YEAST

VOL. 8

95-106 (1992)

The Small Heat-Shock Protein Hsp26 of Saccharomyces cerevisiae Assembles into a High Molecular Weight Aggregate NICOLA J. BENTLEY, IAN T. FITCH* AND MICK F. TUITEt

Biological Laboratory, University of Kent, Cunterbury Kent CT2 7NJ. U.K. Received 8 July 1991

Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiue,yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26)was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under nonstress conditions and such high level synthesis, prior to a lethal heat shock (SOOC), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat-shock granule, HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (1 5-25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules. heat-shock protein; Hsp26 overexpression; yeast (Succhuromyces cerevisiue); heat-shock proteins; Hsp26-containing high molecular weight aggregate.

KEY WORDS -Small

INTRODUCTION Cells and whole organisms respond to elevated temperatures and other forms of stress by synthesizing a group of proteins known as heat-shock proteins (Hsps). This transient cellular response is conserved in both eukaryotes and prokaryotes (Lindquist, 1986) and results in the synthesis of a number of proteins belonging to one or other of the four major families of Hsps, grouped accordingly to their molecular weight, namely the Hsp90 family (8396 kDa), the Hsp70 family (68-78 kDa), the GroEL family (58-65 kDa) and the small Hsp family (1640kDa) (Lindquist and Craig, 1988). The total number of Hsps synthesized is species-specific, but in all organisms studied to date, the synthesis of a 70 kDa Hsp, Hsp70, has been invariably identified. The primary structure of this protein is highly conserved amongst organisms as diverse as Escherichia coli, Drosophila melanogaster and man (Bardwell and Craig, 1984; Hunt and Morimoto, 1985). *Current address: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, U.S.A. ?Addressee for correspondence. 0749-503X/92/020095-12 $06.00 0 1992 by John Wiley & Sons Ltd

The small Hsp family is the most diverse group of Hsps and although they have been identified in all eukaryotes studied to date, their cellular function remains a mystery. The complexity of the small Hsp family varies greatly among different groups of organisms. For example, in the yeast Saccharomyces cerevisiae, two major small Hsps have been described: Hsp26 (McAlister et al., 1979; Petko and Lindquist, 1986)and Hspl2 (Praekelt and Meacock, 1990), while in plants a complex array of up to 30 small Hsps with molecular weights in the range of 15-27 kDa have been described (Nagao et al., 1985; Mansfield and Key, 1987). There is no simple relationship between the number of small Hsps and the genetic complexity of an organism, since in humans only one small Hsp has been described (Hsp28, Hickey et al., 1986; Arrigo and Welch, 1987), while the fruit fly Drosophila melanogaster synthesizes at least six (Ayme and Tissieres, 1985), of which four are closely related, namely Hsp22, Hsp23, Hsp26 and Hsp27 (Voellmy et al., 1981; Ingolia and Craig, 1982). In spite of the observed species diversity in numbers of small Hsps synthesized, small Hsps do

96 share a number of structural and regulatory properties. For example, as well as being induced by heat shock they can also be expressed in non-stressed cells in response to normal developmental cues (Lindquist and Craig, 1988).The best understood in this respect is Hsp26 of S. cerevisiae, which is also synthesized in cells that are transitioning from the exponential phase of growth to the stationary phase, and during sporulation (Kurtz et al., 1986). At the structural level small Hsps show limited regions of amino acid identity and furthermore show significant homolom with mammalian a-crvstallins (Ingolia and ydraig, 1982; de Jong et a/.: 1988), particularly with the C-terminus of the polypeptide. One other unusual property of small Hsps in higher eukaryotes is their apparent ability to assemble into high molecular weight aggregates termed 'heat-shock granules' (HSGs) following their expression under stress conditions. Such HSGs have been described in mammalian (Arrigo and Welch, 1987), plant (Never et al., 1983, 1989), avian (Collier et a/., 1988) and Drosophila (Arrigo and Welch, 1987) cells and have sedimentation coefficients of between 15-20s and molecular weights between 20&800 kDa. The functional significance of the HSG is unknown. We have begun a study into the function and cellular distribution of Hsp26, the major small Hsp of S. cerevisiue, in both stressed and non-stressed cells. We report here the cellular consequences of overexpressing this protein in both stressed and nonstressed yeast cells and demonstrate that, like its higher eukaryote counterparts, Hsp26 also selfassembles into HSGs of molecular weight 550 000.

