Mol Oen Genet (1990) 223:9%106 © Springer-Verlag 1990

HSP12, a new small heat shock gene of Saccharomyces cerevMae: Analysis of structure, regulation and function Uta M. Praekelt and Peter A. Meacock Leicester Biocentre, University of Leicester, University Road, LeicesterLEI 7RH, England Received March 23, 1990 Summary. We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural similarity to other small heat shock proteins. H S P 1 2 gene expression is induced several hundred-fold by heat shock and on entry into stationary phase. H S P 1 2 m R N A is undetectable during exponential growth in rich medium, but low levels are present when cells are grown in minimal medium. Analysis of H S P t 2 expression in mutants affected in cAMP-dependent protein phosphorylation suggests that the gene is regulated by cAMP as well as heat shock. A disruption of the H S P 1 2 coding region results in the loss of an abundant 14.4 kDa protein present in heat shocked and stationary phase cells. It also leads to the induction of the heat shock response under conditions normally associated with low-level H S P 1 2 expression. The H S P 1 2 disruption has no observable effect on growth at various temperatures, nor on the ability to acquire thermotolerance. Key words: Heat shock - Saccharomyces cerevisiae - Stationary phase - cAMP - Thermotolerance

Introduction The heat shock response is a universal phenomenon observed in all organisms. Exposure to elevated temperatures or other types of stress, such as anoxia and starvation, leads to the synthesis of so-called heat shock proteins (hsps) (Lindquist 1986). The accumulation of hsps is associated with the acquisition of thermotolerance, an increased ability to survive short exposures to otherwise lethal temperatures (McAlister and Finkelstein 1980). However, as yet there is no direct evidence for the protective function of hsps during heat stress (Schlesinger 1986). Offprint requests to ."U.M. Praekelt

The hsps are divided into two major size classes of 70-100 kDa and 17-30 kDa, which in many organisms are encoded by multigene families (Lindquist 1986). The yeast Saccharomyces cerevisiae has multigene families encoding the large hsps (Werner-Washburne et al. 1987; Borkovich et al. 1989), however, only one small hsp of Mr 26 kDa (hsp26) has so far been identified (Petko and Lindquist 1986). The large hsps are highly conserved between species, and mutational analysis in yeast has shown that they perform vital functions during normal growth (Normington etal. 1989; Chirico et al. 1988; Deshaies etal. 1988; Ostermann etal. 1989). In spite of their massive accumulation after heat shock, their role in the acquisition of thermotolerance is not clear, since gene inactivations had no effect on the induction of thermotolerance by a brief heat shock (Craig and Jacobsen 1985; Borkovich et al. 1989). Consistent with these observations, work with Drosophila melanogaster and Dictyostelium discoideum has shown a strong correlation between the accumulation of the small hsps and the acquisition of thermotolerance (Berger and Woodward 1983; Loomis and Wheeler 1982). The small hsps show only limited sequence conservation between species. In higher eukaryotes and yeast, they are synthesised during certain developmental stages as well as after heat shock (Lindquist 1986). The yeast hsp26 accumulates to high levels during sporulation and in stationary phase. Whilst the induction of heat shock genes by heat shock is coordinately regulated by heat shock factor (HSF) interacting with conserved upstream sequences (Sorger and Pelham 1987, 1988), developmental control apparently involves separate mechanisms. In D. melanogaster, small hsps are under hormonal regulation (Berger and Woodward 1983), and evidence is accumulating that developmental regulation of yeast hsps may be mediated by cAMP (Tanaka et al. 1988; WernerWashburne et al. 1989). The role of hsp26 in sporulation, stationary phase and thermotolerance is not clear, since inactivation of the hsp26 gene had no apparent effect on performance or survival under these conditions (Petko and Lindquist

98 1986). These observations have led to the suggestion that yeast may possess (an)other, as yet unidentified, small hsp(s), which could compensate for the lack of hsp26 function (Petko and Lindquist 1986; Bossier et al. 1989). One of the genes encoding ubiquitin (UBI4) was recently found to be heat-inducible (Finley et al. 1987) and, although it was important for survival of chronic heat stress, it too could not be implicated in the ability to acquire thermotolerance to acute heat stress (Tanaka et al. 1988). We report here the isolation and characterisation of a new small heat shock gene, HSP12, from yeast. Its expression is strongly induced by heat shock and on entry into stationary phase, and appears to be affected by cAMP-mediated protein phosphorylation. We present results obtained with an HSP12 disruption strain and discuss the role of this new small heat shock gene in thermotolerance.

