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Journal of Bacteriology, December 1998, p. 6484-6492, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Suppression of an Hsp70 Mutant Phenotype in Saccharomyces
cerevisiae through Loss of Function of the Chromatin
Component Sin1p/Spt2p
Bonnie K.
Baxter and
Elizabeth A.
Craig*
Department of Biomolecular Chemistry,
University of Wisconsin, Madison, Wisconsin 53706
Received 29 May 1998/Accepted 9 October 1998
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ABSTRACT |
The Ssa subfamily of Hsp70 molecular chaperones in the budding
yeast Saccharomyces cerevisiae has four members, encoded by SSA1, SSA2, SSA3, and
SSA4. Deletion of the two constitutively expressed genes,
SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that
an extragenic suppressor of the temperature sensitivity of ssa1
ssa2 strains, EXA1-1, is a loss-of-function mutation
in SIN1/SPT2, which encodes a nonhistone component of
chromatin. Loss of function of Sin1p leads to overexpression of
SSA3 in the ssa1 ssa2 mutant background, at a
level which is sufficient to mediate suppression. In a strain
which is wild type for SSA genes, we detected no effect of
Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of SSA3 in the
ssa1 ssa2 mutant background as well as in
heat-shocked wild-type strains is mediated by the heat shock
transcription factor HSF. Our findings suggest that it is HSF-mediated
induction of SSA3 which is modulated by Sin1p. The
EXA1-1 suppressor mutation thus improves the growth of
ssa1 ssa2 strains by selectively increasing HSF-mediated
expression of SSA3.
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INTRODUCTION |
The Hsp70s (heat shock proteins of
70 kilodaltons) comprise a highly abundant and well-conserved family of
molecular chaperones, involved in facilitating a wide variety of
cellular processes. In the budding yeast Saccharomyces
cerevisiae, one of the most abundant subfamilies of Hsp70 is the
cytosolic Ssa subfamily. There are four Ssa proteins, which have more
than 80% amino acid identity with each other. Together, the Ssa
proteins are essential for vegetative growth; at least one must be
expressed at high levels in order for cells to be viable
(39). The four subfamily members have distinct patterns of
regulation. During exponential growth, only Ssa1p and Ssa2p are
detectable; expression of Ssa3p and Ssa4p requires induction by heat
shock or other stressors (38). Processes in which the Ssa
proteins have been implicated include translocation of substrates into
mitochondria and the endoplasmic reticulum (3, 11, 13),
thermotolerance (33), and protein folding (24).
Ssa proteins also function in the autoregulation of the heat shock
response, as was demonstrated by the observation that overexpression of
either Ssa1p or Ssa4p interferes with induction of a reporter gene
under the control of a heat shock promoter element (HSE)
(37).
The importance of Ssa protein in the regulation of heat shock genes has
also become apparent in the analysis of ssa1 ssa2 double
mutant strains. ssa1 ssa2 strains, lacking functional copies of the two constitutively expressed subfamily members, are unable to
form colonies at elevated temperatures and exhibit slow growth at
permissive temperatures (12). The physiology underlying this phenotype is likely to be complex. The presence of a wild-type copy of
SSA4 allows the strain to survive, as indicated by the inviability of an ssa1 ssa2 ssa4 triple mutant
(39). Neither SSA3 nor SSA4 would
ordinarily be expressed during exponential growth; their expression in
ssa1 ssa2 mutant strains (which appears to be stronger for
Ssa4p than for Ssa3p) is mediated by a generalized, constitutive
induction of HSE-mediated transcription (5, 7). This
induction is not specific to SSA3 and SSA4, as an
HSE-regulated marker gene is also induced (7) and a number
of heat shock-regulated genes are constitutively expressed in this
background (12, 12a; this report). ssa1
ssa2 cells can therefore be thought of as constitutively heat-shocked.
The complexity of this situation became apparent in the previous
analysis of an extragenic suppressor of the slow growth of ssa1
ssa2 cells, a suppressor called EXA3-1 (for
extragenic suppressor of Hsp70 subfamily A). EXA3 is
allelic to HSF1, which encodes the heat shock
transcription factor HSF (28). The
EXA3-1 mutation creates an amino acid substitution in
the DNA-binding domain of HSF, a change which reduces HSF-mediated
expression under both basal and heat-shock conditions. Ssa protein is
thus lower in ssa1 ssa2 EXA3-1 cells than in the parental
ssa1 ssa2 strain. Furthermore, increasing HSF-mediated
expression by introducing an extra copy of HSF1 into
ssa1 ssa2 cells causes further impairment of growth rather
than suppression (16). These data clearly demonstrate that a
cytosolic deficiency in Ssa protein in just one aspect of the phenotype
of the ssa1 ssa2 strain. The constitutive expression of heat
shock genes which allows these cells to survive (by enabling expression
of SSA3 and SSA4) introduces a secondary problem:
likely the overexpression of another heat-inducible protein(s) which is
detrimental for growth. The EXA3-1 mutation represents a
delicate solution to this problem, adjusting heat shock gene expression so that the toxicity of the response is minimized while Ssa
protein is maintained at levels which, though reduced, are
apparently sufficient for growth.
