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(Received for publication, August 10,
1995; and in revised form, November 7, 1995) From the
Rap1p is a transcriptional regulator of Saccharomyces
cerevisiae, which plays roles in both transcriptional activation
and silencing. To identify proteins involved in Rap1p-dependent
regulation of transcription, we used the two-hybrid system to screen
for Rap1p-interacting proteins. Two of the clones isolated from this
screen encode a truncated protein with homology to small heat shock
proteins (HSPs). Here we present an analysis of this novel S.
cerevisiae HSP, which we name Hsp42p. Expression of HSP42 is regulated by a range of stress conditions similar to S.
cerevisiae HSP26, with which Hsp42p shares most homology. However, HSP42 expression is more sensitive to increased salt
concentration and to starvation and, in contrast to HSP26 is
expressed in unstressed cells. Hsp42p interacts with itself in the
two-hybrid assay. This interaction is dependent on a hydrophobic region
which is conserved among small HSPs. Using bacterially expressed Hsp42p
fusion proteins, we demonstrate that this is a direct interaction.
Fractionation of yeast protein extracts by size demonstrates that all
of the Hsp42p in these extracts is present in complexes with a
molecular mass of greater than 200 kDa, suggesting that Hsp42p exists
in high molecular mass complexes.
The repressor/activator protein (Rap1p) of Saccharomyces
cerevisiae plays an important role in transcriptional silencing at
both HM loci and
telomeres(1, 2, 3) . Rap1p is also able to
activate gene expression and is essential for viability(4) ,
presumably because of its role in the activation of glycolytic and
ribosomal protein genes(5, 6, 7) . We were
interested in identifying other proteins involved in these processes,
in an attempt to gain further insight into the different functions of
Rap1p. We have previously used the two-hybrid system to identify
proteins which play a role in the silencing functions of Rap1p. Sir3p
and Sir4p interact with the carboxyl terminus of Rap1p in a two-hybrid
assay(8) , and the silencing protein, Rif1p, was identified in
a similar way(9) . We decided, therefore, to extend this search
for Rap1p-interacting proteins. Of the clones identified by this
screen, two encoded the same truncated protein, with homology to small
heat shock proteins. When eukaryotic cells are exposed to conditions
of stress, such as increased temperature, the expression of proteins
known as heat shock proteins (HSPs) ( In S. cerevisiae, the major
small HSP is Hsp26p, the expression of which is rapidly induced when
cells are transferred to higher temperatures. Hsp26p is one of the
major polypeptides produced on heat
shock(18, 19, 20, 21) . To date, one
other small HSP has been identified from S. cerevisiae, a
12-kDa protein with no homology to Hsp26p(22) . In addition to
heat shock, HSP26 expression is induced under other conditions
of stress, such as increased salt concentration and
starvation(20, 23) . However, no phenotype has been
observed on disruption of HSP26(18, 19) ,
suggesting that the function of Hsp26p in stress tolerance may overlap
with the functions of other HSPs. The identification of other S.
cerevisiae HSPs may, therefore, provide a greater insight into the
function of the small HSPs in yeast. Here we present the
identification and analysis of a novel small HSP of S.
cerevisiae. This HSP (Hsp42p) is most similar to S. cerevisiae Hsp26p. HSP42 expression is up-regulated by all stress
conditions tested. In contrast to HSP26, HSP42 is
expressed at a relatively high level in cells growing exponentially at
25 °C. By sucrose gradient fractionation, it has been demonstrated
that Hsp26p is present in large complexes (21) . We show that
Hsp42p is present in high molecular mass complexes, which are
heterogeneous in size. Our results also demonstrate that Hsp42p
interacts with itself and that this interaction is direct. The
interaction of Hsp42p with itself is dependent on the carboxyl-terminal
region of the protein including the conserved hydrophobic region.
