From the Departments of Internal Medicine and § Biochemistry,
¶ Biochemistry and Molecular Biology Graduate Program, University
of Texas Southwestern Medical Center, Dallas, Texas 75390-8573
Received for publication, December 4, 2000
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ABSTRACT |
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The Gal system of Saccharomyces
cerevisiae is a paradigm for eukaryotic gene regulation.
Expression of genes required for growth on galactose is regulated by
the transcriptional activator Gal4. The activation function of Gal4 has
been localized to 34 amino acids near the C terminus of the protein.
The gal4D allele of GAL4 encodes a truncated
protein in which only 14 amino acids of the activation domain remain.
Expression of GAL genes is dramatically reduced in gal4D
strains and these strains are unable to grow on galactose as the sole
carbon source. Overexpression of gal4D partially relieves the defect in
GAL gene expression and allows growth on galactose. A search for
extragenic suppressors of gal4D identified recessive
mutations in the SUG1 and SUG2 genes, which encode ATPases of the 19S regulatory complex of the proteasome. The
proteasome is responsible for the ATP-dependent degradation of proteins marked for destruction by the ubiquitin system. It has been
commonly assumed that effects of SUG1 and SUG2
mutations on transcription are explained by alterations in the
proteolysis of gal4D protein. We have investigated this assumption.
Surprisingly, we find that SUG1 and SUG2
alleles that are unable to suppress gal4D cause a larger
increase in gal4D protein levels than do suppressing alleles. In
addition, mutations in genes encoding subunits of the proteolytic 20S
sub-complex of the proteasome increase the levels of gal4D protein but
do not rescue its transcriptional activity. Therefore, an alteration in
the proteolysis of gal4D by the proteasome cannot explain the effects
of mutations in SUG1 and SUG2 on expression of
GAL genes. These findings suggest that the 19S regulatory complex may
play a more direct role in transcription.
In the yeast Saccharomyces cerevisiae, expression of
genes required for the metabolism of galactose is controlled by the
positive regulator Gal4 and the negative regulator Gal80. Gal4 is a
transcriptional activator with an N-terminal DNA binding domain and a
C-terminal activation domain. Partial deletion of the activation domain
of Gal4 in the gal4D allele leads to a dramatic loss in the
ability to activate transcription of GAL genes. Using a reporter gene assay, the gal4D protein was found to activate transcription from the
GAL1/10 promoter to ~4% of the level driven by wild-type Gal4 (1).
The gal4D protein does not activate the GAL genes sufficiently to allow
growth on galactose as the sole carbon source (1). Recessive mutations
in SUG1 and SUG2 have been identified that partially restore the ability of gal4D to activate transcription (1-3). In strains carrying the sug1-1 or
sug2-1 alleles, reporter gene activity was restored to
~55% and 70%, respectively (1, 4). Both the sug1-1 and
sug2-1 alleles allow gal4D strains to grow on
galactose as the sole carbon source (1, 3). In the work reported here
we have investigated the mechanism for this suppression.
Sug1 (1) and Sug2 (3) are members of the ATPases
Associated with diverse cellular Activities
(AAA)1 family. Members of
this family share a 230-amino acid conserved region known as the AAA
module (5), which contains Walker A and B nucleotide-binding motifs
(6). Both Sug1 and Sug2 are components of the 19S regulatory complex
(regulatory particle) of the yeast 26S proteasome (3, 7, 8), along with
four other AAA proteins (Ref. 9 and references therein). The finding that Sug1 and Sug2 are components of the proteasome suggested that
sug1-1 and sug2-1 might suppress gal4D
by stabilizing the mutant protein and allowing it to accumulate.
Indeed, several authors have argued that the transcriptional phenotypes
associated with mutations in ATPases of the proteasome are indirect and
due to alterations in proteolysis (7, 10-14). Consistent with this possibility, high level overexpression of gal4D from a multicopy vector
rescues the ability to activate a reporter gene to
~60%.2
If suppression of gal4D is dependent on stabilization of an
unstable protein, then levels of gal4D protein in the cell should correlate with suppression. To address this question, we made a set of
congenic yeast strains carrying wild-type and mutant SUG1
and SUG2 alleles. We used these strains to compare levels of
gal4D protein in strains that suppressed the transcriptional phenotype
of gal4D and those that did not. In addition to comparing sug1-1 and sug2-1 to their respective wild-types,
we also wished to evaluate other alleles of SUG1 and
SUG2. Alleles of SUG1 have been identified
independently by Xu et al. (15) as mutations that suppressed
a temperature-sensitive allele of CDC68, but their interaction with gal4D has not been defined. We chose one of
these alleles, sug1-20, to compare with sug1-1.
We also wanted to compare sug2-1 to an allele isolated in a
different manner. However, until recently, no SUG2 allele
besides sug2-1 was known. In 1988 McCusker and Haber (16)
isolated mutants exhibiting both cycloheximide resistance and
temperature-sensitive lethality (crl). These strains have
hypersensitivity to amino acid analogs and fail to arrest in
G1 under some starvation conditions (17). After
SUG2 was cloned (3) it became clear from mapping data that
crl13 was an allele of SUG2
(16).3 We have determined the
mutation in the crl13 allele of SUG2, which we
have designated sug2-13, and have compared its phenotype to
that of sug2-1.