Here we report the characterization of another extragenic suppressor of
the temperature sensitivity of ssa1 ssa2 strains, EXA1 (28). We show that EXA1 is
allelic to SIN1/SPT2, which encodes a nonhistone component
of chromatin. Sin1p/Spt2p is an abundant, nonessential,
nucleus-localized protein with the ability to bind DNA nonspecifically
in vitro and to affect expression of a variety of loci in vivo
(21). SIN1 (for Swi-independent transcription) is
so named because loss of Sin1p function allows constitutive expression
of a group of genes normally dependent on the multiprotein Swi/Snf
complex (36). The Swi/Snf complex from both yeast and human
cells has the ability to alter nucleosomal structure in chromatin,
rendering promoter templates more accessible to activators such as
Gal4p and general transcription factors such as TATA-binding protein
(8, 18, 19, 22, 30). Conversely, Sin1p is thought to act at
some promoters to maintain chromatin in a repressive state,
inaccessible to the transcription machinery. Mutations in
SIN1 also allow increased expression of marker genes whose
promoters have been disrupted by the transposable element Ty; hence,
its alternate name is SPT2 (for suppressor of Ty insertion) (32, 40). For simplicity, we will use the name
SIN1 throughout this report.
We show that loss of function of Sin1p mediates the suppression
conferred by EXA1-1 and increases the HSF-mediated
expression of Ssa3p. Expression of other heat-shock-regulated
genes, including Ssa4p and Hsp104, is not increased. In fact,
overexpression of Ssa3p in the EXA1-1 suppressor strain may
serve to downregulate other heat shock-responsive genes, thus
simultaneously increasing levels of Ssa protein while decreasing
expression of any heat-inducible proteins which are detrimental for growth.
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MATERIALS AND METHODS |
Strains, media, and genetic techniques.
Escherichia
coli cells were grown in LB (0.5% yeast extract, 1% tryptone,
1% NaCl) supplemented with 100 µg of ampicillin per ml as necessary
for plasmid selection. E. coli cells were transformed by
electroporation with a Gene Pulser apparatus (Bio-Rad Laboratories,
Hercules, Calif.) according to the manufacturer's instructions or by a
CaCl2-based protocol (25). Restriction enzymes
and buffers were from New England Biolabs (Beverly, Mass.), Promega
(Madison, Wis.), or Boehringer Mannheim (Indianapolis, Ind.) and were
used according to manufacturer's instructions.
Yeast strains were grown in YPD (1% yeast extract, 2% peptone, 2%
dextrose) or in selective medium (0.67% yeast nitrogen base without
amino acids, 2% dextrose, supplemented with required amino acids as
necessary). Liquid selective medium used for immunoblot analysis and
determination of growth curves contained an elevated level of dextrose
(6%) to delay the diauxic shift. Yeast strains were transformed by
electroporation using the Gene Pulser apparatus (Bio-Rad Laboratories)
or according to a modified lithium acetate protocol (14).
Matings were done on YPD, with selection of zygotes by
micromanipulation. Sporulation plates contained 1% potassium acetate,
0.1% yeast extract, 0.05% dextrose, and amino acid supplements.
Yeast strains are shown in Table
1.
ssa1 and
ssa2 disruptions in MW116 and MW163 were
constructed by inserting an auxotrophic
marker gene into the coding
region, replacing codons 307 to 386;
ssa3 and
ssa4 disruptions in these strains are insertions of the
markers indicated at positions near the 5' end of each gene.
ssa1-3::HIS3 and
ssa2-2::URA3
mutants each carry a replacement of codons 10
to 160 with the
respective marker gene. A comparison of strains
carrying the original
ssa1 and
ssa2 disruptions (those used in
MW116
and MW163) with those carrying the more extensive deletions,
ssa1-3 and
ssa2-2, found them to be
phenotypically indistinguishable
(
39).
Isolation of extragenic suppressors of the
ssa1 ssa2
high-temperature growth defect, including
EXA1-1, has been
previously
described (
28). To identify and clone
EXA1-1, a high-copy-number
genomic library was prepared from
an
ssa1-3 ssa2-2 EXA1-1 strain
(
28) by
Sau3A partial digestion and ligation into
BamHI-digested
YEp351 (
17). This library was
transformed into an
ssa1 ssa2 strain, and transformants were
screened for improved high-temperature
growth.
Disruption of
SIN1 by linear transformation was conducted
with plasmid construct WB51 (
21), a generous gift of Ira
Herskowitz.
This construct replaces an 805-bp
PstI-
HindIII fragment of
SIN1 (which encodes amino acids [aa] 48 to 315 of 333 aa) with the
marker
gene
TRP1. Disruption was confirmed by PCR and restriction
analysis.
RNA analysis.
Cells were either grown to mid-log phase at
32°C or grown to mid-log phase at 23°C and subjected to a 30-min
heat shock at 39°C as indicated and harvested by centrifugation.
Total cellular RNA was isolated by the heat-freeze extraction method as
previously described (34) and quantitated spectrophotometrically.
S1 nuclease protection assays were carried out essentially as described
previously (
2). For detection of
SSA3 mRNA, an
oligonucleotide was synthesized which is complementary to nucleotides
(nt) +29 to
23 (where the AUG start codon is nt +1 to +3) and
which
carries an 8-nt 3' overhang. (Synthesis was by Genosys,
Inc., The
Woodlands, Tex.) The oligonucleotide was purified by
sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
eluted into 0.3 M sodium acetate, phenol extracted, ethanol precipitated,
and
spectrophotometrically quantitated before labeling with polynucleotide
kinase and hybridization to total RNA. For standardization, a
second
oligonucleotide, which is complementary to nt 295 to 258
of the mature
actin RNA and which carries a 26-nt 3' overhang,
was used in parallel.