MATERIALS AND METHODS Escherichia coli and yeast strains used For all plasmid DNA transformation and manipulation, the E. coli strain DH5a (supE44 lucV169 (A80 lucZM15) hsdR17 recA1 endAl gyrA96 thi-1 relA1) was used. For M13 transfections and preparation of single-stranded DNA, the E. coli strain TG1 (supE hsdS thi (lac- proAB) F (traC36 proAB' lacPlacZM15) was used. The S. cerevisiue strain MD40/4c (MATa ura2 trpl leu2-3, -112, his3-11, -15) was used throughout this study. E. coli and S. cerevisiae strains were grown on standard L-Broth and YEPD- or YNB-based media respectively as previously described (Spalding and Tuite, 1989).

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Recombinant DNA methods All DNA manipulations were performed essentially as described by Sambrook et al. (1989) using restriction and modification enzymes (BCL, BRL) in accordance with manufacturer's instructions. Plasmid DNA was transformed into E. coli, using essentially the CaC1,-based method originally described by Cohen et al. (1972), and analysed using the rapid mini-procedure described by Birnboim and Doly (1979). Yeast transformation was by the method of Beggs (1978). P/asmidconstructions Plasmid pUKC360 (Figure la) was constructed by cloning a 2.6 kb BamHI-HindIII restriction fragment, containing the Hsp26 gene and at least I500 bp upstream of the HSP26coding region from the PUC18-based plasmid pHSP26, into the multiCOPY YEP plasmid pMA3a (Spalding and Tuite, 1989). This involved linearization of pMA3a with BamHI followed by treatment with alkaline phosPhatase, then Partial digestion with Hind11 (pMA3a contains two Hind11 sites). The desired 7.3 kb BamHI-HindIII fragment was electroeluted from a 1% agarose gel and ligated to the 2.6 kb BamHI-Hindll fragment frorn pHSP26 to generate pUKC360. Plasmid pUKC370 (Figure b, was constructed a 0'7 kb Bgfll-BamH1 fragment, encornby passing the entire HSP26 coding region and 57 bp 5' and 32 bp 3' to the Hsp26 region, into the PGK-based expression plasmid pMA91 (Mellor et al.9 19831, first digested with and treated with alkaline phosphatase as recommended by the suppliers (BCL). Analysis of thermotolerance S. cerevisiae strain MD40/4C was grown at 30°C until mid-exponential phase (ca. 5 x lo6cells ml-I). Two 10 ml aliquots of this culture were then transferred to separate sterile 100 ml flasks allowing one

culturetogrowat30"Cwhiletheotherwasincubated at 42°C. After 30min both cultures were rapidly transferred to a 50°C water bath and incubation was continued for a further 20 min, during which time 1 ml aliquots were removed at 5-min intervals, seriallydiluted inYEPD( 10-4dilution)and plated in triplicate onto YEPD agar. After incubation at 30°C for 2-3 days, the numbers of colonies were counted and the survival of the heat-stressed cells was calculated as a % of the non-heat-stressed (30°C) cells viable immediately prior to the shift to 50°C.

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a

EHP

P P Figure 1 . Restriction maps of plasmids constructed to overexpress Hsp26 in S. cerevisiue. (a) plasmid pUKC360 containingtheentireHSP26 gene and its promoter and terminator regions.(b) plasmid pUKC370 containing the HSP26 coding region flanked by the promoter and terminator of the PGKl gene. Restriction enzyme sites shown are: B, BumHI; Bg, BglII; H, HindIII; R, EcoRI; P, PstI; X, XbaI. The bacterial plasmid sequences are indicated by the thin line, the 2 pm plasmid DNA fragment carrying the LEU2-d gene, by the open box, the HSP26 gene sequences by the filled-in boxes and the PGKZ gene promoter (5') and terminator (3') sequences by the stippled box. The direction of transcription of the HSP26 gene is indicated by the arrow.