CTY10-5D was co-transformed with
LexA/RAP1(635-827) and a G Plasmids that activated the
LexA operator-lacZ reporter in the presence of
LexA/RAP1(635-827), but not LexA/lamin or LexA alone, were
further tested for their ability to derepress a TRP1 reporter
gene at HMR(31) . At the silent mating-type loci,
Rap1p functions to recruit silencing factors such as Sir3p. Proteins
which interact with Rap1p and play a role in silencing at the HM loci might be expected to derepress these loci when fused to the
Gal4p activation domain, as this activation domain would be recruited
to the silent loci. Of the clones that activated the LexA
operator-lacZ reporter in a LexA/RAP1(635-827)-dependent
manner, and derepressed hmr
Figure 1:
Derepression of
LexA/RAP1(635-827) by orf721. A, the orf721-containing plasmids are shown schematically, together
with the structures of pK721 and pD215. B, CTY10-5D
cells containing the LexA fusions indicated and either pRS423, pK721
(containing orf721), or pD215 (containing HSP42) were
grown in liquid culture for 2 days and assayed for
To determine whether the protein encoded
by this open reading frame (orf721) was responsible for
activation of the LexA operator-lacZ reporter, it was cloned
into the 2-µm vector pRS423(25) . In this plasmid (pK721)
the 3` junction resulted in the addition of a single codon (His) after
amino acid 353 of orf721, followed by a termination codon. To
ensure that the promoter was also present, 560 bases of sequence 5` to
the ATG were included in this construct. Either pK721 or pRS423 lacking
an insert was cotransformed with LexA/RAP1(635-827) or
LexA/RAP1(653-827) into CTY10-5D, and Searching the GenBank(TM) and EMBL data bases with the orf721 sequence revealed that it was contained within an open reading
frame encoding 375 amino acids, located on chromosome IV. To
generate the full-length gene, we amplified the 3` region (encoding
amino acids 329-375 and 105 bases of 3` sequence) by PCR from
genomic DNA. This sequence was ligated into pK721 to generate the
full-length gene (creating pD215). Surprisingly, this construct was
unable to activate the LexA operator-lacZ reporter in the
presence of LexA/RAP1(635-827). Additionally, pK721, but not
pD215, was able to derepress the TRP1 gene at HMR (Fig. 1C). Similar results were observed with a
telomeric URA3 reporter gene (data not shown). Thus,
overexpression of the truncated protein encoded by orf721 was
able to derepress transcription. However, no effect on transcription
was observed on overexpression of the full-length protein.
Figure 2:
Amino acid sequence alignment of small
HSPs. A, the predicted amino acid sequence of Hsp42p is shown,
aligned with the sequence of Hsp26p. Identities are indicated by lines and similarities by colons. The boxed sequence indicates the region that is conserved among small HSPs.
The arrow indicates the final amino acid encoded by hsp42t. B, an alignment of the hydrophobic regions of
small HSPs from several species (S. c., S.
cerevisiae; N. c., Neurospora crassa; A.
t., Arabidopsis thalania; T. a., Triticum
aestivum; H. s., Homo sapiens; D. m., Drosophila melanogaster) is shown (see text for references). A
consensus (6/7 or greater identical) is shown below, with X representing a hydrophobic residue. Amino acids shown in lowercase are conserved only among the upper five
sequences.
Figure 3:
HSP42 expression is induced by increased
temperature. 10 µg of RNA isolated from cells grown at 25 °C (lane 1) and shifted to 30 °C (lanes 2-4),
37 °C (lanes 5-7), or 39.5 °C (lanes
8-10) for 20-60 min were separated by electrophoresis
through 1% agarose. Following electrophoresis and Northern blotting,
membranes were hybridized sequentially with probes for HSP42, HSP26, and actin.
HSP26 mRNA levels are increased when
cells are transferred to medium containing high concentrations of
NaCl(23) . Cells were grown at 25 °C in rich medium (YPD)
and then transferred to YPD containing 0.7 M NaCl for 20 min
to 3 h, after which RNA was isolated. As shown in Fig. 4A, the level of HSP42 mRNA starts to
increase within 20 min of the addition of NaCl and continues to
increase for at least 1 h. The observed increase in HSP26 mRNA
appears to be less rapid (Fig. 4A). To compare more
directly the relative increases in HSP42 and HSP26 mRNA levels, RNA isolated from cells grown at 25 °C, cells
shocked at 39.5 °C for 20 min, and cells incubated in 0.7 M NaCl for 40 min was electrophoresed in adjacent lanes (Fig. 4B, lanes 1-3). In comparison to HSP42, the increase in HSP26 mRNA expression is
greater on heat shock than on addition of NaCl. Up-regulation of HSP42 expression appears to be more sensitive, than HSP26 expression, to increased salt concentration, whereas HSP26 expression is more sensitive to increased temperature. However,
these differences are relatively subtle and may simply reflect
differences in the rates of induction of expression.
Figure 4:
Induction of HSP42 expression
under conditions of stress. Northern analysis was carried out as in Fig. 3, with: A, RNA from cells incubated in 0.7 M NaCl for 20 min to 3 h (lanes 2-6); and B,
RNA from cells grown at 25 °C, cells heat-shocked at 39.5 °C
for 20 min, and cells incubated in 0.7 M NaCl for 40 min
electrophoresed in lanes 1-3. RNA was analyzed from
cells incubated in sporulation medium (YPAc; lanes 5-8)
and from cultures grown to stationary phase for 2 or 6 h (Stat; lanes 10 and 11).