Data from four separate sets of S1 digestion
reactions gave similar
results and were combined to give mean
values (see Fig.
4A).
Primer extension analysis was performed essentially as described
previously (
2), with an 18-nt primer complementary to
nt 52 to 35 of
SSA4 to generate a 106-nt product. For
standardization,
an oligonucleotide complementary to the snRNA U4 was
used to generate
a 44-nt product. Three sets of similar primer
extension results
were combined to give mean values (see Fig.
4B).
SSA3 promoter fusion plasmids.
The promoter
fusion vectors used to express SSA3 at various levels were
constructed by Mumberg et al. (26). PCR was used to create
an SpeI site immediately upstream of the start codon of
SSA3 for fusion to the various promoters. All PCR-generated sequences were confirmed by dideoxy sequencing using Sequenase (United
States Biochemical, Cleveland, Ohio) to verify that no PCR-induced
mutations had been introduced. Ligation of a 2.2 kb SpeI-HindIII SSA3 fragment into
the expression vectors put SSA3 under the control of the
ADH1, TEF2, or GPD promoter on either a low or a high-copy-number vector, as indicated in Table
2. The plasmids are ordered from 1 to 6 (Table 2) according to the level of 
-galactosidase activity
expressed from a similar fusion of lacZ to each construct,
as reported by Mumberg et al. (26). These expression levels
correlated well with our observations for Ssa3p (see Fig. 5A).
Growth tests.
Growth tests on agar plates were routinely
conducted by growing cells overnight at permissive temperatures and
then diluting them into sterile water as a series of 10-fold serial
dilutions. Aliquots of each dilution were spotted onto agar plates
which were incubated at the temperatures indicated. Liquid growth tests were conducted by growing cultures overnight at the temperature indicated, verifying that they were in exponential growth, and monitoring their growth through optical density (OD) measurements (at
600 nm) taken approximately every 45 to 75 min over a period of 6 to
12 h. These measurements were used to construct an exponential growth curve, which was used to calculate doubling time. As indicated in the figure legends, each doubling time calculation was repeated for
multiple transformants and multiple cultures, and these data were
combined to obtain an average population doubling time and the standard
error of measurement for each strain.
Anti-Ssa3p/Ssa4p antibodies.
PCR was used to generate a
BamHI site immediately upstream of codon 539 and an
EcoRI site immediately downstream of codon 603 of both
SSA3 and SSA4. Each
BamHI-EcoRI fragment was cloned into pGEX-KT
(15) to create a fusion of
glutathione-S-transferase to a 65-aa peptide of either Ssa3p
or Ssa4p. Each fusion protein was expressed in E. coli and
purified by adsorption to glutathione-agarose beads (Sigma Chemical
Co., St. Louis, Mo.). Eluate from the beads was used to inoculate rabbits.
Rabbit antisera generated against the Ssa3 fusion protein and the Ssa4
fusion protein had indistinguishable reactivities.
Neither showed any
reactivity against Ssa1p or Ssa2p, and both
showed a similar reactivity
with Ssa3p and Ssa4p. This result
is not surprising, given that 49 of
the 65 Ssa amino acids in
the fusion constructs are identical between
Ssa3p and Ssa4p and
many of the remaining residues are conservative
substitutions.
The antiserum raised against the Ssa3 fusion protein was
arbitrarily
chosen for use in these
experiments.
Immunoblot analysis.
For immunoblot analysis, protein
extracts were prepared by vortexing cells in the presence of glass
beads in a buffer containing 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris (pH 8.0), and 1 mM EDTA; boiling the lysates in Laemmli sample
buffer (4); and clarifying the extracts by centrifugation in
a microcentrifuge at top speed for 5 min. For each experiment, a
preliminary polyacrylamide gel was run and stained with Coomassie blue,
and the intensity of Coomassie staining in each lane was quantitated by
densitometric scanning and used to normalize loadings. Equivalent
amounts of each extract were then loaded on a second polyacrylamide gel
and transferred to nitrocellulose. Membranes were blocked in
Tris-buffered saline (20 mM Tris [pH 7.4], 150 mM NaCl) containing
0.5% polyoxyethylene-sorbitan monolaurate (Tween 20; Sigma Chemical
Co.) as a blocking agent; this solution was also used for incubations
with both primary and secondary antibodies. Detection utilized
secondary anti-rabbit immunoglobulin G antibody conjugated to
horseradish peroxidase and the ECL Western blotting detection kit
(Amersham Corp., Arlington Heights, Ill.)
Ssa3p and Ssa4p are similar in predicted size (70,554 versus 69,657 Da)
and are not always separated by SDS-PAGE. To achieve
reliable
separation, we used 7.5% polyacrylamide gels which contained
0.3%
bisacrylamide, rather than the more standard 0.2%. Gels (11
cm by 16 cm by 0.75 mm) were electrophoresed at 30 mA for approximately
4 h, at which point the Ssa proteins had migrated about halfway
through
the separating gel. When this procedure was used, Ssa3p
consistently
migrated more slowly than Ssa4p (see in Fig.
2 to
4). Use of this
protocol allowed us to determine that the protein
which was originally
observed to accumulate in
ssa1 ssa2 EXA1-1 strains
(
28) is in fact Ssa3p and that this protein comigrates
with
the Ssa3p expressed in strains which are wild-type at the
EXA1/SIN1 locus.
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RESULTS |
Identification of EXA1-1.