Two-dimensional SDS-PAGE analvsis

Cells were pulse-labelled in YNB-based medium for 15 minwith2.5 pCiml-' ['4C]aminoacidmixture and the proteins separated on a two-dimensional non-equilibrium pH gradient gel system essentially as described by O'Farrell et al. (1977). The firstdimension urea-polyacrylamide gel had a PIrange of 3.5-1 0.0, the second dimension was by SDS-PAGE using a 12.5% polyacrylamide gel. For autoradiography, dried gels were exposed to X-ray film (Fuji) for 14-28 days at -70°C. Pur$cation of HSGs

The method used was adapted from one originally described by Arrigo et al. (1987). 500ml YEPD- or YNB-based cultures of yeast were grown at 30°C to the desired cell density. Where appropriate, the culture was heat-shocked at 42°C for 30min. Cells were then harvested (10min at 1200 x g), resuspended in 2 ml ice-cold lysis buffer (10 mM-Tris-HC1, pH 7.5, 10 mM-NaC1, 5 mMMgCl,, 1 mM-P-mercaptoethanol, I mM-PMSF) and placed on ice. A similar volume of glass beads (0-4-0.45mm BDH) were then added to the meniscus of the cell slurry and vortexed for 2 x 2.5min periods with the samples being placed on ice for 1 min between each vortexing. The resulting cell

lysate was transferred to a fresh tube and the iemaining glass beads were washed once with a further 1 ml fresh lysis buffer. NP-40 was then added to the cell lysate to a final concentration of 0.5% (v/v), vortexed briefly, then centrifuged for 10 min at 16 000 x g . The resulting supernatant was transferred to a fresh centrifuge tube and further centrifuged for 3 h at 180 000 x g at 4°C. The resulting pellet was resuspended in buffer A (1 0 mM-TrisHCl, pH 7.5, 10mM NaCl, 10 mM-MgCl,, 1 mMP-mercaptoethanol) and layered onto a 10 ml 0.51.0 M-sucrose gradient in 10 mM-Tris-HC1 (pH 7.5), 10 mM-NaC1, 5 mM-MgC1,. The gradient was centrifuged for 17 h at 35 000 rpm and the resulting gradient fractionated into 10 x 1 ml fractions. To identify fractions containing Hsp26, 10 pg of protein from each fraction was separated by onedimensional SDS-PAGE (Laemmli, 1970) using 12.5% (w/v) polyacrylamide, and the resulting gel stained with 0.1% (w/v) Coomassie Blue in 40% (v/v) methanol, 15% (v/v) acetic acid and destained in 10% (v/v) methanol, 7.5% (v/v) acetic acid. Analysis of the HSGs 100 PgofpurifiedHSGs wereincubated with 10 pg of either a-amylase (Sigma), chymotrypsin (Sigma) or ribonuclease A (BCL) respectively for 30 min at room temperature in buffer A. The resulting samples

98 were then fractionated on 0.5-1 .O M-sucrose gradients as described above and the resulting fractions analysed by one-dimensional SDS-PAGE. A control gradient containing 100 pg untreated purified HSGs was always run in parallel. To calculate the molecular weight of the purified Hsp26-containing HSGs, a Sephacryl S-400 (Pharmacia) column (I .5 cm x 40 cm) was prepared and calibrated at 4°C with standard proteins of known molecular weight (thyroglobulin, 669 kDa; 0-galactosidase tetramer, 465 kDa; catalase, 232 kDa in buffer X (50 mMNaCl, 10 mM-NaH,PO, 1 mM-EDTA, 1 mM-NaN,, 0.2 mM-dithiothreitol, pH 7.0)). The column was then washed for a further 2 h with buffer X at a flow rate of 2.5 ml h-' cmP3 before a sample of HSG (1 mg in 1 ml) was applied in buffer X and 0.5 ml fractions eluting from the column were monitored spectrophotometrically at A,,,. Molecular weight of the eluting HSG was calculated against a calibration curve generated from the flow-rate of the three standard proteins eluted under identical conditions.