In this work, we have produced a set of congenic strains carrying
wild-type and mutant alleles of both SUG1 and
SUG2. Using these strains we have shown that there is allele
specificity to suppression of gal4D. Mutations in alleles of
SUG1 and SUG2 not selected for their ability to
suppress gal4D do not do so. This has allowed us to
explicitly test the hypothesis that the sug1-1 and
sug2-1 mutations lead to alterations in the level of gal4D protein and that this accounts for rescue of the gal4D
phenotype. We find that the levels of gal4D protein are higher in
strains carrying sug1 and sug2 mutations that do
not suppress gal4D than in strains carrying the
sug1-1 and sug2-1 mutations. Therefore, changes
in the proteolysis of gal4D by the proteasome cannot account for the
transcriptional effects of the sug1-1 and sug2-1 mutations.
Identification of the SUG2 Mutation in the crl13
Strain--
James Haber and John McCusker supplied us with Y55-297
(crl13) and its congenic wild-type Y55 (CRL13).
The SUG2 gene from 85 bp before the start codon to 200 bp
after the stop codon was amplified by PCR from both strains and cloned
using the TA cloning kit to produce pSJR187 (crl13) and
pSJR188 (CRL13). The sequence of the entire gene was
then determined by fluorescence-automated sequencing. There were
several silent polymorphisms as compared with the data base sequence
present in the SUG2 alleles amplified from both the
crl13 and wild-type strains. However, only one mutation in
the crl13 strain resulted in an amino acid change. The codon for amino acid 231 was changed from CTA to CGA to create an L231R substitution. We designated this mutation sug2-13.
Production of Congenic Strains Containing the sug2-1 and sug2-13
Mutations--
The SUG2 locus from 208 bp before the start
codon to 54 bp after the stop codon was amplified with oligos
containing the ClaI site and blunt cloned into pUC118 to
produce pSJR79. The sug2-1 mutation was inserted into this
plasmid by site-directed mutagenesis to create
pUC118-sug2-1. This plasmid was sequenced to confirm that
only the desired mutation was present. A fragment containing the gene
was liberated from the plasmid by digestion with ClaI. The
plasmid containing the PCR-amplified and TA-cloned SUG2 gene containing the sug2-13 mutation (pSJR187) was digested with
Age1 and SnaB1 to liberate a fragment from 42 bp
before the start codon to 191 bp after the stop codon.
These fragments were purified and transformed into Sc530
(SUG2::URA3 pMTL-SUG2), which is
congenic to W303 (3, 18). The transformants were plated to glucose
medium. The plasmid pMTL-SUG2 expresses SUG2
under control of the GAL1/10 promoter. Because SUG2 is an essential gene, this strain can only survive on
galactose medium, which induces the GAL1/10 promoter. If a
gene replacement event occurs that reintroduces SUG2 into
its native location, the strain recovers its ability to grow on
glucose. Transformants able to grow on glucose were patched and
replica-plated to medium lacking uracil. Clones that could grow on
glucose medium and were uracil auxotrophs were candidates for gene
replacement events, because the deletion marker had been lost. The
SUG2 gene was amplified from these strains by PCR and was
completely sequenced. Sequencing in both directions confirmed that the
L231R mutation was present in the sug2-13 strain Sc677 and
that the E300K mutation was present in the sug2-1 strain
Sc671. GAL4 was deleted from these strains, and from the
congenic wild-type W303, by transforming them with a
GAL4::HIS3 fragment liberated from plasmid pJCS112
with BamHI. His+ colonies were patched and replica-plated to
glucose and galactose media to confirm that they had lost the ability
to use galactose as the sole carbon source. The loss of Gal4 was
confirmed by Western blot of extracts of these strains after growth on
raffinose, which does not repress synthesis of Gal4. The final set of
congenic strains was Sc736 (sug2-1 GAL4::HIS3),
Sc738 (sug2-13 GAL4::HIS3), and Sc748 (SUG2
GAL4::HIS3).
Production of Congenic Strains Carrying the sug1-1 and sug1-20
Mutations--
The starting material for the production of these
strains was pJS159, which contains the SUG1 locus as a
XhoI/BamHI fragment. The sug1-1 and
sug1-20 mutations were inserted into this plasmid by
site-directed mutagenesis to produce pSJR171 and pSJR173, respectively. Fragments containing the wild-type and mutant genes were liberated from
the plasmid with EcoRI/KpnI digestion and
transformed into Sc500 (SUG1::URA3
pMTL-SUG1), which is congenic to W303. Gene replacement
events were selected as described above. The SUG1 gene was
amplified from these strains by PCR and completely sequenced. Sequencing of the genes confirmed that the desired mutations had been
introduced and that no other mutations were present. Sc654 contained
the sug1-1 mutation, Sc658 contained the sug1-20
mutation, and Sc507 was the congenic wild-type strain. GAL4
was deleted from these strains as described above to give Sc729
(sug1-1 GAL4::HIS3), Sc733 (sug1-20
GAL4::HIS3), and Sc727 (SUG1
GAL4::HIS3), respectively.