EXA1-1 (Extragenic
suppressor of Hsp70 subfamily A) was isolated as a dominant suppressor
of the temperature sensitivity of an ssa1 ssa2 double
deletion strain (28). To clone the gene responsible for
suppression, a genomic library was constructed from an ssa1 ssa2
EXA1-1 haploid strain by using a high-copy-number yeast vector
(see Materials and Methods). Library plasmids were transformed into an
ssa1 ssa2 strain and screened for the ability to confer
improved high-temperature growth. One class of plasmids identified in
this screen were those carrying either of the remaining two
SSA gene family members, SSA3 or SSA4,
indicating that when present in high copy number, either
SSA3 or SSA4 is able to functionally substitute
for the lack of SSA1 and SSA2. Suppression by
another library plasmid, p24, is shown in Fig.
1A.
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FIG. 1.
Suppression of the ssa1 ssa2 growth defect by
library plasmid p24. (A) ssa1 ssa2 strain BB363 was
transformed with either a vector control or with library plasmid p24.
Transformants were selected, grown at permissive temperatures, and then
tested for improved growth by spotting 10-fold serial dilutions onto an
agar plate containing a medium selective for the plasmid. The plate
shown was incubated at 34°C for 3 days. (B) Subclone analysis of p24.
The yeast insert contained on each plasmid tested is represented
schematically by a rectangular box, with the ability (+) or
inability () of each construct to cause suppression indicated to the
right. Filled-in boxes represent disruption of the NdeI site
in RAD4 (by filling in the 2-nt 5' overhang) or the
PstI site in SIN1 (by deletion of the 4-nt 3'
overhang). Both disruptions alter the reading frame of the affected
gene. (C) Deletion of SIN1 causes suppression. Haploid
progeny of diploid strain BB358 were isolated, tested for the indicated
mutations by replica plating, and spotted as 10-fold serial dilutions
to test growth. The YPD plate shown was incubated at 34°C for 3 days.
In both panels A and C, approximately equal numbers of cells were
spotted for each strain. WT, wild type.
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As diagrammed in Fig.
1B, suppressor plasmid p24 contains a 4.77-kb
insert which includes the 3' end of
RAD4 (approximately
50%
of the coding region), the entire
SIN1/SPT2 open reading
frame,
and approximately 24% of an adjacent full-length Ty1
transposable
element. Truncation of the p24 insert fragment at a
HindIII site
at the 3' end of
SIN1/SPT2 had
no effect on suppression (Fig.
1B), indicating that the Ty1 element is
not relevant to suppression.
Similarly, disruption of the
RAD4 coding region at an internal
NdeI site had
no effect. By contrast, disruption of the
SIN1/SPT2 coding
region at a
PstI site near the 5' end eliminated
suppression,
demonstrating that it is
SIN1/SPT2 which
is responsible for the
ability of p24 to confer improved
growth. Interestingly, truncation
at the
HindIII site
removes the last 17 codons of
SIN1/SPT2 without
affecting
suppression. This result was explained when the
SIN1/SPT2 region of the
EXA1-1 library plasmid was subcloned and
sequenced
and a nonsense mutation which introduces a stop codon 5' of
the
HindIII site, at codon 227 of 333, was
revealed.
We predicted that a nonsense mutation at codon 227 of
SIN1
would generate a dominant negative allele, since a previous genetic
analysis of
SIN1 found that truncation of the protein coding
region
at positions corresponding to aa 179, 213, 271, 303, 318, or 324
results in a dominant negative phenotype (
23). To verify
that
suppression of the
ssa1 ssa2 growth phenotype resulted
from a
loss of Sin1p function, we generated a chromosomal deletion of
SIN1 in a diploid strain already heterozygous for deletion
of
both
SSA1 and
SSA2, creating strain BB358 (see
Materials and Methods
for details). Haploid progeny of BB358 were then
isolated and
tested for growth. Deletion of
SIN1 has no
discernible effect
on growth of an otherwise wild-type strain, as
previously reported
(
40). However, growth of
ssa1
ssa2 strains is significantly
improved by deletion of
SIN1 (Fig.
1C). When compared side by
side, the level of
growth improvement achieved by deletion of
SIN1 was
indistinguishable from that generated by introduction
of suppressor
plasmid p24 (data not shown), strongly suggesting
that the allele of
SIN1 carried on the suppressor plasmid has
a dominant
negative
phenotype.
To confirm that
EXA1 is allelic to
SIN1,
ssa1 ssa2 EXA1-1 strain BB360 was crossed to
ssa1 ssa2
sin1 strain BB361. The resulting
diploid strain, BB366, was
sporulated and dissected, and haploid
progeny were tested for growth at
34°C. All progeny of 22 four-spore
tetrads showed growth comparable
to the parental strains and significantly
better than that of
ssa1 ssa2 strains, indicating that
EXA1-1 is
tightly linked to
SIN1. We conclude that the
EXA1-1 suppressor
mutation is the mutation identified on
library plasmid p24: the
introduction of a premature stop codon in
SIN1 in place of codon
227.
Loss of function of Sin1p causes overexpression of SSA3.
Sin1p is an abundant nuclear protein with nonspecific DNA-binding
activity whose function affects the expression of a wide variety of
genes (21, 31, 36, 40). We surmised that loss of Sin1p
function causes improved growth of ssa1 ssa2 strains by
altering expression of a gene or genes whose product is important for
growth of this strain. Given our isolation of both SSA3 and SSA4 as high-copy-number suppressors of the ssa1
ssa2 strain, altered regulation of one or both of these loci
through loss of function of Sin1p was an obvious hypothesis.