As cells are
transferred to sporulation medium or move into stationary phase, HSP26 mRNA expression is up-regulated(20) . As shown
in Fig. 4B (lanes 4-8), when cells were
transferred to YPAc, the mRNAs for both HSP26 and HSP42 were up-regulated, although the increase in HSP42 mRNA
was more obvious. Similarly, when cells were grown to high density,
both RNAs were up-regulated (Fig. 4B, lanes
9-11). In this case, HSP26 expression increased
more dramatically. Thus, expression of the HSP42 gene responds
to the same range of stress conditions as HSP26, although
there are clear differences in the relative increases in expression
levels of the two genes.
When equal numbers of
cells that had been stored at 4 °C for 5 months on YPD were plated
onto fresh YPD plates, cells lacking HSP42 showed a slightly
reduced viability compared to wild-type W303-1B cells. In a
representative experiment, 10.8% of wild-type cells produced colonies,
whereas only 6.7% of hsp42 cells were viable. Interestingly,
the viability of hsp26 cells (10.5%) was similar to that of
wild-type, and the viability of the double mutant (7.0%) was similar to
that of cells lacking HSP42 alone.
Two types of G
Figure 5:
Hsp42p interactions in the two-hybrid
system. A, the interactions of LexA, LexA/lamin, and
LexA/HSP42(22-353) with G
To narrow down the region of
Hsp42p which is required for it to interact with itself, fusion
constructs were created encoding different amounts of Hsp42p fused to
LexA (Fig. 5B). Using the two-hybrid assay, these
constructs were tested for interaction with
G To determine whether the interaction observed in the
two-hybrid assay is direct, Hsp42 fusion proteins were expressed in E. coli. As shown in Fig. 6A, two His-tagged,
DHFRS/HSP42 fusions (within pQE40), encoding amino acids 50-375
or 148-375 of Hsp42p (6H-hsp(50-375) and
6H-hsp(148-375); Fig. 6A) were created. A third
construct expressed amino acids 148-375 of Hsp42p, tagged with an
epitope recognized by a T7-specific antibody (T7-hsp(148-375); Fig. 6A). His-tagged fusion proteins were isolated on
nickel-agarose. Crude bacterial extract containing the T7-tagged Hsp42p
was incubated with either of the nickel-agarose-bound His-tagged Hsp42p
fusions or with 6xHis-DHFRS. The nickel-agarose was washed extensively
and bound proteins were separated by SDS-PAGE, Western blotted, and
incubated with a T7-specific monoclonal antibody. As shown in Fig. 6B, a T7-reactive protein was present in the lanes
containing proteins isolated in the presence of both of the His-tagged
Hsp42p fusion proteins, expressed from 6H-hsp(50-375) and
6H-hsp(148-375). A small amount of T7-tagged Hsp42p was retained
on the 6xHis-DHFRS (pQE40) nickel-agarose; however, this was probably
the result of nonspecific binding to the nickel-agarose. Thus, the
bacterially produced T7-tagged Hsp42p was capable of interacting
specifically with His-tagged Hsp42p in the absence of other yeast
proteins, strongly suggesting that this interaction is direct.
Figure 6:
Biochemical analysis of Hsp42p
interaction. A, the bacterial expression constructs are shown. HSP42 sequences were fused to a T7 antibody epitope or to
His-tagged DHFRS. B, isolation of T7-tagged Hsp42p using
His-tagged Hsp42p. Expression of recombinant proteins was induced, and
His-tagged proteins were partially purified on nickel-agarose.
Bacterial lysate containing T7-tagged Hsp42p was incubated with the
His-tagged fusion proteins (bound to nickel-agarose), encoding either
amino acids 50-375 or 148-375 of Hsp42p as indicated, for 1
h at 4 °C. The nickel-agarose beads were washed extensively.
Proteins were analyzed by SDS-PAGE and Western blotting, using a
T7-specific antibody, to detect T7-tagged proteins retained on the
nickel-agarose. Crude bacterial lysate containing the T7-tagged Hsp42p
was also loaded (Input). The arrow indicates the
position of the T7-tagged Hsp42p; the positions of molecular mass
markers are also shown (69, 46, 30, and 21
kDa).
Figure 7:
Size
fractionation of Hsp42p from yeast cells. Soluble proteins from cells
expressing Hsp42p tagged with three HA epitopes were size-separated by
FPLC, using a Superdex 200 column. Marker proteins with known molecular
sizes of 443, 200, 66, and 12.4 kDa had previously been used to
calibrate the column. These proteins eluted in fractions 11, 13, 18,
and 24, respectively. The numbers above each lane indicate the
fraction numbers loaded in each lane, 5 µl of each 500-µl
fraction were separated on a 12% polyacrylamide gel. The blot was
incubated with an HA-specific antibody to detect the HA-tagged Hsp42p.
The positions of molecular mass markers (69, 46, 30, and 21 kDa) are
shown.