Production of cdc68-1 Strains--
To test the interaction of
SUG1 alleles and the cdc68-1 mutation, strains
Sc729 (sug1-1), Sc733 (sug1-20), and Sc727
(SUG1) were transformed with YepDE68-1 (a multicopy plasmid
carrying the URA3 marker and expressing cdc68-1).
These strains were then transformed with the BamHI fragment
of pBM10 (LEU2) to disrupt the chromosomal CDC68
gene. Transformants were selected on medium lacking leucine to select
for integration events. To ensure that the chromosomal CDC68
gene was disrupted, transformants were tested for their ability to grow
on medium containing 5-FOA. Because CDC68 is an essential
gene, yeast that carry CDC68 only on the URA3
plasmid should not survive on 5-FOA. Accordingly, 5-FOA+ clones were
discarded. The remaining strains were deduced to have the chromosomal
CDC68 locus deleted and to carry cdc68-1 on the multicopy plasmid. The congenic set of strains produced was Sc761 (SUG1 CDC68::LEU2 YEp352-cdc68-1),
Sc766 (sug1-1 CDC68::LEU2
YEp352-cdc68-1), and Sc769 (sug1-20
CDC68::LEU2 YEp352-cdc68-1).
Production of pre Mutant Strains--
Strains WCG4-11/22a
(pre1-1 pre2-2) and yH129/14 (pre1-1 pre4-1) (19,
20) were provided by Dieter Wolf, Wolfgang Hilt, and Wolfgang
Heinemeyer. GAL4 was deleted from these strains as described above to
produce.Sc774 (pre1-1 pre2-2 GAL4::HIS3), Sc779 (pre1-1 pre4-1 GAL4::HIS3), and Sc782 (PRE1
PRE2 PRE4 GAL4::HIS3).
Assay of Cycloheximide Resistance and Temperature
Sensitivity--
Strains were grown in Yeast
extract-peptone-dextrose (21) to stationary phase. The
A600 values of the cultures were normalized to
0.1 by dilution in water. Serial 10-fold dilutions were performed, and
10 µl of each dilution was spotted onto plates. Cycloheximide plates
were prepared as described previously (16). Temperature sensitivity
assays were done on YEPD plates. Cycloheximide plates and control
plates were grown for 3 days at 30 °C. Plates for the temperature
sensitivity experiment were grown at either 30 °C or 37 °C for 3 days.
Assay of gal4D Suppression--
Yeast strains were
transformed with single-copy plasmids (derived from pSB32) expressing
either gal4D (pSJR261) or wild-type Gal4 (pSJR263), or with a multicopy
plasmid (derived from Yep351) expressing gal4D (pSJR268). In each case
the encoded proteins were tagged at their N termini with three tandem
copies of the T7 epitope tag (Novagen), and the GAL4 gene
was expressed from its own promoter. The transformed strains were grown
to stationary phase in complete medium lacking leucine (21), to select
for the plasmid, with raffinose as the carbon source. The use of
raffinose ensured that there was no selection for suppressors of
gal4D. Glucose was not used, because it represses the
synthesis of Gal4. The cell suspensions were diluted as above and
spotted to complete-leucine plates with glucose or galactose as
the carbon source.
Assay of Gal4 and gal4D Protein Levels--
Transformed strains
were grown to stationary phase as above, then diluted into a larger
volume of complete-leucine medium with raffinose as the carbon source.
They were grown to an A600 of 0.8 (mid- to
late-log for these strains), and 15 ml was harvested by centrifugation.
The cells were washed once in ice-cold water, then suspended in 100 µl of 2× SDS loading buffer and snap-frozen in liquid nitrogen. At
the same time the cells were harvested, another aliquot of the culture
was diluted 1:10 with water containing 0.02% sodium azide. The
A600 of these samples was used to calculate a
volume of 2× SDS loading buffer to add to the frozen samples so that
they had equal concentrations of cells. After the appropriate dilution,
the cells suspended in SDS loading buffer were boiled for 10 min. They
were spun briefly to remove cell debris, then equal amounts of the
supernatant were loaded onto an SDS-PAGE gel. A three-stage
polyacrylamide gel (stacking 4%, top separating 10%, bottom
separating 12.5%) was run in a Bio-Rad Protean apparatus overnight at
70 V with cooling by circulating water at 16 °C. The high percentage
bottom stage of the gel was used to retard the low molecular mass
cyclophilin (used as a loading control) during the long run required to
separate Gal4 protein from a cross-reacting band with a similar
electrophoretic mobility. Proteins were transferred to a nitrocellulose
membrane (Millipore) in a Genie blotting apparatus (Idea Scientific)
for 50 min at 24 V. The membranes were blotted with anti-T7 monoclonal
antibody (Novagen) to detect Gal4 and with rabbit anti-cyclophilin
antibody (a gift of K. Sykes). Membranes were developed with
horseradish peroxidase-conjugated secondary antibodies and the
Renaissance chemiluminescence reagent.