To facilitate analysis of Ssa3p/Ssa4p expression, antibodies were
raised against a protein fusion of glutathione-
S-transferase
and a peptide from the carboxyl terminus of Ssa3p (aa 539 to 603;
see
Materials and Methods). This region of Ssa3p has 75% amino
acid
identity with Ssa4p but only 48 and 45% identity with Ssa1p
and Ssa2p,
respectively. The resulting antibodies do not cross-react
with Ssa1p or
Ssa2p, as indicated by the absence of reactive bands
in an
extract from a wild-type strain in exponential growth (e.g.,
Fig.
2, lane 1). Previous studies using
[
35S]methionine and two-dimensional gel electrophoresis
have shown
that under these conditions, Ssa1p and Ssa2p are abundant,
while
Ssa3p and Ssa4p are undetectable (
39). The antibodies
do react
with both Ssa3p and Ssa4p, as demonstrated by analysis of
extracts
from strains MW163 and MW116, in which Ssa3p or Ssa4p,
respectively,
is the only Ssa protein expressed (lanes 17 and 18). This
analysis
also demonstrated that Ssa3p and Ssa4p are separable
under the
conditions used, with Ssa3p migrating slightly more slowly
than
Ssa4p (lanes 16 to 18).
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FIG. 2.
Loss of function of Sin1p increases Ssa3p expression in
ssa1 ssa2 strains. Cells were grown in YPD to mid-log phase
at the temperature indicated. Extracts were probed by immunoblot
analysis using an antiserum reactive against Ssa3p and Ssa4p (see
Materials and Methods). Extracts from MW163 and MW116 were used as
markers and were adjusted to give equivalent signals, requiring
approximately 1/15 as much MW163 extract as MW116 extract. WT, wild
type.
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Our immunoblot analysis confirmed previous observations (
39)
that Ssa3p and Ssa4p expression is induced in
ssa1 ssa2
cells
compared to wild-type cells (for example, compare lane 8 to lane
6). This effect is considerably more dramatic for Ssa4p, but Ssa3p
is
clearly detectable in
ssa1 ssa2 cells upon longer film
exposures.
Comparison of an
ssa1 ssa2 strain (lane 8) with
either an
ssa1 ssa2 sin1 strain or an
ssa1 ssa2
EXA1-1 strain (lanes 9 and 10)
demonstrated that loss of function
of Sin1p leads to further elevated
expression of Ssa3p. This effect was
evident during steady-state
growth at all temperatures tested, but was
only detectable in
strains carrying deletions of
ssa1 and
ssa2. Ssa3p was not detectable
under steady-state growth
conditions in strains which were wild-type
at
SSA1 and
SSA2, regardless of the presence or absence of function
of
Sin1p (for example, compare lane 7 with lane 6). It is possible
that
loss of Sin1p increases the expression of Ssa3p under these
conditions,
and yet the level of Ssa3p remains below the limit
of detection.
Alternatively, expression of Ssa3p in wild-type
cells during
exponential growth may be repressed by a mechanism
that does not
require Sin1p. This finding thus led us to the question
of whether we
could detect an influence of Sin1p on
SSA3 expression
in
cells which are wild-type for
Ssa.
In wild type cells, Ssa3p expression is normally induced in response to
two distinct physiological signals: heat shock and
the transition into
stationary phase. Heat shock induction is
mediated by an HSE in the
SSA3 promoter, a binding site for the
heat shock
transcription factor HSF (
5). During the transition
to
stationary phase, induction of
SSA3 is mediated by an
adjacent
element, the post-diauxic shift upstream activation
sequence,
whose activity is increased by the presence of the HSE.
No induction
occurs without this upstream activation sequence,
but the final
level of stationary-phase expression is increased by the
presence
of the HSE. (
6). To assess whether Sin1p plays a
role in modulating
Ssa3p expression in wild-type as well as
ssa1
ssa2 cells, we used
extracts from stationary-phase and
heat-shocked
cultures.
In stationary-phase extracts, Ssa3p was detectable in all
strains tested (Fig.
3A). In an
ssa1 ssa2 background, loss of function
of Sin1p led to
increased levels of Ssa3p, just as was seen in
extracts from
exponentially growing cultures (compare lanes 4
and 5 to lane 3). In
cells which were wild-type for
SSA, however,
loss of Sin1p
function had no detectable effect (compare lane
2 to lane 1). In
heat-shocked extracts, the picture was different
(Fig.
3B). Ssa3p
expression was again higher in
ssa1 ssa2 sin1 and
ssa1 ssa2 EXA1-1 strains than in an
ssa1 ssa2
strain, as was
seen for the other conditions tested (compare lanes 4 and 5 to
lane 3). Under conditions of heat shock, however, Ssa3p
expression
was also higher in a
sin1 strain than in a
wild-type strain (compare
lane 2 to lane 1 and lane 8 to lane 7). Since
both the elevated
SSA3 expression seen in an
ssa1
ssa2 strain and the induction
of
SSA3 by heat shock are
mediated by the HSE (
5), these findings
suggest that loss of
Sin1p function potentiates the ability of
the heat shock transcription
factor HSF to stimulate expression
of
SSA3. Interestingly,
although induction of
SSA4 is also mediated
by HSF
(
7), no increase in Ssa4p levels due to loss of function
of
Sin1p was detected under any conditions tested (Fig.
2 and
3).
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FIG. 3.
The effect of Sin1p function on Ssa3p expression is
apparent in heat-shocked but not stationary-phase cells. (A) As in Fig.