We have isolated a novel S. cerevisiae gene that
encodes a small HSP. Hsp42p shares a high degree of homology with other
HSPs over a conserved hydrophobic region present in many small HSPs (19) . Analysis of the regulation of HSP42 mRNA
expression demonstrates that this gene is responsive to conditions of
stress. HSP42 expression is up-regulated by increases in
temperature and salt concentration, as well as by conditions of
limiting growth and overgrowth of cell cultures. Interestingly,
although HSP26 expression is also up-regulated under all these
conditions, there are differences in the responses of these two genes
to the various conditions of stress. This may reflect slightly
differing functions of these two proteins. Thus, Hsp42p may play a more
important role in the response to increased salt concentration, whereas
Hsp26p may be required for tolerance of high temperatures. Several
small HSPs, including Hsp26p, have been shown to aggregate within
cells(21, 33, 34, 35) . When a
G Disruption of the HSP26 gene
does not result in any detectable phenotype, even under conditions of
stress. As Hsp42p is the protein most similar to Hsp26p identified so
far in S. cerevisiae, it was possible that disruption of both
genes would result in a discernible phenotype. However, we were unable
to detect any significant difference in viability between cultures of
wild-type and hsp42hsp26 double mutant cells incubated either
at high temperature or in high concentrations of NaCl. It is possible
that Hsp42p and Hsp26p play no role in the tolerance of increased
temperature or salt concentration, their up-regulation under these
conditions being due only to overlapping response mechanisms. However,
we favor the alternative explanation that the response to these stress
conditions is dependent on multiple proteins, with Hsp42p and Hsp26p
playing some role. In common with other species, S. cerevisiae may have multiple small HSPs, with HSP26 and HSP42 representing the first members of a family of small HSPs in yeast. We isolated clones encoding a truncated Hsp42p as proteins that
potentially interact with Rap1p. However, we have been unable to show a
direct biochemical interaction between Rap1p and Hsp42p. The
interaction observed in the two-hybrid system is, therefore, likely to
be indirect. One possibility is that overexpression of the truncated
Hsp42p alters the interaction of silencing factors with Rap1p, allowing
the LexA/RAP1(635-827) fusion to become an activator. This type
of effect has been observed on deletion of SIR3, SIR4 or RIF1(8) . Furthermore, the genetic interaction
we observed appears to be dependent on the carboxyl-terminal truncation
of Hsp42p. Truncation of Hsp42p to amino acid 353 removes a region of
the protein with a high proportion of acidic residues. In contrast, the
truncated protein has a relatively basic region at its carboxyl
terminus. This suggests either that the interaction is artifactual or
that the truncation has uncovered a function of Hsp42p that is tightly
regulated within the context of the entire protein. We have been unable
to demonstrate any effect of the overexpression of full-length Hsp42p
on transcriptional activation or silencing. Thus, it is possible that hsp42t encodes a dominant negative form of Hsp42p or,
alternatively, a constitutively active form of the protein. On first
consideration, the association of Hsp42p with Rap1p and a specific
effect on silencing seems unlikely to reflect this HSP's normal
function. However, Hsp42p may play some more general role in regulating
transcriptional activation and silencing. The effects observed on
Rap1p-dependent silencing may only be obvious because of the
sensitivity of this system. It is of interest, in this regard, that
recent work has demonstrated a possible mechanistic link between
stress, aging, and silencing in yeast(36) . Specifically, both
stress resistance and life-span are regulated, at least in part, by the
putative Sir protein silencing complex. Given the clearly-established
role of Rap1p in HM locus and telomeric silencing, it is not
unreasonable to speculate that the effect of Hsp42 on Rap1p described
here may have consequences for both stress response and life span in
yeast.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2717-2723
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is induced. HSPs can
be divided into four classes; the hsp90 and hsp70 families, the
GroEL-related HSPs, and the small HSPs, which are typically up to 40
kDa in size(10) . Some of these HSPs, such as hsp70, are highly
conserved between organisms as divergent as mammals, yeast and bacteria (11, 12) . However, the small HSPs, share far less
sequence similarity between species, with the main region of homology
being a hydrophobic stretch of about 35 amino acids, located near the
carboxyl terminus of the protein(13, 14) . The number
of small HSPs identified in different species varies greatly. For
example, in many species of plants such as the soybean, more than 20
small HSPs have been identified(14) , whereas in Drosophila six small HSPs are known (15) and in humans only one has
been identified(16) . The function of small heat shock proteins
remains unclear. However, in Dictyostelium a mutation that
abolishes induction of small HSP gene expression causes reduced stress
tolerance, suggesting that these proteins do indeed play a role in
stress resistance(17) .
Plasmids
The LexA/RAP1 fusions are as
described(8) . The G fusion library screened with
LexA/RAP1 was created in pGAD3(24) . The G
fusion
library screened with LexA/HSP42 was created in pGAD424 (24) by
P. James (Wisconsin University). orf721 was subcloned into
pRS423 (25) to create pK721. The 3` region of HSP42 was generated by PCR and subcloned into pK721, creating pD215.
