availability of
L-methionine. Furthermore, Rouillon et al. did not report
methionine auxotrophy upon MET30 overexpression. Interestingly, these authors used homocysteine rather than sulfate as a
nonrepressing sulfur source. Cells mutant for MET4 can grow on homocysteine (39), suggesting that full derepression of
MET genes is not required for homocysteine metabolism.
Additionally, we measured Met30p half-life when cells were in
steady-state sulfate or methionine levels, a situation that may be
different from that when cells make the transition from a nonpreferred
sulfur source to a preferred sulfur source.
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FIG. 8.
Proposed model for the regulation of
SCFMet30p complex activity. Met30p abundance is regulated
by methionine availability. In the presence of methionine, Met30p
stability increases to permit SCFMet30p complex formation
and subsequent repression of methionine biosynthetic gene expression by
mediating Met4p degradation. In the absence of methionine, Met30p
abundance is decreased, lowering the activity of SCFMet30p
and, thus, derepressing methionine biosynthetic gene expression (expr)
by allowing the accumulation of Met4p.
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|
How does the methionine-dependent effect on Met30p relate to SCF
complex formation? We reveal that the interaction of Met30p with Skp1p
is important in order to maintain Met30p stability (Fig. 5). This is
similar to our results with Cdc4p, which demonstrated that the
Skp1p-F-box interaction was important to stabilize Cdc4p (24). Possibly, Skp1p stabilizes F-box proteins by masking
adjacent degradation motifs. Our data reported here are consistent with previous reports that have similarly shown that the F-box-Skp1p interaction is important for maintaining Met30p stability (29, 32). When cells are grown in the absence of methionine, Skp1p may
be removed from SCFMet30p, resulting in Met30p degradation.
However, in the presence of L-methionine, growth at 37°C
did not result in a loss of Met30p. There are a number of possible
explanations for this effect. Possibly, the decision to degrade Met30p
is made before Met30p enters the SCF complex. Alternatively, the
presence of L-methionine could change the interaction
between Skp1p and Met30p such that Skp1p is less susceptible to
inactivation. Finally, in the presence of L-methionine, an
increase in Met30p might be sufficient to increase Skp1p function,
similar to what is seen when CDC4 levels are increased in
skp1 mutants (1).
Paradoxically, we have also demonstrated that removal of a region
containing the F box of Met30p results in a protein whose abundance is
unaffected by the availability of L-methionine. How can the
apparently contradictory nature of these observations be reconciled?
Possibly, the region of Met30p that contains the F box carries out two
functions. One function is to bind Skp1p and thus link Met30p with the
Ub ligase machinery. A second function may be to target Met30p for
degradation. In the absence of L-methionine, loss of Skp1p
activity results in Met30p degradation. However, removal of the region
containing the F box will also remove the Met30p degradation sequence
and result in a protein whose stability is unaffected by the
availability of L-methionine. A similar phenomenon is seen
in Neurospora crassa. Loss of SCON1, a homologue of Skp1p, results in constitutive activation of the sulfate assimilation pathway.
However, mutations of conserved F-box residues within SCON2, the Met30p
homologue, lead to constitutive repression of the sulfate assimilation
pathway (19). Our data support the notion that the F-box
region is important in regulating Met30p abundance in addition to its
role of coordinating SCF complex formation.
The SCFMet30p complex is not the only SCF complex that
regulates metabolic processes in yeast. SCFGrr1p mediates
glucose repression, and SCFCdc4p mediates general control
of amino acid biosynthesis by targeting the transcriptional activator
Gcn4p for degradation (15, 20). Additionally, both
SCFGrr1p and SCFCdc4p are also involved in
regulating cell cycle progression. Potentially, by regulating F-box
protein abundance, environmental conditions would impact on both
metabolism and cell cycle progression. Indeed, Met30p has a positive
influence in G1 (29; C. Dixon and N. Mathias, unpublished data), and thus methionine availability could
determine the length of the G1 phase of the cell cycle by
regulating Met30p stability.
Finally, we have identified a region on Met30p residues which is
necessary and sufficient to alter the abundance of the protein dependent upon on the availability of L-methionine.