Materials and methods

Yeast strains and culture media. The haploid S. cerevisiae strain $288C (e, mal, gal2) was used for cDNA and genomic cloning, and for studies of gene expression by Northern blotting. The diploid strain 842 (ale, ade2-1/ade2-1, trpl-1/ trp l-1, leu2-3/leu2-112, his3-11/his3-15, ura3/ura3, canrl IO0/CAN) was used for the HSP12 gene disruption. Mutants affected in cAMP-dependent protein phosphorylation were: AM-110-4C (Mata, cyrl-2, leul); AM-180-2B (Mata, bcyl, his7); AM9-10A (Mate, cyrl1, bcyl), and were obtained from Dr. A. Wheals, Bath University. Yeast media were prepared as described (Sherman et al. 1986). Pre-sporulation medium consisted of 0.8% bacto yeast extract, 0.3% bacto peptone and 10% dextrose, and sporulation medium contained 1% potassium acetate, 8 lag/ml adenine, 4 lag/ml each of tryptophan, histidine and uracil, and 12 lag/ml leucine. Isolation and analysis of cDNA and genomic clones. The isolation of cDNA clones will be described elsewhere (U.M. Praekelt and P.A. Meacock, in preparation). Two of the cDNA clones containing HSP12 sequences were subcloned into the EcoRI sites of pUC18 and M13rap18 vectors (Vieira and Messing 1982; Yanisch-Perron et al. 1985). Total yeast DNA was partially digested with Sau3A and cloned into 2EMBL4 digest with BamHI (Frischauf etal. 1983). Recombinant clones were screened with HSP12 cDNA, and the positive clone mapped by Southern blotting as described by Maniatis et al. (1982). A 1.7 kb EcoRI fragment containing the HSP12 gene was subcloned into pUC18, and overlapping restriction fragments from this cloned into M13mpl9 and rap18 vectors. Sequencing was carried out by the method of Sanger et al. (1977). Purification of RNA and Northern blotting. Cells were broken by vortexing with glass beads in buffer containing 1% tri-iso-propylnaphthalene sulphonate (Kodak),

6% 4-amino salicylate, 50 mM TRIS-HC1 pH 8.3 and 6% phenol equilibrated with 50 mM TRIS-HC1 pH 8.3, and extracted with phenol: chloroform: isoamyl alcohol 50: 50: 1 (v: v: v) three times. Nucleic acids were precipitated with 0.2 M lithium acetate pH 6.0 and 2.5 volumes ethanol at - 2 0 ° C and dissolved in sterile distilled water (SDW). RNA was precipitated by addition of an equal volume of 6 M lithium acetate pH 6.0 and stored on ice for 2 h. The precipitate was washed twice in 3 M lithium acetate pH 6.0, dissolved in SDW, ethanol precipitated as above, and dissolved in SDW at a concentration of approximately 2 rag/m1. R N A was electrophoresed on agarose gels containing formaldehyde (Maniatis et al. 1982) and blotted onto Hybond-N (Amersham) membrane. Blots were pre-hybridised in BLOTTO (Makara and Henson 1985) for 2 h at 65 ° C, and hybridised to the probe overnight at the same temperature. Probes were prepared by the random hexamer priming method (Feinberg and Vogelstein 1983) using e-[32P]dCTP. Washing was carried out in 3 x SSC (SSC is 0.15 M NaC1, 15 mM sodium citrate) at 65 ° C. For autoradiography Kodak X-OMAT AR film was used.

Primer extension. Fifty nanograms of the oligonucleotide TCCTTTTCTACCTGCG (obtained from J. Keyte, Biochemistry Department, Leicester University), which is complementary to bases 9-24 of the coding strand, was labelled in kinase I buffer (Maniatis et al. 1982) with 50 laCi 7-[32p]-ATP and 10 units T4 polynucleotide kinase (total volume 10 lal) for 30 min at 37 ° C, ethanol precipitated, and dissolved in 5 gl SDW. The primer was heated at 65° C for 3 rain and annealed in a final volume of 20 lal containing 1 x RT buffer (50 mM TRIS-HC1 pH 8.3, 50 mM NaC1, 8 mM MgC12), 25 mM dithiothreitol, 1 lal RNasin (Promega Biotec), 50 lag total R N A at 42 ° C for 1 h. Primer extension was carried out by adding 5 lal eachof 10 mM dATP, dCTP, dGTP, dTTP, 3 lal 10 x RT buffer, 0.5 lal actinomycin D (2.5 mg/ml, 20 units AMV reverse transcriptase in 2 lal and 4.5 lal SDW, and incubating at 42°C for 2 h. The reaction was stopped by addition of 1 lal 10% SDS and 5 lal 0.5 M EDTA. R N A was hydrolysed by adding 30 lal SDW and 20 lal NaOH and boiling for 5 rain, and the solution neutralised with 20 lal 1 M HC1. The D N A was precipitated at - 7 0 ° C after addition of 5 lag carrier tRNA and 300 lal ethanol. After centrifugation the pellet was dissolved in 3 lal TE (10 mM TRIS-HC1 pH 7.5, 1 mM EDTA), and electrophoresed in parallel with sequencing reactions which had been primed with the same oligonucleotide. Analysis of proteins. Proteins were extracted from 0.5 ml aliquots of culture by vortexing cell pellets with glass beads in 100 lal lysis buffer (50 mM TRIS-HC1 pH 7.2, 1% deoxycholate, 1% Triton, 0./% SDS and 2raM phenylmethylsulfonyl fluoride). Proteins were denatured by boiling with an equal volume of 2 x sample buffer (25 mM TRIS-HC1 pH 6.8, 2% SDS, 10% glycerol, 0.2 M /~-mercaptoethanol, 0.002% bromophenol blue) and electrophoresed in denaturing 15% polyacrylamide

99

gels as described by Haines (1981). For in vivo labelling of proteins, 20 gCi of [aSS]methionine was added to 0.5 ml aliquots of cell suspension in minimal SD medium, incubated at 30° C for 10 rain and washed with cold SDW twice; proteins were extracted as above.