LexA/HSP42(22-353) was created by subcloning an MscI to BamHI fragment from pK721 into the LexA fusion plasmid
pBTM116(24) . LexA/HSP42(22-332) was created by cloning
an MscI to AccI fragment of HSP42 into
pBTM116. LexA/HSP42(148-375) was created by transferring HSP42 sequences from the shorter G
fusion into
pBTM116. LexA/HSP42(182-375) was generated by PCR. HA-tagged
Hsp42p was expressed from within pRS423 (pD238), in which the 3` region
of the gene was generated by PCR, to include a unique NotI
site 5` of the termination codon. A NotI fragment encoding
three copies of the HA peptide (YPYDVPDYA), recognized by the 12CA5
monoclonal antibody (BABCO), was ligated into this construct.
His-tagged bacterial expression constructs were generated within pQE40
(Qiagen) and T7 epitope-tagged constructs in pET21a (Invitrogen). The HSP42 disruption was created by replacing sequences between an XhoI site (406 base pairs 5` to the ATG) and an EcoRV
site (at codon 81) with the HIS3 gene. HSP26 was
disrupted by replacing a BglII to NruI fragment
containing the entire coding sequence with a 2.2-kilobase pair LEU2 fragment. Disruption constructs were integrated using standard
techniques(26) . Sequencing was carried out using the dideoxy
chain termination method, using Sequenase (Amersham).
Yeast Strains
Growth and manipulation of yeast
strains was carried out using standard procedures(26) .
Screening of the G fusion library with LexA/RAP1 was
carried out in CTY10-5D (MATaade2-1
trp1-901 leu2-3, 112 his3-200 gal4 gal80 URA3::LexA
op-lacZ). The pGAD424 library was screened using L40 (24) and HLY655 (MAT
L40). All other strains were
derived from W303-1B (HML MAT HMRaade2-1 can1-100 his3-11, 15
leu2-3, 112 trp1-1 ura3-1). LacZ assays were
performed as described(8) . Spot assays were performed by
spotting 10-fold serial dilutions of a saturated overnight culture onto
the appropriate selective media. Plates were photographed after
2-3 days.Northern Analysis
Cell pellets from 5-ml cultures
were resuspended in 0.2 ml of extraction buffer (0.5 M NaCl,
0.2 M Tris-HCl, pH 7.6, 10 mM EDTA, 1% SDS). 0.4 g of
acid-washed glass beads (0.45-0.5 mm) and 0.2 ml of
phenol/chloroform/isoamyl alcohol (25:24:1) were added. Cells were
vortexed for 3 min and centrifuged for 5 min, and the aqueous phase was
re-extracted twice with phenol/chloroform/isoamyl alcohol and
precipitated with ethanol. 10 µg of RNA (per lane) were
electrophoresed through 1% agarose with formamide, and transferred to
nylon membranes (Hybond). Membranes were hybridized with random-primed
DNA probes in 0.5 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 30% formamide at 42 °C, washed in 2 
SSC, 1% SDS
at 65 °C, and exposed to Kodak X-AR5 film.Western Blotting
Proteins were fractionated on 12%
polyacrylamide and electroblotted onto nitrocellulose membranes.
Membranes were blocked in PBS, 5% nonfat milk, 0.1% Tween 20 and
incubated with the appropriate antibody (anti-HA from BABCO, or anti-T7
from Invitrogen) in PBS, 1% nonfat milk, 0.1% Tween 20, for 1 h at room
temperature. After washing in PBS, 0.1% Tween 20 four times, membranes
were incubated with peroxidase-conjugated mouse Ig-specific antibody
for 1 h. Following further washing, blots were developed using ECL
(Amersham).Size Separation of Yeast Protein Extracts by
FPLC
The cell pellet from a 1-liter culture (A > 1.0) of W303-1B with pD238 was resuspended in 5 ml lysis
buffer (20 mM HEPES, pH 6.8, 150 mM KOAc, 2 mM MgOAc, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). 1 ml of glass beads was added, and
cells were vortexed for 1 min and left on ice for 1 min. This was
repeated eight times. Cell debris was removed by centrifugation at 7000
rpm for 15 min at 4 °C. The supernatant was centrifuged at 50,000
rpm for 30 min at 4 °C and filtered through a 0.2-µm filter,
and 3.2 mg of protein were loaded onto a Superose 200 column that had
been calibrated with proteins of known size. 40 fractions of 0.5 ml
were collected, and 5 µl of each fraction were analyzed by SDS-PAGE
and Western blotting.
Expression of Bacterial Fusion Proteins
Proteins
were expressed from within pQE40 in M15(pREP4) cells (Qiagen). pET21a
fusions were expressed in BL21 cells (Invitrogen). A saturated culture
was diluted 1:10 and grown at 37 °C for 90 min. Protein production
was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside to 2 mM.