Characterization of the crl13 Mutation and Production of Congenic
Strains Carrying Different sug2 Alleles--
Despite the fact that
mutations in SUG1 have been isolated in multiple different
screens (1, 15, 22), until recently no mutant alleles of
SUG2 besides sug2-1 were known. We wanted to
investigate the allele specificity of gal4D suppression by alleles of SUG2. Therefore, we decided to identify the
mutation in the crl13 allele of SUG2. The
SUG2 open reading frames were amplified from the
crl13 strain and its parental wild-type strain and were
completely sequenced. Both of these strains were kindly provided by J. E Haber and J. H. McCusker. Two nucleotide changes were found in
the SUG2 gene from the crl13 strain. Only one of these resulted in an amino acid change, substituting an arginine for a
leucine at position 231. We will refer to this novel sug2 allele as sug2-13. The mutation in sug2-1 results
in a substitution of glycine 300 by a lysine (3). Unlike the G300K
substitution encoded in sug2-1, the L231R substitution
encoded in sug2-13 is within the Walker A nucleotide-binding
motif of Sug2 (6, 23).
We produced a set of congenic strains carrying either the
sug2-1, sug2-13, or wild-type SUG2
alleles. These strains were produced by a two-step gene replacement
strategy so that the mutant sug2 alleles were integrated at
their native chromosomal loci (see "Experimental Procedures"). We
verified that Sug2 protein was expressed at identical levels in each of
these strains, demonstrating that neither of the mutant sug2
alleles destabilized the Sug2 protein (data not shown). We also tested
the sub-cellular localization of Sug2 and found that it was primarily
nuclear in each strain as it is in wild-type yeast (data not shown)
(24). So that the interaction of the sug2 alleles with
different GAL4 variants could be tested, we deleted the
GAL4 gene from each of these strains (see "Experimental
Procedures").
Temperature Sensitivity and Cycloheximide Resistance of sug2 Mutant
Strains--
The crl mutants were selected for temperature
sensitivity and cycloheximide resistance. Therefore, the set of
congenic strains we produced were tested for temperature sensitivity at
37 °C. These results, presented in Fig.
1A, are consistent with
earlier reports. The sug2-13 strain is extremely
temperature-sensitive just as reported for the crl13 strain
(16). In contrast, sug2-1 does not cause temperature
sensitivity, consistent with previous results (3). The other reported
phenotype of the crl13 strain was resistance to
cycloheximide. Therefore, we tested the panel of congenic strains for
this phenotype, but found no evidence for cycloheximide resistance in
either sug2 strain (Fig. 1A). Indeed, both
sug2 strains may be more sensitive to cycloheximide than the
congenic wild-type strain. This was not surprising, as McCusker and
Haber previously found that cycloheximide resistance did not segregate
2:2 when the crl13 strain was crossed into a different
genetic background, the commonly used S288c strain (16). In fact, only
a very small proportion of segregants from this cross displayed
cycloheximide resistance. The authors concluded that there must be at
least two suppressors of cycloheximide resistance in S288c, perhaps due
to differences in permeability of the drug (16). It seems likely that
the W303 background of our congenic strains also contains such
suppressors. In contrast, temperature sensitivity segregated 2:2 when
the crl13 was crossed into the S288c background (16),
consistent with our finding of temperature sensitivity in the W303
background.
Suppression of gal4D by sug2 Alleles--
To test the suppression
of gal4D by sug2 alleles we transformed each
strain with a single-copy, centromeric vector carrying the
gal4D gene. As controls, each strain was also transformed with a multicopy, 2 µM plasmid that overexpresses gal4D
and a centromeric plasmid expressing wild-type GAL4. In each
case the expressed Gal4 protein was epitope-tagged with three tandem
copies of the T7 epitope tag at its N terminus to facilitate
immunologic detection. Because the chromosomal copy of GAL4
had been deleted in each of these strains, the epitope-tagged proteins
expressed from the plasmids were the only source of Gal4 activity in
these strains.
Cultures were grown to stationary phase in selective medium with
raffinose as the carbon source. After the densities of the cultures
were normalized, serial dilutions were performed and the diluted cells
were spotted to selective plates with either glucose or galactose as
the carbon source. Fig. 2A
shows that each of the strains grew well on glucose medium regardless
of which plasmid they contained. This is expected, because Gal4 is dispensable for growth on glucose. As expected, each of the strains could grow on galactose medium when transformed with the wild-type GAL4 plasmid or the plasmid overexpressing gal4D. However,
only the sug2-1 strain could grow when transformed with
gal4D on a single-copy plasmid. The finding that
sug2-13 does not suppress gal4D demonstrates
allele specificity in suppression of gal4D by
SUG2 alleles.