2, except cultures were grown at 30°C for 4 days to stationary
(stat.) phase. Combined extracts from MW116 and MW163 (log-phase
cultures [shown in Fig. 2]) were used as markers for Ssa3p and Ssa4p.
(B) Cultures were grown to mid-log phase at 23°C and then transferred
to 39°C for 75 min before harvest. Lanes 7 and 8 are the same as
lanes 1 and 2, except with twice as much protein (2×) loaded. WT, wild
type.
|
|
Given existing data about Sin1p and its role in transcriptional
regulation and chromatin structure (
21,
31,
36,
40),
we
predicted that alteration of
SSA3 expression by Sin1p occurs
at the level of transcription. To test this prediction, we monitored
SSA3 mRNA levels by S1 nuclease protection analysis in
ssa1 ssa2 strains with or without a functional copy of
SIN1 (Fig.
4A). As
expected,
loss of Sin1p function correlates with elevated levels
of
SSA3 message:
SSA3 mRNA levels were 3- to
4.5-fold higher in
ssa1 ssa2 sin1 or
ssa1 ssa2
EXA1-1 strains compared to an
ssa1 ssa2 strain. In
contrast,
SSA4 mRNA levels are decreased approximately
twofold by loss of Sin1p function, as assessed by primer extension
analysis (Fig.
4B).
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|
FIG. 4.
Sin1p affects SSA3 expression at the level of
mRNA. Cultures were grown to mid-log phase at 32°C. For comparison, a
wild-type culture was grown to mid-log phase at 23°C and subjected to
a 30-min heat shock at 39°C [WT(HS)]. RNA was prepared
and analyzed by S1 nuclease protection (A) or primer extension (B)
assay as described in Materials and Methods. Results were normalized to
those with strain ssa1 ssa2 and are reported as a mean of
three to four experiments, with error bars representing ± 2 standard errors.
|
|
Elevated Ssa3p expression is sufficient to explain
suppression.
To study the effects of Ssa3p expression on the
growth of an ssa1 ssa2 strain more closely, we constructed a
set of expression constructs in which the SSA3 protein
coding region was regulated by a series of three heterologous
promoters, each of which could be expressed from either a low- or a
high-copy-number plasmid (26). For simplicity, we will refer
to these plasmid constructs by the numbers 1 to 6, in order of the
level of expression they provide (Table 2). To assess Ssa3p expression
from these constructs in comparison with that generated by loss of
Sin1p function, cultures were grown to early log phase in a selective
liquid medium at 34°C. Extracts from these cultures were used in
immunoblot analysis, as shown in Fig. 5A.
As expected, Ssa3p expression increased progressively through the
series of promoter constructs. Promoter constructs 2 and 3 gave levels
of Ssa3p which were comparable to that generated by loss of Sin1p
function, while constructs 4 to 6 gave much higher levels of
expression.
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|
FIG. 5.
Expression of varying levels of Ssa3p in an ssa1
ssa2 strain. Cultures were grown at 34°C in a liquid medium
selective for plasmids 1 to 6 (Table 2). Plasmid numbers are given in
brackets; where no plasmid is indicated, strains were carrying the
vector plasmid pRS315. (A) Anti-Ssa3/4p immunoblot. Cells were
harvested in early log phase. Extracts were prepared and subjected to
SDS-PAGE and immunoblot analysis using antibodies reactive against
Ssa3p and Ssa4p. (B) Growth at 34°C throughout early log phase was
monitored by OD measurements. For each strain, average doubling time
was determined from at least two independent plasmid transformants and
from a total of at least three separate cultures. Error bars represent
±2 standard errors. WT, wild type.
|
|
To assess the effect of Ssa3p overexpression, growth tests were
performed on liquid cultures, and the cultures' optical densities
at
600 nm (OD) were used to monitor growth. OD measurements were
taken
throughout early log phase and used to calculate the doubling
time of
each strain. Combined measurements taken from multiple
transformants
are presented in Fig.
5B. In these assays, the plasmid
constructs
conferred varying degrees of growth, ranging from no
significant
improvement over the vector control for construct
1 to growth
approaching that of the wild type for construct 6.
Plasmid construct 2, which generated a level of Ssa3p expression
similar to that conferred
by lack of Sin1p function (Fig.
5A),
also conferred a degree of growth
improvement similar to that
seen in
ssa1 ssa2 sin1 or
ssa1 ssa2 EXA1-1 strains (Fig.
5B).
This result demonstrates
that the overexpression of Ssa3p caused
by lack of Sin1p function is
sufficient to mediate significant
improvement of
growth.
The improvement of growth conferred by each
SSA3 plasmid to
a population of cells in liquid culture under selective conditions
(Fig.
5B) does not correlate directly with the average level of
Ssa3p
expressed (Fig.
5A). This finding likely reflects the lower
stability of plasmids containing the 2µ origin of replication
compared to centromere-containing plasmids (
27) and a
resultant
underestimate of the growth improvement conferred by
2µ-based
plasmids 3, 5, and
6.
In the course of these experiments, we noted that overexpression of
Ssa3p from the promoter fusion constructs correlates with
lowered
expression of Ssa4p (Fig.
5A). This effect, although more
subtle, is
also detectable upon loss of function of Sin1p: Ssa4p
expression is
decreased slightly as Ssa3p expression is increased.