After 4 h, cells were collected by centrifugation, lysed by sonication
in 500 µl of NETN (100 mM NaCl, 10 mM Tris, pH 8,
1 mM EDTA, 0.5% Nonidet P-40), and insoluble proteins were
removed by centrifugation. Histidine-tagged fusions were partially
purified on nickel-agarose beads. 50 µl of nickel-agarose, with
protein from 0.5 ml of cell culture, was incubated with 20 µl of
bacterial extract containing T7-tagged Hsp42p at 4 °C in 500 µl
of NETN for 1 h. The beads were washed with 4 ml of NETN and boiled in
2 SDS-PAGE loading buffer. Proteins were analyzed by SDS-PAGE
and Western blotting.Screening of G
The
pGAD3-based library was screened with LexA/RAP1 in CTY10-5D as
described previously(8, 24) . The pGAD424 library was
transformed into L40 cells(27) , transformants were grown in
SC-Leu, collected by centrifugation, and frozen in SC-Leu with 15%
glycerol at -70 °C. After spreading onto SC-Leu plates and
incubation at 30 °C for 2 days, they were replica-plated onto YPD
plates that had been spread with 100 µl of an overnight culture of
HLY655 containing the LexA fusion. After mating overnight, cells were
replica-plated to SC-Leu-Trp-His, to select for both plasmids
(diploids) and activation of the LexA operator-HIS3 reporter(28) . His Fusion Libraries
colonies were picked
after 2 or 3 days and tested for their ability to activate the LexA
operator-lacZ reporter on a filter assay as described
previously(8) . G
plasmids were recovered from
His
, LacZ
diploids.
Overexpression of orf721 Causes
LexA/RAP1(635-827) to Become an Activator
The Rap1 protein
has been shown to play important roles in both transcriptional
activation and silencing(1) . To identify other proteins
involved in these processes which may interact with Rap1p, we screened
a two-hybrid library with a construct containing RAP1 sequences fused to LexA. The fusion protein produced contained
amino acids 635-827 of Rap1p. This region has previously been
shown to contain a domain involved in transcriptional silencing at the
mating-type loci HML and HMR, and can interact with
the silencing factors Sir3p and Sir4p(8, 29) . In
addition, most of the Rap1p activation domain, which has been mapped to
amino acids 630-695, is contained within this
region(30) . However, the LexA/RAP1(635-827) fusion does
not activate transcription of reporter constructs containing multiple
LexA operators. fusion library.
Transformants were then assayed for
-galactosidase activity on
nitrocellulose filters. Plasmids were isolated from positive colonies
and assayed for the ability to confer the activating phenotype on
re-transformation into CTY10-5D.
A::TRP1, two contained
overlapping inserts. Sequence analysis demonstrated that in neither
case was an in-frame fusion with G sequences present.
Thus, these clones appeared to encode proteins capable of activating
independently of G
. Further analysis revealed that they
contained the same DNA sequences, cloned in opposite orientations, and
that activation was dependent on an open reading frame of 353 amino
acids. This open reading frame contained no translational stop codon,
but was fused either to the G
sequences, in
reverse orientation, or to the termination sequence (Fig. 1A).
-galactosidase
activity. Activity is shown, normalized for protein concentration. C, W303-1B cells with the hmrA::TRP1 reporter
were transformed with pRS423, pK721, or pD215 and grown overnight, and
dilutions plated onto SC-his and SC-his-trp plates. Plates were
photographed after 2 days at 30 °C.
-galactosidase
assays were performed. As shown in Fig. 1B,
LexA/RAP1(635-827), gave a low level of -galactosidase
activity with pRS423. However, the activity increased by greater than
17-fold in the presence pK721, suggesting that orf721 may
contain an activation domain. To test this possibility, a construct
expressing LexA fused to amino acids 22-353 of orf721 was constructed. This fusion protein was not able to activate the
LexA operator-lacZ reporter, thus it is unlikely that orf721 encodes an activation domain. As shown in Fig. 1B, pK721 was unable to activate
LexA/RAP1(653-827), a fusion in which the activation domain of
Rap1p is further truncated. These results suggest that overexpression
of the truncated protein encoded by orf721 causes the
LexA/RAP1(653-827) hybrid to become an activator, possibly by
affecting interactions with proteins involved in Rap1p-dependent
silencing. Previous results have demonstrated that the loss of
expression of Rap1p-interacting proteins involved in silencing, such as
Sir3p or Sir4p, causes LexA/RAP1(635-827), but not
LexA/RAP1(653-827), to become an activator(8) . orf721 Encodes a Small Heat Shock
Protein
Comparison of the predicted amino acid sequence of orf721 to GenBank(TM) and EMBL data bases revealed that
amino acids 305-339 of orf721 have homology with a
conserved hydrophobic region found at the carboxyl terminus of many
small heat shock proteins (Fig. 2). This region also shows
homology to -crystallin (19) . The protein most similar to
that encoded by orf721 is S. cerevisiae Hsp26p (Fig. 2A), sharing 46% identity (66% similarity) with
amino acids 305-339 of orf721 (Fig. 2A).