Levels of gal4D in sug2 Mutant Strains--
To directly test the
hypothesis that defective protein degradation by proteasomes allows
gal4D protein accumulation and that this is responsible for suppression
of gal4D by sug2-1, we determined the levels of
the gal4D and Gal4 proteins in each strain. Strains were grown to mid-
to late-log phase and harvested quickly. Equal numbers of cells were
boiled directly in 2× SDS loading buffer. The samples were centrifuged
briefly to remove cell debris and then separated by SDS-PAGE. Western
blots were performed with anti-T7 epitope antibodies for detection of
gal4D and Gal4 proteins and with anti-cyclophilin antibodies as an
internal loading control. The levels of both the Gal4 and gal4D
proteins were slightly increased compared with wild-type in the
sug2-1 strain as shown in Fig. 2B. Surprisingly,
the levels of Gal4 and gal4D were more dramatically increased in the
sug2-13 strain. Similar results were obtained in three
separate experiments. These data show that suppression of
gal4D does not positively correlate with the levels of gal4D protein in the sug2 mutant strains. Of note, the
dramatically increased levels of gal4D seen in the sug2-13
strain are still much lower (~20-fold) than those seen in strains
carrying the plasmid overexpressing gal4D (Fig. 2B). As
previously shown, overexpression of gal4D on a multicopy plasmid
(~50-fold overexpression) conditions 60% of wild-type activation as
assayed by Temperature Sensitivity and Suppression of cdc68-1 by sug1 Mutant
Alleles--
The sug1-1 and sug1-20 alleles of
SUG1 have been previously characterized. As summarized in
the introduction, sug1-1 suppresses the mutant phenotype of
gal4D (1). The sug1-20 allele, in contrast, was
identified as a suppressor of the transcription factor
cdc68-1 (15). Both alleles have been found to confer
temperature sensitivity at 37 °C. Consistent with this, we found
that both the sug1-1 and sug1-20 alleles
conferred temperature sensitivity in the W303 background (Fig.
3A). To test suppression of
cdc68-1 by these sug1 alleles in the W303
background, a plasmid expressing cdc68-1 was transformed
into each strain of the congenic set. The chromosomal CDC68
gene was then deleted, and the strains were characterized as described
under "Experimental Procedures." These strains were then tested for
temperature sensitivity (Fig. 3B). Consistent with the
findings of Xu et al. (15), the sug1-20 allele
suppresses the temperature sensitivity of cdc68-1 at the
intermediate temperature of 35 °C. In contrast, sug1-1
does not. Therefore, the allele specificity of cdc68-1
suppression noted by Xu et al. is reproduced in the W303
background.
Suppression of gal4D by sug1 Alleles--
The ability of mutations
in sug1 to suppress the transcriptional phenotype of
gal4D was assayed in the same way as for sug2 alleles. Wild-type, sug1-1, and sug1-20 strains
from which GAL4 had been deleted were transformed with a
centromeric vector carrying the gal4D gene, a multicopy, 2 µM plasmid that overexpresses gal4D, or a centromeric
vector expressing wild-type GAL4. The Gal4 proteins produced
were epitope-tagged with three tandem copies of the T7 epitope tag as
previously described. The epitope-tagged proteins expressed from the
plasmids were the only source of Gal4 activity in the cell. Spotting
tests for growth on glucose and galactose were performed as described
above. As shown in Fig. 4A,
each strain was able to grow on glucose and galactose when either
wild-type Gal4 was expressed at normal levels or gal4D was
overexpressed. In contrast, gal4D was unable to support growth in the
wild-type and sug1-20 backgrounds. Only the strain carrying
the sug1-1 mutation was able to grow on galactose when gal4D
expressed at wild-type levels was the activator. Therefore, there is
allele specificity in the suppression of gal4D by
sug1 alleles.
Levels of gal4D in sug1 Mutant Strains--
If a defect in protein
degradation by the proteasome is responsible for accumulation of gal4D
and growth of strains expressing ga4D on galactose, we would expect
gal4D levels be higher in the sug1-1 background than the
wild-type and sug1-20 strains. To test this hypothesis, the
levels of the gal4D and Gal4 proteins in each strain were determined by
Western blot as described above. The levels of both the Gal4 and gal4D
proteins were elevated in the sug1-1 strain compared with
wild-type strain (Fig. 4B). However, the level of gal4D
protein was more dramatically elevated in the sug1-20
background. Similar results were obtained in three separate experiments. Therefore, suppression of gal4D does not
positively correlate with the levels of gal4D in the sug1
mutant strains. Although the levels of gal4D are increased in the
sug1-20 strain, they are not as high as in the
gal4D-overexpressing strain. For the same reasons noted above regarding
the sug2-13 strain, the lack of gal4D
complementation in the sug1-20 strain is not surprising. We conclude
that stabilization of gal4D due to a defect in proteasomal proteolysis
cannot explain suppression of gal4D by sug1-1.
This is entirely consistent with the results obtained for
sug2-1 (Fig. 2).