Immunoblot
analysis of extracts from the promoter fusion series
demonstrated that
Hsp104 expression is also inversely correlated
with Ssa3p
expression (data not shown). These observations suggest
that
Ssa3p, like Ssa1p and Ssa4p (
37), can serve as a negative
regulator of the heat shock
response.
Effect of loss of function of Sin1p on expression of chaperones
functionally related to Ssa proteins.
The data shown in Fig. 5
demonstrate that elevated expression of Ssa3p is sufficient to explain
suppression of the high-temperature growth defect of ssa1
ssa2 cells by EXA1-1. However, as reported in the
original characterization of the EXA suppressors
(28), EXA1-1 is able to confer improved growth to
ssa1 ssa2 mutant cells in the absence of a functional copy
of SSA3. Given this finding, we decided to investigate the
regulation of other genes which might be important to the growth of
ssa1 ssa2 strains. As an initial search for such genes, we
analyzed the expression of various proteins which are known to
cooperate with the Ssa proteins in cellular functions.
Hsp104 and Sti1p are both heat-inducible proteins with
demonstrated functional interactions with Ssa. Hsp104 is
important
for the acquisition of thermotolerance in yeast. Genetic
studies
have demonstrated that the Ssa proteins are important for
thermotolerance
in the absence of Hsp104, and that Hsp104 is important
for the
vegetative growth of cells deficient in Ssa protein
(
33). Similarly,
deletion of
sti1 causes a severe
growth defect in combination
with deletions of
ssa1 and
ssa2 (
29). Sti1p is the yeast homolog
of
mammalian Hsp70 organizing protein, or Hop (for a review, see
reference
20). Both Hsp104 and Sti1p showed elevated
expression
in strains lacking
ssa1 and
ssa2, but
expression was not further
elevated by loss of function of Sin1p (Fig.
6A and B). In fact,
Hsp104 is sensitive
to feedback regulation of the heat shock response
by Ssa3p (see above),
and its levels, if anything, are slightly
decreased upon loss of
function of Sin1p.
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|
FIG. 6.
Effect of loss of Sin1p function on expression of
various chaperones. Cells were grown to mid-log phase at 30°C in YPD,
separated by SDS-PAGE, and subjected to immunoblot analysis using
antibodies reactive against Hsp104 (A), Sti1p (B), or Ydj1p (C). WT,
wild type.
|
|
In contrast, immunoblot analysis revealed that expression of Ydj1p was
somewhat elevated in an
ssa1 ssa2 sin1 strain and
an
ssa1 ssa2 EXA1-1 strain compared to an
ssa1 ssa2
strain, a
pattern similar to that seen for Ssa3p (Fig.
6C, compare
lanes
4 and 5 to lane 3). Ydj1p is an
S. cerevisiae homolog
of the
E. coli protein DnaJ, which cooperates with the
Hsp70 DnaK in facilitating
protein refolding and in lambda replication
(
24,
41). Deletion
of
YDJ1 causes poor growth in
otherwise wild-type cells (
1,
9,
10) and is
synthetically lethal with the quadruple mutation
ssa1-45 (Ts)
ssa2 ssa3 ssa4 (
3).
Compared to Ssa3p, the effect
of loss of function of Sin1p on
Ydj1p expression was minor and
varied from experiment to experiment;
the result shown in Fig.
6C is from an experiment in which the effect
was particularly
clear.
We wanted to determine whether overexpression of Ydj1p could improve
the growth of an
ssa1 ssa2 strain, either alone or in
combination with an elevated level of Ssa3p. To test this possibility,
we began with two
ssa1 ssa2 strains. One of these was
carrying
the vector plasmid pRS315 and the other was carrying
SSA3 promoter
fusion plasmid 2 (Table
2), which leads to
Ssa3p expression comparable
to that seen in the context of
EXA1-1-1 (Fig.
6A). We transformed
each of these strains
with either a vector control or a high-copy-number
plasmid carrying
YDJ1 under the control of its own promoter. Introduction
of
the
YDJ1 plasmid into either strain caused a significant
increase
in expression of Ydj1p as assessed by immunoblot (Fig.
7A) and
yet had no discernible effect on
growth (Fig.
7B and C). We conclude
that while expression of Ydj1p may
be slightly affected by loss
of function of Sin1p in
ssa1
ssa2 strains, elevated Ydj1p expression
does not play a role in
improved growth.
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|
FIG. 7.
Overexpression of Ydj1p does not improve growth.
ssa1 ssa2 strain BB363 carrying either a vector
plasmid (a1 a2) or promoter fusion plasmid p415TEF:SSA3
(plasmid 2 [Fig. 5]; a1 a2 [2]) was
transformed with either pRS424 (vector) or pYW2, a multicopy
plasmid carrying YDJ1 (YDJ1). Transformants were
selected at a permissive temperature, 23°C. (A) Immunoblot analysis.
Cultures were grown at 34°C, and extracts were prepared and subjected
to immunoblot analysis using antibody reactive against Ydj1p. (B)
Colony growth tests. Transformants were grown at 23°C and then
spotted as 10-fold serial dilutions onto an agar plate containing a
medium selective for pRS424 or pYW2. The plate shown was incubated for
3 days at 34°C. Approximately equal numbers of cells were spotted for
each strain. (C) Growth tests in liquid. Cultures were grown overnight
in a medium selective for pRS424 or pYW2 at 34°C, and their growth
throughout early log phase was monitored by OD measurements. For each
strain, the average doubling time (indicated atop each bar) was
determined from three independent plasmid transformants, and from a
total of nine separate cultures. Error bars represent ±2 standard
errors.
|
|
| |
DISCUSSION |
We have shown that the previously identified
extragenic suppressor of ssa1 ssa2 strain temperature
sensitivity, EXA1-1, is a dominant loss-of-function mutation
in SIN1. Loss of Sin1p function causes overexpression of
SSA3 in the context of HSF-mediated induction. SSA3 is able to functionally complement the loss of
SSA1 and SSA2, and overexpression of
SSA3 is sufficient for suppression.
