(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) ()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) .
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 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.
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 hmrA::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).
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 -galactosidase
activity. Activity is shown, normalized for protein concentration. C, W303-1B cells with the hmr
A::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.
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 -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) .
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/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.
Figure 5:
Hsp42p interactions in the two-hybrid
system. A, the interactions of LexA, LexA/lamin, and
LexA/HSP42(22-353) with G 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.
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/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.
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 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.
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.