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based expression plasmid pMA91 (Mellor et al., 1983) to generate the plasmid pUKC370 (Figure 1b), which otherwise has the same yeast selection and replication sequences as pUKC360. Regulation of HSP26 expression on a multicopy plasmid

To determine whether the HSP26 gene carried on a multicopy plasmid was regulated in a normal manner, its expression was studied in MD40/4c [pUKC360] transformants by two-dimensional SDS-PAGE analysis of ['4C]-labelledproteins synthesized either during a mild heat shock (22-36°C) or in cells that have entered stationary phase (approximately 2 h after cessation of growth). In unstressed exponentially growing cells, Hsp26 is not detectable unless cells have been subjected to a heat shock (McAlister er al., 1979; Lindquist et al., 1982), a result we confirmed for the untransformed MD40/ 4c strain (Figure 2a, b), although under the relatively mild heat-shock conditions used (22-36"C), Hsp26 represented only a minor proportion of the total Electron microscopic analysis of HSGs protein synthesis. In the pUKC360 transformant, HSGs, purified by two sequential 0.5-1 .O M-sucrose very low levels of Hsp26 synthesis were detectable in gradients as described above, were adsorbed unstressed exponentially growing cells (Figure 2c) onto freshly glow-discharged carbon-coated grids although, based on the intensity of the labelled (Polaron G200). Following absorption ofexcess pro- Hsp26 polypeptide detected by autoradiography, it tein, the grids were stained with 5% (w/v) uranyl represented only between 0.1-1.0% of the total proacetate, dried and examined under a Phillips 410 teins synthesized. However, following a 22-36°C electron microscope. heat shock, very high levels of Hsp26 synthesis were detectable (Figure 2d) and, in addition, the overexpression of at least three other low molecular RESULTS weight (24 000-27 000) polypeptides was also detectEngineering overexpression of Hsp26 in S. cerevisiae able, with one of these proteins (labelled b in Figure To facilitate the overexpression of the small heat- 2d) having an identical molecular weight to Hsp26 shock protein Hsp26, either during or prior to a heat (but different isoelectric point). Since these shock, two multicopy plasmids were constructed. In additional polypeptides a-c did not label with the plasmid pUKC360 (Figure la) the entire HSP26 [35S]methionine(data not shown), in common with gene and its promoter region (up to - 1500 bp) were Hsp26 (Bossier et al., 1989), this suggests that these inserted into the multicopy plasmid pMA3a that polypeptides could represent differentially modified contains the LEU2-d selectable marker and the forms of Hsp26, possibly phosphorylated (or 2 pm plasmid ORI and REP3 sequences (Spalding dephosphorylated) forms, as have been described for and Tuite, 1989). To allow for overexpression of mammalian Hsp28 (Arrigo and Welch, 1987). In the Hsp26 prior to a heat shock, a derivative of the pUKC360 transformant there was no indication that HSP26 gene containing a natural BglII restriction overexpression of the Hsp26 polypeptide during a site at position -56 with respect to the HSP26 mild heat shock had any qualitative or quantitative translation start codon (Bossier et al., 1989) and a effect on the synthesis of other Hsps (compare Figure BamHI linker introduced at the NruI site + 33 bp 3' 2b and 2d). to the HSP26, translational termination codon (P. In the untransformed MD40/4c strain, Hsp26 Bossier and M. F. Tuite, unpublished data) were represented one of the major polypeptides syntheused. This allowed for the insertion of the HSP26 sized 2 h after cells had reached stationary phase coding region into the BglII site of the PGK- (Figure 3a), whereas in the pUKC360 transformant

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1

Figure 2. Two-dimensional SDSPAGE (non-equilibrium pH gradient) analysis of radiolabelled proteins synthesized after a mild heat shock (22-36°C) in either an untransformed strain (panels a, b) or in the same strain transformed with the plasmid pUKC360 (panels c, d). The cells were pulse-labelled with ['4C]-labelledtotal amino acids as described in Materials and Methods. The exposed autoradiographs (28 days) are shown as follows: (a) untransformed, 22°C; (b) untransformed after transferring from 22°C to 36°C for 1 h; (c) pUKC360 transformant, 22°C; (d) pUKC360 transformant after transferring from 22°C to 36°C for 1 h. The Hsp26 polypeptide is indicated by the large arrow, the three potentially Hsp26-related polypeptides are indicated by the small arrows labelled a d .

Hsp26 represented greater than 90% of the total protein synthesis continuing in such stationary phase cells (Figure 3b). All three of the potentially Hsp26-related polypeptides, detected in the pUKC360 transformant after a mild heat shock (Figure 2d), were also synthesized in the pUKC360 transformaht in stationary phase (Figure 3b). These results suggest that the HSP26 gene is regulated normally on a multicopy plasmid both by a mild heat shock and by entry into stationary phase, although there was some evidence that Hsp26 synthesis occurred, albeit at a low level, in non-stressed exponentially growing cells.