Although GST-Met30(184-277)p confers methionine auxotrophy when
overproduced, we were unable to demonstrate that this mutant more
efficiently repressed MET gene expression than did wild-type
Met30p. Possibly, repression by wild-type Met30p could not be improved
upon. Another likely reason is that GST-Met30(185-277)p lacks the
F-box motif which is necessary for Skp1p binding and proper complex
architecture. Thus, GST-Met30(185-277)p activity may be slightly
compromised. Indeed, GST-Met30(185-277)p can complement the
met30-6 temperature-sensitive strain only when overproduced
and is unable to complement a MET30 null strain (data not
shown). The region identified as regulating Met30p stability in a
methionine-dependent manner is highly conserved in the Met30p family of
proteins (Fig. 7). In N. crassa and Aspergillus nidulans, Met30p activity is most likely conferred by SCON2 and sconB, respectively (18, 27). In silico analysis revealed that these proteins, as well as an uncharacterized
Schizosaccharomyces pombe protein, contain a motif highly
similar to the region of Met30p that imparts
L-methionine-regulated changes in Met30p stability. Thus,
we have described a novel mechanism for SCF regulation by the
environment; methionine-dependent stabilization of an F-box component
of an SCF complex. This mechanism may be conserved for methionine
biosynthesis among fungi and may have implications for SCFs controlling
glucose and amino acid biosynthesis and transport.
We are indebted to Yolande Surdin-Kerjan, Phil Hieter, Steve
Reed, and Mike Tyers for plasmids and yeast strains. We thank Kelly
Tatchell and Lucy Robinson for reading the manuscript and for comments.
We also thank an anonymous reviewer for significant editorial changes
to the manuscript.
This work was supported by start-up funds to N.M. from Louisiana State
University Health Sciences Center. Initial studies were funded by NSF
award MCB 9728069 to M.G.G.
| 1.
|
Bai, C.,
P. Sen,
K. Hofmann,
L. Ma,
M. Goebl,
J. W. Harper, and S. J. Elledge.
1996.
SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box.
Cell
86:263-274[CrossRef][Medline].
|
| 2.
|
Barral, Y.,
S. Jentsch, and C. Mann.
1995.
G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast.
Genes Dev.
9:399-409[Abstract/Free Full Text].
|
| 3.
|
Burton, E. G., and R. L. Metzenberg.
1972.
Novel mutation causing derepression of several enzymes of sulfur metabolism in Neurospora crassa.
J. Bacteriol.
109:140-151[Abstract/Free Full Text].
|
| 4.
|
Conklin, D. S.,
C. Kung, and M. R. Culbertson.
1993.
The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae.
Mol. Cell. Biol.
13:2041-2049[Abstract/Free Full Text].
|
| 5.
|
Craig, K. L., and M. Tyers.
1999.
The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction.
Prog. Biophys. Mol. Biol.
72:299-328[CrossRef][Medline].
|
| 6.
|
Deshaies, R. J.
1999.
SCF and cullin/ring H2-based ubiquitin ligases.
Annu. Rev. Cell Dev. Biol.
15:435-467[CrossRef][Medline].
|
| 7.
|
Elbe, R.
1992.
A simple and efficient procedure for transformation of yeasts.
BioTechniques
13:18-20[Medline].
|
| 8.
|
Feldman, R. M.,
C. C. Correll,
K. B. Kaplan, and R. J. Deshaies.
1997.
A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p.
Cell
91:221-230[CrossRef][Medline].
|
| 9.
|
Flick, J. S., and M. Johnston.
1991.
GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.
Mol. Cell. Biol.
11:5101-5112[Abstract/Free Full Text].
|
| 10.
|
Hershko, A., and A. Ciechanover.
1998.
The ubiquitin system.
Annu. Rev. Biochem.
67:425-479[CrossRef][Medline].
|
| 11.
|
Hinnebusch, A. G., and G. R. Fink.
1983.
Positive regulation in the general control of Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA
80:5347-5378.
|
| 12.
|
Hochstrasser, M.
1996.
Ubiquitin-dependent protein degradation.
Annu. Rev. Genet.
30:405-439[CrossRef][Medline].
|
| 13.
|
Hope, I. A., and K. Struhl.