This conserved region is within an extended region of weaker homology
between the carboxyl termini of Hsp42p and Hsp26p. As shown in Fig. 2B, within the conserved region (amino acids
305-339 of orf721) amino acids at several positions are
absolutely conserved between divergent species (13, 16, 19, 32) . The observed
homology suggests that orf721 encodes a small HSP, of
predicted molecular mass 42.8 kDa. We, therefore, name this gene HSP42 and the truncated protein, encoded by orf721,
hsp42t.
Regulation of HSP42 mRNA Expression
To determine
whether expression of HSP42 mRNA is sensitive to conditions of
stress, such as increased temperature or salt concentration, Northern
analysis was undertaken. Expression of HSP26 and actin mRNAs
was also analyzed for comparison. RNA was isolated from W303-1B cells
grown at either 25 °C or at 25 °C and shifted to 30, 37, or
39.5 °C for 20 to 60 min. Incubation of cells at 37 °C for 20
min resulted in an increase in the amount of HSP42 mRNA. The
increase in HSP42 mRNA levels was even more dramatic when
cells were transferred from 25 °C to 39.5 °C (Fig. 3).
Little change in the level of HSP42 mRNA was observed in cells
transferred to 30 °C. When the same blot was re-hybridized with an HSP26-specific probe, a similar pattern of induction was
observed (Fig. 3). However, there was no detectable level of HSP26 expression at 25 °C, whereas HSP42 was
expressed at a relatively high level in cells grown at 25 °C. Actin
mRNA expression was unaffected by temperature change. Thus, the levels
of HSP42 and HSP26 mRNAs appear to be regulated
similarly by heat shock, confirming that HSP42 is a heat
shock-responsive gene.
Phenotypic Analysis of Cells Lacking a Functional HSP42
Gene
To determine whether expression of Hsp42p was necessary for
stress tolerance, we disrupted the HSP42 gene in W303-1B
cells. This mutation replaced 405 base pairs of the promoter and the
first 243 base pairs of coding sequence with the HIS3 gene.
Using a probe containing the entire HSP42 coding sequence, no
expression of HSP42 mRNA was detectable by Northern analysis
(data not shown). A deletion of the entire HSP26 coding
sequence was also created. Wild-type and mutant cells were subjected to
a range of stress conditions, and viability was assessed. No
significant differences in the tolerance of elevated temperature were
observed between wild-type cells and cells lacking the HSP42 gene or both HSP42 and HSP26 (data not shown).
Incubation of wild-type and hsp42hsp26 cells in YPD containing
1 M NaCl greatly reduced the viability of the cultures.
However, when cells were plated onto YPD plates after 3 or 4 h in 1 M NaCl, no significant differences in the viability of
wild-type and mutant cultures were observed.Hsp42p Interacts with Itself via a Conserved
Carboxyl-terminal Domain
In an attempt to identify proteins
which interact with Hsp42p, we screened a two-hybrid (G fusion) library with a LexA/HSP42 construct encoding amino acids
22-353 of Hsp42p. LexA/HSP42(22-353) was transformed into
HLY655 cells, which contain integrated LexA operator-lacZ and
LexA operator-HIS3 reporters. The G
library was
transformed into L40 cells (L40 differs from HLY655 only in its mating
type). Cells containing the G
library were then mated with
LexA/HSP42-containing cells. Colonies able to grow on medium lacking
leucine, tryptophan, and histidine were selected. G
plasmids were recovered from 20 of the His
clones, 17 of which activated both the LexA operator-lacZ and LexA operator-HIS3 reporters in the presence of
LexA/HSP42. By sequence analysis, we determined that 11 of these 17
clones encoded in-frame G
fusions with HSP42.
/HSP42 fusions were
observed, encoding amino acids 50-375, or 148-375 of
Hsp42p. To confirm that the interaction with LexA/HSP42 was specific,
the G
/HSP42 fusions were tested for interaction with LexA,
LexA/lamin and LexA/HSP42 (Fig. 5A). Both
G
/HSP42 fusions activated the LexA operator-lacZ reporter in the presence of LexA/HSP42, but not LexA or
LexA/lamin. No activation was observed with LexA/HSP42 and a plasmid
expressing G
alone. These results suggest that Hsp42p
specifically interacts with itself.
and the
G
/HSP42 fusions are shown. L40 cells containing G
fusions and HLY655 cells containing LexA plasmids were mated on
YPD, replica-plated onto SC-leu-trp, and after 2 days assayed for lacZ activity on a nitrocellulose filter. + indicates an
interaction (detectable lacZ activity); -,, no
detectable interaction after 24 h. B, G
alone and
the two G
/HSP42 fusions were tested for interaction with
LexA fusions containing the amino acids of Hsp42p shown. The hatched box indicates the position of the conserved
hydrophobic region (amino acids 305-339). Interactions were
assayed in liquid as described previously(8) ; the
-galactosidase activity is shown, corrected for protein
concentration.