Thermotolerance in strains overexpressing Hsp26

A potential role for small Hsps is in the acquisition of thermotolerance (Lindquist and Craig, 1988). While gene deletion studies with HSP26 in S. cerevisiae have convincingly demonstrated that Hsp26 is required neither for viability nor for thermotoleranceper se (Petko and Lindquist, 1986), we used the plasmids pUKC360 and pUKC370 to investigate whether elevating the levels of this small Hsp, either as a consequence of a heat shock (in pUKC360 transformants) or by replacing the normally regulated HSP26 promoter with the consti-

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Figure 3. Two-dimensional SDS-PAGE (non-equilibrium pH gradient) analysis of proteins synthesized by cells 2 h after entry into stationary phase. The cells were pulse-labelled with ['4C]-labelledtotal amino acids as described in Materials and Methods. The exposed autoradiographs (28 days) are shown as follows: (a) untransformed strain; (b) pUKC360 transformant. The Hsp26 polypeptide is indicated by the large arrow, the three potentially Hsp26-related polypeptides are indicated by the small arrows labelled a x .

tutively expressed promoter from the PGKl gene (in pUKC370 transformants), significantly enhanced thermotolerance. Thus in pUKC370 transformants, elevated levels of Hsp26 would be present prior to the heat shock. Thermotolerance was measured as acquired resistance to thermal killing at 50°C subsequent to growth at 30°C. While the expected thermotolerance was observed in control cells that had been preincubated at 42°C for 30 minprior to the shift to 50"C, there was no dramatic enhancement of thermotolerance in the pUKC360 transformant subject to the lethal heat shock without prior preincubation at 42°C (Figure 4). There was, however, a reproducible increase in thermotolerance in the pUKC370-transformed cells in the absence of a42"C preincubation, indicating that elevating the levels of Hsp26 prior to a severe heat shock can provide a degree of thermotolerance for the cells, which is not observed if overexpression of Hsp26 is the result of a prior heat shock. These results therefore support the notion that changes in the cellular levels of Hsp26 in heat-shocked cells do not affect thermotolerance (Petko and Lindquist, 1986). Hsp26 forms a high molecular weight complex In strains transformed with pUKC360 or pUKC370, very high levels of expression of Hsp26 were observed in stationary phase cells, with Hsp26 representing approximately 17% of the steady-state

0

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15

20

time at 50°C (min) Figure 4. Thermotolerance in strains overexpressing Hsp26. Killing at 50°C over a 20-min time period was measured in the following four strains; MD40/4c transformed with pMA3a (0); MD40/4c transformed with pMA3a incubated at 42°C for 30 min ( 0 ) MD40/4c ; transformed with pUKC360 (0);and MD40/4c transformed with pUKC370 (A). The error bars indicate the standard deviation for the % viability values obtained.

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I 23456 7

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45

-

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Figure 5. The overexpression of Hsp26 in pUKC360 and pUKC370 transformants. Total protein samples (50 pg) were prepared either from exponential phase cells (lanes 1 , 3 , 5 , 7 ) or from stationary phase cells (lanes 2 , 4 , 6 , 8 ) and analysed by one-dimensional SDS-PAGE. Shown is the Coomassie Bluestained gel. Lanes I , 2, MD40/4c untransformed; lanes 3, 4 MD40/4c [pUKC360] transformant; lanes 5, 6 MD40/4c pUKC3701 transformant; lanes 7, 8 MD40/4c [pMA3a] transformant. M, molecular weight markers (kDa). The position of Hsp26 is indicated by the arrow.