1985.
GCN4 protein, sunthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast.
Cell
43:177-188[CrossRef][Medline].
|
| 14.
|
Iraqui, I.,
S. Vissers,
F. Bernard,
J. O. de Craene,
E. Boles,
A. Urrestarazu, and B. Andre.
1999.
Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease.
Mol. Cell. Biol.
19:989-1001[Abstract/Free Full Text].
|
| 15.
|
Jaquenoud, M.,
M. P. Gulli,
K. Peter, and M. Peter.
1998.
The Cdc42p effector Gic2p is targeted for ubiquitin-dependent degradation by the SCFGrr1 complex.
EMBO J.
17:5360-5373[CrossRef][Medline].
|
| 16.
|
Kaiser, P.,
R. A. Sia,
E. G. Bardes,
D. J. Lew, and S. I. Reed.
1998.
Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1.
Genes Dev.
12:2587-2597[Abstract/Free Full Text].
|
| 17.
|
Kamura, T.,
D. M. Koepp,
M. N. Conrad,
D. Skowyra,
R. J. Moreland,
O. Iliopoulos,
W. S. Lane,
W. G. Kaelin, Jr.,
S. J. Elledge,
R. C. Conaway,
J. W. Harper, and J. W. Conaway.
1999.
Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.
Science
284:657-661[Abstract/Free Full Text].
|
| 18.
|
Kumar, A., and J. V. Paietta.
1995.
The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.
Proc. Natl. Acad. Sci. USA
92:3343-3347[Abstract/Free Full Text].
|
| 19.
|
Kumar, A., and J. V. Paietta.
1998.
An additional role for the F-box motif: gene regulation within the Neurospora crassa sulfur control network.
Proc. Natl. Acad. Sci. USA
95:2417-2422[Abstract/Free Full Text].
|
| 20.
|
Li, F., and M. Johnston.
1997.
Grr1 of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skp1: coupling glucose sensing to gene expression and the cell cycle.
EMBO J.
16:5625-5638.
|
| 21.
|
Liu, Y.,
N. Mathias,
C. N. Steussy, and M. G. Goebl.
1995.
Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.
Mol. Cell. Biol.
15:5635-5644[Abstract].
|
| 22.
|
Mathias, N.,
S. L. Johnson,
M. Winey,
A. E. M. Adams,
L. Goetsch,
J. R. Pringle,
B. Byers, and M. G. Goebl.
1996.
Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.
Mol. Cell. Biol.
16:6634-6643[Abstract].
|
| 23.
|
Mathias, N.,
C. N. Steussy, and M. G. Goebl.
1998.
An essential domain within Cdc34p is required for binding to a complex containing Cdc4p and Cdc53p in Saccharomyces cerevisiae.
J. Biol. Chem.
273:4040-4045[Abstract/Free Full Text].
|
| 24.
|
Mathias, N.,
S. Johnson,
B. Byers, and M. Goebl.
1999.
The abundance of cell cycle regulatory protein Cdc4p is controlled by interactions between its F box and Skp1p.
Mol. Cell. Biol.
19:1759-1767[Abstract/Free Full Text].
|
| 25.
|
Meimoun, A.,
T. Holtzman,
Z. Weissman,
H. J. McBride,
D. J. Stillman,
G. R. Fink, and D. Kornitzer.
2000.
Degradation of the transcription factor gcn4 requires the kinase pho85 and the SCF(CDC4) ubiquitin-ligase complex.
Mol. Biol. Cell.
3:915-927.
|
| 26.
|
Mitchell, D. A.,
T. K. Marshall, and R. J. Deschenes.
1993.
Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast.
Yeast
9:715-723[CrossRef][Medline].
|
| 27.
|
Natorff, R.,
M. Piotrowska, and A. Paszewski.
1998.
The Aspergillus nidulans sulphur regulatory gene sconB encodes a protein with WD40 repeats and an F-box.
Mol. Gen. Genet.
257:255-263[CrossRef][Medline].
|
| 28.
|
Patton, E. E.,
A. R. Willems, and M. Tyers.
1998.
Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis.
Trends Genet.