/HSP42(50-375), G
/HSP42(148-375)
and G
alone. CTY10-5D cells were grown in liquid
culture and
-galactosidase assays were performed after 2 days. As
shown in Fig. 5B, only very low levels of activity were
observed when the LexA/HSP42 fusions were assayed in the presence of a
G plasmid lacking an insert. LexA fusions containing amino
acids 148-375 (equivalent to the shorter fusion isolated from the
G
library) and 182-375 of Hsp42p interacted with
both G
/HSP42 fusions. Two further truncations (to amino
acid 201 and 243) resulted in a loss of this interaction. However, as
judged by Western blot, neither of these fusions produced a protein of
the expected size. We were therefore unable to define further the
amino-terminal boundary of the interacting region. The LexA fusion with
which the G
library was screened, encoding amino acids
22-353, was able to interact with both G
/HSP42
fusions. Truncation to amino acid 332, removing seven amino acids from
the carboxyl-terminal end of the conserved hydrophobic region, resulted
in a loss of this interaction (Fig. 5B). Expression of
this fusion protein was confirmed by Western blotting. Thus, the region
required for interaction of Hsp42p with itself is located between amino
acids 182 and 353, and it appears that the presence of the conserved
hydrophobic region (amino acids 305-339) is required for this
interaction.
Hsp42p Forms High Molecular Mass Complexes
To
determine whether the Hsp42 protein in yeast cells is present in
monomeric form or as higher molecular mass complexes, HSP42 (within pRS423) was tagged at the carboxyl terminus with three
copies of the HA epitope. Soluble proteins extracted from W303-1B cells
expressing HA-tagged Hsp42p were size-separated by FPLC, using a
Superdex 200 column. Forty fractions were collected and subjected to
analysis by SDS-PAGE and Western blotting. As shown in Fig. 7,
no HA-tagged Hsp42p was detected in the fractions containing proteins
below 66 kDa, where monomeric Hsp42p would be expected to be found.
Hsp42p was present exclusively in fractions 7-14, suggesting that
it is present in complexes ranging upwards from 200 kDa in size (Fig. 7). Given the apparent molecular mass of HA-tagged Hsp42p
(approximately 52 kDa), the size range in which Hsp42p eluted from the
Superdex 200 column suggests that, if these complexes consist of Hsp42p
alone, it is at least tetrameric. The broad range over which Hsp42p
eluted suggests that the Hsp42p complexes are heterogeneous in size.
fusion library was screened with LexA/HSP42, the
majority of the interacting clones isolated encoded in-frame fusions of
the Gal4p activation domain with Hsp42p. Thus, as with other HSPs,
Hsp42p appears to interact with itself. Using bacterial fusion
proteins, we have demonstrated that this Hsp42p-Hsp42p interaction is
direct. Analysis of the high molecular mass complexes containing
Hsp26p, by sucrose gradient and SDS-PAGE fractionation, has
demonstrated that Hsp26p is the predominant protein within these
complexes(21) . However, the presence of less abundant or
otherwise undetectable proteins cannot be ruled out. The demonstration
that the interaction of Hsp42p with itself is direct lends weight to
the idea that small HSPs form high molecular mass complexes by
homo-multimerization. The interaction of Hsp42p with itself appears to
be dependent on the conserved hydrophobic region. Therefore, it seems
likely that the aggregation of Hsp26p within the cell is via a direct
interaction dependent on the analogous region of Hsp26p. An additional
possibility is that Hsp42p and Hsp26p interact with each other. To test
this, we created a LexA/HSP26 fusion. Although a fusion protein of the
expected size was produced at high levels, no interaction with
G
/HSP42 fusions was observed (data not shown). We were
unable to test the interaction of Hsp26p with a LexA/HSP42 fusion
because G
/HSP26 fusions were toxic to the cells. In this
context, it is of interest that HSP42 is expressed at
relatively high levels in unstressed cells whereas HSP26 expression is undetectable. Thus, Hsp42p may function in both
stressed and unstressed cells.
)
We thank Paul Bartel and Stan Fields for the generous
gift of the pGAD3 library and Philip James for generously providing the
pGAD424 library. We also thank Heike Laman for providing yeast strains
and Heike Laman and David Vannier for useful discussion. We thank Lucy
Pemberton and Michael Rexach for help with FPLC.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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