Levels of Gal4 Protein and Growth on Galactose in Strains with
Defects in 20 S Proteasome Function--
The experiments above suggest
that a change in the proteolytic function of the proteasome is not
responsible for the suppression of gal4D by
sug1-1 and sug2-2. Both Sug1 and Sug2 are
components of the 19S regulatory complex of the proteasome. As an
additional test of this hypothesis, we examined the levels of gal4D and
growth on galactose in strains carrying mutations in subunits of the 20S proteasome. Strains carrying mutations in 20S proteasome subunits have been previously described. WCG4-11/22a (pre1-1 pre2-2)
and yH129/14 (pre1-1 pre4-1) each have mutations in two 20S
subunits (19, 20). They both have severe defects in protein degradation by the proteasome. We deleted the chromosomal copy of GAL4
from each of these strains and from the congenic wild-type strain
WCG4a. We then examined the levels of Gal4 and gal4D proteins expressed from centromeric vectors in the strains produced. As shown in Fig.
5A the levels of both gal4D
and Gal4 were significantly increased in both 20 S mutant strains when
compared with the wild-type. They were not, however, increased to the
level of gal4D expressed from a multicopy (2 µm) vector. These
strains were also assayed for the ability to grow on galactose as the
sole carbon source. As shown in Fig. 5B, all three strains
were able to grow on glucose and galactose when transformed with a
plasmid expressing wild-type Gal4 or a multicopy plasmid expressing
gal4D. However, when gal4D was expressed from a single-copy centromeric
vector, none of the strains were able to grow on galactose as the sole
carbon source. Therefore, even though these strains have a severe
defect in proteolysis, the increase in the levels of gal4D due to this
defect is not sufficient to allow gal4D to function for galactose
metabolism. We conclude that the transcription phenotypes of
sug1-1 and sug2-1 cannot be explained by
alterations in proteasome-mediated proteolysis of gal4D protein.
The finding that Sug1 and Sug2 are components of the proteasome
suggested a simple explanation for the suppression of gal4D by
sug1-1 and sug2-1: A defect in degradation by the
proteasome in sug mutant strains might lead to an
accumulation of gal4D protein. Although gal4D lacks much of its
activation domain, at high levels it might activate sufficient
transcription to allow growth on galactose. This argument was given
credence by the finding that high level overexpression of gal4D from a
high-copy-number plasmid does partly rescue its function2
(Figs. 2A and 4A). However, we have shown here
that there is no positive correlation between the levels of gal4D
conditioned by the sug1 and sug2 mutant alleles
and their ability to suppress the gal4D transcriptional
defect. In fact, the correlation is reversed. Although
sug2-13 stabilizes gal4D much more than sug2-1, it fails to suppress gal4D phenotype. Likewise,
sug1-20 stabilizes gal4D more than sug1-1, but
only sug1-1 suppresses gal4D. These results imply
that if inefficient proteolysis is responsible for the suppression of
gal4D by sug1-1 and sug2-1, then gal4D
protein is not the proteolytic target responsible for the phenotype.
Reinforcing this conclusion, strains that have very severe defects in
proteolysis due to mutations in 20S subunits are not able to grow on
galactose when gal4D is the activator (Fig. 5B).
We propose two possible explanations for these data. The
rescue of gal4D by sug1-1 and sug2-1
may be due to altered proteolysis of an as yet unidentified
transcription factor, which enhances the weak activity of gal4D
protein. This model would imply that this putative factor is stabilized
by some mutations in SUG1 and SUG2, but not by
others. Furthermore, this putative factor would have to be degraded
normally in the 20 S double mutant strains, even though they have a
severe defect in general degradation of proteins by the proteasome.
Though we feel that this explanation is unlikely, we cannot rule it
out. Such a putative factor could be identified by transforming a
gal4D strain with a multicopy library derived from a
An alternate explanation is that the 26S proteasome or its 19S
regulatory subunit may be involved in the transcriptional process in a
nonproteolytic fashion. It has long been speculated that proteasomal
ATPases are responsible for unfolding substrate proteins so that they
can be threaded into the proteolytic chamber of the proteasome
(25-27). We have suggested, by analogy to activities of the
structurally and functionally related ClpX ATPase in Escherichia coli (28, 29), that the 19S ATPases might also have a
"chaperone-like" role in rearranging protein complexes without
degrading them (30). This proposal was prompted by our findings
suggesting that proteasomal ATPases were important for nucleotide
excision repair (NER) in yeast, but that the proteolytic activity of
the proteasome was not (30). Specifically, we found that mutations in
SUG1 resulted in sensitivity to ultraviolet light in
vivo and that antibodies to Sug1 inhibited NER in
vitro. Surprisingly, mutations in 20S proteasome subunits do not
lead to sensitivity to ultraviolet light and the potent proteasome
inhibitor lactacystin has no effect on NER in vitro (30).
Consistent with a nonproteolytic role for proteasomal ATPases, the 19 S
regulatory complex of the proteasome has now been shown to possess
classically defined chaperone activity using model substrates (31). Of
note, the folding substrate was not degraded, even in the presence of
the 20S proteolytic component of the proteasome (31). This suggests
that activities of the 19S regulatory complex and the 20S proteolytic
core are not obligatorily linked.