14:236-243[CrossRef][Medline].
|
| 29.
|
Patton, E. E.,
A. R. Willems,
D. Sa,
L. Kuras,
D. Thomas,
K. L. Craig, and M. Tyers.
1998.
Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box protein complexes that regulate cell division and methionine biosynthesis in yeast.
Genes Dev.
12:692-705[Abstract/Free Full Text].
|
| 30.
|
Patton, E. E.,
C. Peyraud,
A. Rouillon,
Y. Surdin-Kerjan,
M. Tyers, and D. Thomas.
2000.
SCF(Met30)-mediated control of the transcriptional activator Met4 is required for the G(1)-S transition.
EMBO J.
19:1613-1624[CrossRef][Medline].
|
| 31.
|
Rose, M. D.,
F. Winston, and P. Heiter.
1990.
Methods in yeast genetics: a laboratory course.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 32.
|
Rouillon, A.,
R. Barbey,
E. E. Patton,
M. Tyers, and D. Thomas.
2000.
Feedback-regulated degradation of the transcriptional activator Met4 is triggered by the SCF(Met30p) complex.
EMBO J.
19:282-294[CrossRef][Medline].
|
| 33.
|
Russell, I. D.,
A. S. Grancell, and P. K. Sorger.
1999.
The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex.
J. Cell Biol.
145:933-950[Abstract/Free Full Text].
|
| 34.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 35.
|
Scheffner, M.,
U. Nuber, and J. M. Huibregtse.
1995.
Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.
Nature
373:81-83[CrossRef][Medline].
|
| 36.
|
Seol, J. H.,
R. M. Feldman,
W. Zachariae,
A. Shevchenko,
C. C. Correll,
S. Lyapina,
Y. Chi,
M. Galova,
J. Claypool,
S. Sandmeyer,
K. Nasmyth, and R. J. Deshaies.
1999.
Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.
Genes Dev.
13:1614-1626[Abstract/Free Full Text].
|
| 37.
|
Skowyra, D.,
K. L. Craig,
M. Tyers,
S. J. Elledge, and J. W. Harper.
1997.
F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex.
Cell
91:209-219[CrossRef][Medline].
|
| 38.
|
Skowyra, D.,
D. M. Koepp,
T. Kamura,
M. N. Conrad,
R. C. Conaway,
J. W. Conaway,
S. J. Elledge, and J. W. Harper.
1999.
Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1.
Science
284:662-665[Abstract/Free Full Text].
|
| 39.
|
Thomas, D., and Y. Surdin-Kerjan.
1997.
Metabolism of sulfur amino acids in Saccharomyces cerevisiae.
Microbiol. Mol. Biol. Rev.
61:503-532[Abstract].
|
| 40.
|
Thomas, D.,
L. Kuras,
R. Barbey,
H. Cherest,
P. L. Blaiseau, and Y. Surdin-Kerjan.
1995.
Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD40 repeats.
Mol. Cell. Biol.
15:6526-6534[Abstract].
|
| 41.
|
Unger, M. W., and L. H. Hartwell.
1976.
Control of cell division in Saccharomyces cerevisiae by methionyl-tRNA.
Proc. Natl. Acad. Sci. USA
73:1664-1668[Abstract/Free Full Text].
|
| 42.
|
Willems, A. R.,
T. Goh,
L. Taylor,
I. Chernushevich,
A. Shevchenko, and M. Tyers.
1999.
SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
354:1533-1550[CrossRef][Medline].
|
| 43.
|
Winston, J. T.,
P. Strack,
P. Beer-Romero,
C. Y. Chu,
S. J. Elledge, and J. W. Harper.
1999.
The SCF-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro.
Genes Dev.
13:270-283[Abstract/Free Full Text].
|
| 44.
|
Zhou, P., and P. M. Howley.
1998.
Ubiquitination and degradation of the substrate recognition subunits of SCF ubiquitin-protein ligases.
Mol. Cell.
5:571-580.
|
| 45.
|
Zhu, S., and R. C. Wek.
1998.
Ribosome-binding domain of eukaryotic initiation factor-2 kinase GCN2 facilitates translation control.
J. Biol. Chem.
273:1808-1814[Abstract/Free Full Text].
|
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Abstract
Introduction