levels of protein in cell-free lysates prepared from such transformants, as determined by densitometry of Coomassie Blue-stained one-dimensional SDSPAGE gels (Figure 5). Furthermore, similarly high levels of expression of Hsp26 were observed in nonstressed exponentially growing cells of the pUKC370 transformant, suggesting that there is no increase in the turnover of the Hsp26 polypeptide in non-stressed cells. A common property of small Hsps in higher eukaryotes is an ability to self-assemble into high molecular weight aggregates (often referred to as HSGs; Nover et al., 1983, 1989; Arrigo and Welch, 1987; Collier et al., 1988). To determine whether overexpressed Hsp26 of S. cerevisiae self-aggregated in a similar manner, a subcellular fraction of yeast, enriched for high molecular weight aggregates or structures, was prepared by differential centrifu-

gation as described in Materials and Methods. The resulting fraction was then sedimented through a 0.5-1 .O M-sucrosegradientand the resultinggradient fractionated and analysed by one-dimensional SDSPAGE. In extracts from the strain transformed with thecontrolplasmidpMA3aandallowed togrow into stationary phase, a protein with an apparent molecular weight of 27 000 featured prominently in the lower half of the gradient (Figure 6a). In extracts from the same strain, transformed with plasmid pUKC360 and subjected to a severe heat shock (42°C for 30min), a similar gradient profile was observed except that much higher relative levels of the 27 000 molecular weight polypeptide in the gradient were observed in relation to the pMA3a transformant for a given amount of protein loaded (Figure 6c). That the 27 000 molecular weight polypeptide was Hsp26 was demonstrated in two ways;

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Figure 6. Sucrose gradient analysis of high molecular weight protein aggregates formed in cells overexpressing Hsp26. Samples were prepared as described in Materials and Methods and fractionated on a 0.5 to 1.0~-sucrosegradient. Shown is onedimensional SDS-PAGE analysis of the fractionated gradients with proteins being detected by Coomassie Blue staining. (a) MD40/4c grown to stationary phase; (b) MD40/4c carrying an HSP26::HIS3disruption;(c) MD40/4c [pUKC360] transformant following a heat shock (42°C for 30 min); (d) MD40/4c [pUKC370] transformant grown to exponential phase (i.e. non-stressed). The location of the Hsp26 polypeptide is indicated by the arrow. Lane M contains proteins with molecular weights of 26,36,48,58, 84 and 116 kDa respectively.

by the observation that this polypeptide was missing from the gradient of extracts prepared from MD40/ 4c carrying an HSP26::HIS3 gene disruption and grown to stationary phase (Figure 6b), and by the fact that the protein cross-reacted with an antiHsp26 polyclonal antibody (data not shown). These data therefore confirm that yeast Hsp26, like other eukaryotic small Hsps, is able to form a high molecular weight aggregate both after a heat shock (Figure 6c) and in stationary phase cells (Figure 6a). To determine whether stress-inducing conditions were required for the formation of the Hsp26containing aggregates, extracts from unstressed exponentially growing cells of MD40/4c [pUKC370] transformants were similarly analysed. As shown in Figure 6d, identical results were obtained, indicating that these aggregates can form in nonstressed cells. The growth rate of the pUKC370 transformant did not differ from the untransformed or pMA3a-transformed strain in defined medium. Analysis of the Hsp26-containing aggregates

By subjecting the sucrose gradient fractions containing the Hsp26 aggregates to a second round of

fractionation on a 0.5-1 .O M-sucrose gradient, essentially homogenous aggregate fractions were obtained which, when analysed by one-dimensional SDS-PAGE, appeared to consist entirely of Hsp26 (Figure6and datanot shown). That theseaggregates contained no other class of integral macromolecules was confirmed by subjecting purified aggregates to treatment with either a-amylase or ribonuclease A (see Materials and Methods). Subsequent refractionation ofthe treatedaggregates on a0.5-1.0 M-sucrose gradient demonstrated that, in each case, the Hsp26containing aggregate remained intact, banding in a similar position on the gradient as untreated aggregates (data not shown). This indicates that neither carbohydrate nor RNA are integral components of the Hsp26 aggregate. Treatment of the purified aggregates with the protease chymotrypsin resulted in their complete disappearance from the gradient, confirming their proteinaceous nature. The physical properties of the Hsp26-containing aggregates, purified by two sequential 0.5-1 .O Msucrose density gradients, were determined. The molecular weight of the aggregate was calculated as 550kDa and, given the molecular weight of Hsp26 (27 kDa) and assuming that no other

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Figure 7. Ultrastructural analysis of high molecular weight aggregates containing Hsp26. The aggregates were stained with uranyl acetate and viewed at a magnification of 65 000. The bar represents 50 nm.