Consistent with a nonproteolytic role for the proteasome in
transcription, antibodies to Sug1 inhibit yeast in vitro
transcription, although control antibodies do not. In this same
in vitro transcription system, complete inhibition of
proteasome peptidase activity by lactacystin has no effect on
transcription.4 These
findings are consistent with a role for Sug1 in transcription that is
not dependent on alterations in the proteolytic activity of the proteasome.
In contrast to our results, Molinari et al. (32) have
reported a different connection between the proteasome and
transcriptional activators. They have shown that, at least for chimeric
activators in mammalian cells, impairment of transcription through
mutations of DNA-binding or activation domains leads to an increase in
steady-state levels of the activator. The implication is that
transcriptionally engaged activators are preferentially degraded by the
proteasome. In contrast, we show that levels of the crippled gal4D
activator are less than those of wild-type Gal4. Although production of more gal4D leads to more transcriptional activity, selected mutations in SUG1 and SUG2 can rescue gal4D in a way that
cannot be explained by a simple increase in activator levels. The
implication of our results is that components of the 19 S regulatory
complex may have nonproteolytic roles in transcription. We feel our
results and those of Molinari et al. are probably
manifestations of the proteasome having more than one role in
transcription. In this regard, we have recently found that the 19S
sub-complex of the proteasome functions in transcription elongation in
yeast independent of its proteolytic
activity.5
Another conclusion from the results we report here is that mutations in
the SUG1 and SUG2 genes can produce very
distinctive phenotypes. It had previously been reported that the
sug1-3/cim3 allele did not suppress the gal4D
transcription phenotype (1), although it does suppress the cell cycle
defect of cdc28N-1 (22). Nor did the sug1-1
allele suppress the cdc68-1 defect (which is suppressed by
sug1-20) even though CDC68, like GAL4, is a
transcription factor (15). We now report that neither the
sug1-20 nor the sug2-13 allele can suppress the
gal4D phenotype. It appears that each SUG1 and
SUG2 allele selected has very specific phenotypes. This
allele specificity argues against a common pathway, such as
proteolysis, as an explanation for all of the identified phenotypes. It
further implies that the 19S regulatory complex may have complex regulatory functions beyond regulating the time and manner of protein
degradation by the 20S proteasome.
Clearly, more work is required to define the function of the proteasome
in transcription and DNA repair. The work presented here gives strong
evidence that alterations in proteolysis cannot explain the
transcription phenotypes of sug1-1 and sug2-1. As such, it clears the way for focusing on the exploration of alternative models and provides a second example of the nonproteolytic role of the
proteasome, further expanding the functional repertoire of this complex
protein machine.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (67K):
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Fig. 1.
Temperature sensitivity and cycloheximide
resistance of sug2 mutant strains. Serial 10-fold dilutions of
congenic strains carrying different alleles of sug2 were spotted onto
YEPD plates. A, the sug2-13 allele confers no
detectable cycloheximide resistance in the W303 background. Diluted
cultures were spotted onto control plates or plates containing 1 µg/ml cycloheximide. The W303-derived strains carrying the
sug2-1 and sug2-13 alleles were consistently more
sensitive to cycloheximide than the congenic wild-type strain.
B, the sug2-13 mutation confers dramatic
temperature sensitivity at 37 °C, whereas sug2-1 does
not. Identical plates were incubated at either 30 °C or 37 °C for
2 days. The sug2-13 strain consistently grew less robustly
at 30 °C than the wild-type and sug2-1 strains and showed
no growth at 37 °C, even at low dilution.
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Fig. 2.
Suppression of gal4D is specific for the
sug2-1 allele and does not positively correlate with levels of gal4D
protein. Congenic gal4 strains containing different
alleles of sug2 were transformed with gal4D on
either a single-copy vector (4D) or a multicopy vector
(mc 4D), with wild-type GAL4 on a single-copy
vector (4), or with multicopy vector alone
(vector or
). In each case the gal4D and Gal4 proteins
were tagged at the N terminus with three copies of the T7 epitope tag.
A, the sug2-1 mutation allows gal4D expressed
from a single-copy vector to promote growth on galactose as the sole
carbon source, but the sug2-13 mutation does not. Wild-type
Gal4 and overexpressed gal4D allow growth on galactose in all strain
backgrounds. Yeast strains were grown in liquid culture to log
phase, and serial 10-fold dilutions were spotted onto selective plates
with either glucose or galactose as the sole carbon source. The glucose
and galactose plates were incubated at 30 °C for 2 or 3 days,
respectively. B, the sug2-13 mutation leads to a
large increase in Gal4 and gal4D protein levels, whereas the
sug2-1 mutation has a smaller effect. Yeast strains were
grown in liquid medium, harvested from log-phase cultures, and
boiled directly in 2× SDS-PAGE loading buffer. The extracts were
separated on a long SDS-polyacrylamide gel and analyzed by Western
blotting. The Gal4 and gal4D proteins were detected with a monoclonal
antibody against the T7 epitope tag. The blots were also probed with
anti-cyclophilin antibodies to verify equivalent loading. The lower
band seen on the cyclophilin blot is presumed to be a cyclophilin
degradation intermediate and is more abundant in strains with mutations
in proteasome subunits (see also Figs. 4B and Fig.