Sin1p limits HSF activity at SSA3 but not at related
loci.
Sin1p is a highly abundant protein (~10,000 molecules per
cell) which binds DNA nonspecifically in vitro and is known to affect a
wide variety of loci in vivo, including HO, INO1,
and Ty-disrupted promoters (21, 31, 36, 40). We have added
SSA3 to this growing list. In addition, Sin1p apparently
modulates expression of other cellular components important in
Ssa-deficient strains, as loss of function of Sin1p is able to improve
the growth of an ssa1 ssa2 ssa3 strain (28).
Intriguingly, however, Sin1p's effect is not completely general. Our
data show that loss of Sin1p function increases HSF-mediated expression
of SSA3, and yet HSF-mediated expression of related genes,
including SSA4, HSP104, and STI1 is
not similarly affected. This degree of specificity suggests the
involvement of other regulatory factors which remain to be identified.
Our data suggest an interplay between HSF and Sin1p in the regulation
of
SSA3 in which Sin1p limits the extent to which HSF
can
activate transcription. In yeast, HSF binds constitutively
to its
recognition sequence, the HSE. In promoters such as
SSA1,
this constitutive binding is important to basal expression
(
35).
Upon heat shock, HSF is further activated, elevating
expression
of promoters such as
SSA1 and allowing expression
of promoters
such as
SSA3. In the case of
SSA3
(but not
SSA4,
HSP104, or
STI1),
this
activation may be limited by a compact nucleosomal structure
maintained
by
Sin1p.
The Ssa proteins have a critical role in heat shock gene
regulation.
Our finding that the EXA1-1 mutation leads
to elevated expression of SSA3 is in striking contrast to
data regarding EXA3-1, the other extragenic suppressor of
ssa1 ssa2 cells identified in the same screen.
EXA3-1, a mutation which alters the DNA-binding domain of
HSF and thus lowers its ability to activate transcription, lowers the
expression of heat shock-responsive genes, including SSA3.
While EXA3-1 and EXA1-1 both exert their effects
through altered regulation of HSF-mediated transcription, their effects are different: EXA3-1 lowers HSF-activated transcription in
a generalized fashion, while EXA1-1 elevates HSF-induced
expression of a particular gene, SSA3. These findings
demonstrate that at least two possible mechanisms for improving the
growth of ssa1 ssa2 strains exist: compensating for loss of
Ssa expression by selectively upregulating SSA3 or
alleviating the consequences of chronic heat shock gene overexpression
by downregulating HSF activity. However, these two mechanisms of
suppression may not be as disparate as they first appear. The Ssa
proteins are important in autoregulation of the heat shock response
(37). In the course of the present work, we have noted that
overexpression of Ssa3p from heterologous promoters has the effect of
lowering heat shock gene expression in an ssa1 ssa2 mutant
background, as evidenced by immunoblot analysis of Ssa4p and Hsp104.
Thus, it is possible that the relevant effect of EXA1-1 is
not elevated levels of Ssa3p per se but is the ability of Ssa3p to
autoregulate the heat shock response, decreasing HSF-mediated
expression of some other component. The effect of Ssa3p on heat shock
gene regulation is extremely subtle at the level of Ssa3p
overexpression caused by EXA1-1, but a delicate alteration
in expression may be just what is required.
The original motivation for characterizing extragenic suppressors of
ssa1 ssa2 strain temperature sensitivity was to identify
the
essential role of the Ssa proteins among the many roles which
have been
proposed. By increasing expression of Ssa3p,
EXA1-1 may
alleviate deficiencies in any or all of the cellular pathways
which
require Ssa protein. However,
EXA1-1 has only one obvious
characteristic in common with
EXA3-1: both serve to
downregulate
the constitutive expression of heat shock genes in an
ssa1 ssa2 background.
EXA3-1 does so directly, by
lowering the activity
of HSF.
EXA1-1 does so indirectly, by
increasing the expression
of Ssa3p, which can in turn downregulate heat
shock gene expression.
In both cases, the effect on steady-state levels
of heat shock
proteins is extremely subtle, and yet the improvement of
growth
is quite clear. Whatever other functions Ssa protein may have
in
the cell, its role in regulation of the heat shock response
is clearly
both critical and exquisitely
precise.
| |
ACKNOWLEDGMENTS |
We thank Elizabeth Krainer for her role in the subcloning and
sequencing analysis of p24, the Lindquist laboratory for the antibody
against Hsp104, the Herskowitz laboratory for the SIN1 disruption construct, Jill Johnson and other members of the Craig laboratory for helpful comments during this work, and Cindy Voisine, Chris Pfund, Warren Heideman, and Dave Brow for critical reading of the manuscript.
This work was supported by predoctoral fellowships to B.K.B. from the
Wisconsin Alumni Research Foundation and the National Science
Foundation and by NIH grant GM31107 to E.A.C.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Phone: (608) 263-7105. Fax: (608) 262-5253. E-mail:
ecraig{at}facstaff.wisc.edu.
| |
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Journal of Bacteriology, December 1998, p. 6484-6492, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