macromolecules are associated with the aggregate (see above), this indicated that the aggregates each contain approximately 20 molecules of Hsp26. Electron microscopic analysis of the purified Hsp26-containing aggregates purified from the unstressed pUKC370 transformant grown to midexponential (Figure 7) revealed aggregates of approximately uniform shape and size, being slightly oval in shape with a crinkled surface appearance. The size of the aggregates was estimated as 15-25 nm. There was no evidence of any higher order aggregates. DISCUSSION The cellular role of small Hsps in eukaryotic cells remains something of an enigma. On the one hand they represent one of the major groups of stressinduced proteins, yet on the other hand, no definite cellular role has been assigned to them in either stressed or non-stressed cells. To allow us to probe further the cellular role of Hsp26, the major small Hsp of S . cerevisiae, we constructed genetically engineered strains which massively overexpressed Hsp26 either in response to a stress, such as heat shock or entry into stationary phase, or which con-

stitutively overexpressed Hsp26 in non-stressed exponentially growing cells. In the latter case this was achieved using the highly expressed and widely exploited PGKZ promoter (Mellor et al., 1983). Comparison of the observed steady-state levels of Hsp26 overexpressed from a plasmid-borne gene, either using the natural HSP26 promoter or the PGKZ promoter in stationary phase cells (Figure 5), demonstrated that both promoters are apparently equally effective for Hsp26 overexpression studies. Furthermore, it demonstrated that the HSP26 coding region apparently contains a sequence able to provide the necessary downstream activation sequence function reportedly required for optimal activity of a plasmid-borne PGKZ promoter (Mellor et al., 1987). One possible cellular role for Hsp26 might be in the acquisition of thermotolerance, i.e. the ability to overcome an otherwise lethal temperature. While it is clear from the gene disruption studies of Petko and Lindquist (1986) that an inability to express Hsp26 has no apparent effect on the ability of S. cerevisie to acquire thermotolerance, a fact we have independently confirmed (S. Christodoulou and M. F. Tuite, unpublished data), the Hsp overexpression

104 strains we constructed allowed us to determine whether massive overexpression of Hsp26 either as a consequence of stress, or prior to a stress-inducing condition, could provide the same degree of thermotolerance observed in a wild-type (Hsp26') strain. The results we obtained (Figure 4) indicate that there is a small, but reproducible increase in the degree of thermotolerance only in strains overexpressing Hsp26 prior to the heat stress. Similar results have also been recently reported by Susek and Lindquist (1989) using the galactose-regulated promoter GAL1 to overexpress HSP26 prior to a lethal heat shock. Overexpression of human Hsp28 in rodent cells has also been recently demonstrated to confer thermotolerance to such cells (Landry et al., 1989). These data therefore indicate that artificially elevating the cellular levels of Hsp26 may provide increased thermoprotection, but under normal cellular conditions one or more other Hsps may play a more pivotal role in acquired thermotolerance, e.g. Hspl04 in S. cerevisiae (Sanchez and Lindquist, 1990). One possible explanation for the increased thermotolerance of strains overexpressing Hsp26 might be that this results in overexpression of other Hsps such as Hsp 104. However, both one-dimensional SDS-PAGE (N. J. Bentley and M. F. Tuite, unpublished results) and two-dimensional SDSPAGE (Figure 2) of proteins synthesized in overexpressing strains failed to detect overexpression of other Hsps. The question of whether or not Hsp26 is the only small Hsp in yeast is important in relation to the elucidation of its cellular function. Some eukaryotic cells apparently only express one major small Hsp (e.g. human Hsp28, Arrigo and Welch, 1987), while others (e.g. Drosophila, plant cells) can express anywhere between six and 30 electrophoretically distinct small Hsps. Hybridization analysis and immunological analysis has demonstrated that the S. cerevisiae genome does not contain any other HSP26/Hsp26-related sequences (Petko and Lindquist, 1986). S . cerevisiae does, however, express other low molecular weight Hsps; Hspl2 (Praekelt and Meacock, 1990) and ubiquitin (UB14 gene, Finley et al., 1987) being the two bestcharacterized examples. High resolution twodimensional SDS-PAGE analysis also indicated there are other low molecular weight proteins, possibly related to Hsp26, expressed in S . cerevisie in both a wild-type yet heat-shocked cell (Bossier et al., 1989) and in stressed cells overexpressing Hsp26 (Figures 2, 3). It remains to be determined whether any of these other putative small Hsps can