5B).
-galactosidase assays (8). The threshold for growth on
plates is ~20% of wild-type activity. Therefore, assuming a linear
response of activation to activator levels, the lack of complementation
in the sug2-13 strain is not surprising. We conclude that
stabilization of gal4D protein due to a defect in proteasomal
proteolysis cannot explain suppression of gal4D by
sug2-1.
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Fig. 3.
Temperature sensitivity and suppression of
cdc68-1 in sug1 mutant strains. Serial 10-fold dilutions of
congenic strains carrying different alleles of sug2 were
spotted onto YEPD plates. A, the sug1-1 and
sug1-20 mutations confer temperature sensitivity at
37 °C. Identical plates were incubated at either 30 °C or
37 °C for 2 days. B, the sug1-20 allele
rescues the temperature sensitivity of cdc68-1 in the W303
background but sug1-1 does not. Identical plates were
incubated at either 30 °C or 35 °C for 3 days.
View larger version (64K):
[in a new window]
Fig. 4.
Suppression of gal4D is specific for the
sug1-1 allele and does not positively correlate with levels of gal4D
protein. Congenic gal4 strains containing different
alleles of sug1 were transformed with gal4D on
either a single-copy vector (4D) or a multicopy vector
(mc 4D), with wild-type GAL4 on a single-copy
vector (4), or with multicopy vector alone (
). In each
case the gal4D and Gal4 proteins were tagged at the N terminus with
three copies of the T7 epitope tag. A, the sug1-1
mutation allows gal4D expressed from a single-copy plasmid to promote
growth on galactose as the sole carbon source, but the
sug1-20 mutation does not. Wild-type Gal4 and overexpressed
gal4D allow growth on galactose in all strain backgrounds. Yeast
strains were grown in liquid culture to log phase, and serial
10-fold dilutions were spotted onto selective plates with either
glucose or galactose as the sole carbon source. The glucose and
galactose plates were incubated at 30 °C for 2 or 3 days,
respectively. B, the sug1-20 mutation leads to a
larger increase in Gal4 and gal4D protein levels than the
sug1-1 mutation. Cell extracts were prepared and assayed for
epitope-tagged Gal4 and gal4D proteins and for cyclophilin protein as a
loading control, as described in Fig. 2.
View larger version (58K):
[in a new window]
Fig. 5.
Mutations in 20S proteasome subunits increase
the steady-state levels of gal4D protein but do not suppress
gal4D. Congenic gal4 strains containing either
wild-type 20S proteasome or proteasomes with mutations in two 20S
subunits (pre1-1 pre2-2 or pre1-1 pre4-1) were
transformed with either gal4D on a single-copy
(4D) or a multicopy (mc 4D) vector, or wild-type
GAL4 (4) on a single-copy vector. The gal4D and
Gal4 proteins were tagged at the N terminus with three copies of the T7
epitope tag. A, strains carrying double mutations in 20S
proteasome subunits have higher steady-state levels of gal4D protein
than the congenic wild-type strain. Cell extracts were prepared and
assayed for epitope-tagged Gal4 and gal4D proteins, and for cyclophilin
protein as a loading control, as described in Fig. 2. Note that the
WT mc 4D lane was under-loaded relative to the other lanes.
B, expression of gal4D does not allow growth on
galactose in strains carrying double mutations in 20S proteasome
subunits. Wild-type levels of Gal4 and overexpressed gal4D allow growth
on galactose in all strain backgrounds. Serial 10-fold dilutions of
yeast grown in liquid culture were spotted onto selective plates with
either glucose or galactose as the sole carbon source. The glucose and
galactose plates were incubated at 30 °C for 2 or 3 days,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GAL4 strain. Clones allowing growth on galactose could
then be selected from the library. Overexpression of this putative
factor should suppress gal4D just as its stabilization by
sug1-1 or sug2-1 does.
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ACKNOWLEDGEMENTS |
---|
We thank James Haber and John McCusker for bringing the identity of SUG2 with CRL13 to our attention and for supplying yeast strains; Kathryn Sykes for anti-cyclophilin antiserum; Gerry Johnston for plasmids and strains; Dieter Wolf, Wolfgang Hilt, and Wolfgang Heinemeyer for strains; and Xiang Chen and Eunice Webb for invaluable technical assistance. We thank the Johnston Laboratory for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Internal
Medicine, University of Texas-Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75390-8573. Tel.: 214-648-1415; Fax:
214-648-1450; E-mail: stephen.johnston@utsouthwestern.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010889200
2 J. C. Swaffield and S. A. Johnston, unpublished data.
3 J. E. Haber, personal communication.
4 C. J. Jeon, S. J. Russell, and S. A. Johnston, unpublished data.
5 A. Ferdous, L. Sun, F. Gonzalez, T. Kodadek, and S. A. Johnston, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: AAA, ATPases Associated with diverse cellular Activities family; bp, base pair(s); PCR, polymerase chain reaction; 5-FOA, 5-Fluoro-orotic acid; PAGE, polyacrylamide gel electrophoresis; NER, nucleotide excision repair.
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