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functionally substitute for the loss of an ability to synthesize Hsp26, although a strain carrying a double disruption of both the HSP26 and H SPl2 gene are viable and show a normal degree of thermotolerance (U. Praekelt and P. Meacock, personal communication). The genes for other putative small Hsps from S . cerevisiae have yet to be identified. An unusual property which Hsp26 from S. cerevisiae has in common with other eukaryotic small Hsps is that they can be found in heat-stressed cells as high molecular weight aggregates (20& 800 kDa) termed HGSs. HSGs usually contain the majority of the members of the small Hsp family characteristic of a given species and are mainly located in the perinuclear region of heat-stressed cells (Arrigo et al., 1987; Arrigo and Welch, 1987; Collier et al., 1988; Nover et al., 1989). This apparent ability to self-assemble into high molecular weight multimeric structures is a property small Hsps also share in common with a-crystallin, one of the major structural proteins of the vertebrate eye lens, which also exists as a high molecular weight aggregate (40&900 kDa) composed of two types of related subunits, the aA and a B crystallins (de Jong et al., 1989). It has also recently been shown that expression of the a B gene is heat shock-inducible, suggesting that a B crystallin is a bonajde small Hsp (Klemenz et al., 1991). Small Hsps and the a A and a B crystallins show considerable amino acid identity, particularly within a region in the C-terminal portion of the proteins. This sequence, which forms a distinct hydrophobic region within the polypeptides, has a consensus sequence Gly-Val-Leu-Thr-(X),-Pro with the Gly, Leu and Pro residues being invariant (Nagao et al., 1985;de Jong etal., 1989). The Hsp26 protein from S. cerevisiae has a perfect match to this conserved consensus sequence in its C-terminal region (Bossier et al., 1989). Given that these residues have been implicated as being important in the ternary structure of a-crystallin (Wistow, 1985) would suggest that this hydrophobic region is required for small Hsps to self-assemble. However, it should be noted that, in addition to the small Hsp-containing HSGs, both the yeast mitochondria1 assembly protein Hsp60 and its E. coli homologue GroEL are also able to self-assemble into high molecular weight aggregates (Hendrix, 1979; Bochkareva et al., 1988), but in neither case do these polypeptides show any homology with the short conserved hydrophobic region described above for small Hsps.

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Rossi and Lindquist (1989) had previously pro- other cellular function, a function that remains to be vided preliminary evidence that Hsp26 from s. defined. cerevisiae may exist as a high molecular weight aggregate (i.e. with a molecular weight greater than ACKNOWLEDGEMENTS 500 kDa in cells following a 25-39°C heat shock). N.J.B. and I.T.F. were supported by SERC PhD We have now confirmed this finding and extended studentships. We gratefully acknowledge the assistthe observations, demonstrating that the high ance of D r Helian Boucherie at the University of molecular weight Hsp26-containing aggregate Bordeaux I1 with the SDS-PAGE and financial (Hsp26-HSG) can also form in non-heat-shocked, support to I.T.F. from the Society for General stationary phase cells and also in the absence of any Microbiology to undertake collaborative experstress in cells genetically engineered to overexpress iments in Bordeaux. Finally, we thank Ray Newsam Hsp26 in exponentially growing cells. Electro- and Professor Keith Gull (University of Kent) for phoretic analysis and molecular weight determi- assistance with the electron microscopy and D r nation indicate that the Hsp26-HSG is a multimer Susan Lindquist (University of Chicago, U.S.A.) of approximately 20 identical subunits of Hsp26, for providing us with the HSP26 gene on plasmid and there appears to be no other associated macro- pHSP26. molecules. We have also recently shown that Hsp26 forms identical HSGs when expressed in E. coli REFERENCES (N. J . Bentley and M. F. Tuite, in preparation), Arrigo, A. P. and Welch, W. J. (1987). Characterisation suggesting that Hsp26-HSGs do not require any and purification of a small 28,000 Dalton mammalian other S. cerevisiae proteins for assembly. Electron heat shock protein. J . Biol. Chem. 262,15359-15369. microscopic analysis of the Hsp26-HSGs indicated Arrigo, A. P., Simon, M., Darlix, J. L. and Spahr, P. F. (1987). 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