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HSP12 gene disruption. This was carried out by the onestep disruption method described by Rothstein (1983), as follows. A 1.1 kb HindIII fragment containing the URA3 gene from plasmid YEP24 (Botstein et al. 1979) was blunt-ended and inserted into the StyI site of the shorter HSP12 cDNA clone in pUC18. The cDNA insert was recovered from the vector by EcoRI digestion, and used for transformation of strain 842. URA3 recombinants were selected on minimal medium lacking uracil, and sporulated as described by Sherman et al. (1986). Tetrads were dissected and individual spores grown up on YPD plates. Phenotypes were established by growth on ura- plates, and the gene disruption confirmed by Southern blotting.

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Thermotolerance experiments. Cultures were grown in YPD medium at 25°C to an optical density of 0.1 at 650 rim. Aliquots of 1 ml were dispensed into thin-walled glass tubes, and incubated in a shaking waterbath at 51 ° C for the times indicated, then immediately cooled on ice. Appropriate dilutions were plated onto YPD plates, incubated at 30° C for 2 days, and the number of colony forming units counted. In the case of pretreatment to induce the heat shock response, cultures were incubated at 37° C for 1 h and aliquots of 1 ml treated as above.

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Results

Isolation of cDNA and genomic HSP12 clones We have identified a new yeast heat shock protein gene, which we have named HSPt2, from a collection of genes which were isolated on the basis of their induction on entry into stationary phase of a culture of S. cerevisiae grown in molasses (U.M. Praekelt and P.A. Meacock, manuscript in preparation). Four clones containing HSP12 sequences were isolated by differential screening of a stationary phase cDNA library, using exponential and stationary phase cDNA probes. Three of the clones had inserts of approximately 540 bp while that of the fourth was slightly larger at 600 bp. Northern blot analysis (not shown) confirmed differential expression of HSPt2, and showed the size of the mRNA transcript (560 nucleotides) to correspond closely to the size of the cDNA inserts. Results from a Southern blot analysis of total yeast DNA suggest that HSP12 is a single copy gene (Fig. 2A). A genomic clone containing the HSP12 gene was isolated from a genomic library of $288C DNA. The complete coding region as well as flanking sequences were found on a 1.7 kb EcoRI fi'agment within a 7 kb insert, and this was subcloned into pUC18 for further analysis.

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Fig. 1 A and B. Structure of the HSPI2 gene. A Restriction map of the 1.7 kb genomic EcoRI fragment containing the HSPI2 gene. Indicated sites are: A, AccI; E, EcoRI; H, HindIII; Hp, HpaII; R, RsaI; S, Sau3A; St, StyI; X, XbaI. Above this is shown the position of the mRNA transcript with open reading frame (ORF), and below a summary of the sequencing strategy. B DNA sequence of the HSP12 gene, and deduced amino acid sequence of the ORF, indicating the 5' and 3' ends of the longest cDNA clone (solid circles), putative TATA boxes (boxed), transcript initiation sites (triangles; solid, major site; open, minor site), heat shock elements (HSE, indicating matches to the consensus), and putative polyadenylation signals (solid line). Repeated hexamer motifs are indicated by arrows

DNA sequence analysis The DNA sequences of two cDNA clones and 1275 bp of the 1.7 kb genomic subclone were determined as indicated in Fig. 1. An EMBL database search of the DNA sequence revealed no homology to available sequences. The two cDNA clones, one extending to position + 1 and the other to position - 3 9 , were identical to the genomic sequence in corresponding regions. The orientation of the mRNA transcript was confirmed, and initia-

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Fig. 2 A and B. Copy number and transcript mapping of the HSP12 gene. A Southern blot of yeast DNA, digested with EcoRI, E; BarnHI, B; HindIII, H; PstI, P; KpnI, K; and SalI, S, and hybridised to the insert of an HSP12 cDNA clone. Hybridisation was at 65° C in BLOTTO, and washing was carried out at 65° C in 3 x SSC. B Transcript mapping by primer extension. Autoradiography of lane P, containing the primer extension product, was for 30 min, and that of the adjacent lanes containing sequencing reactions, was for 16 h

tion sites were determined by primer extension analysis (Fig. 2B). Two major initiation sites were found at positions - 5 9 and - 6 0 , of which the latter was used approximately twice as frequently as the former. Surprisingly the two cDNAs had no poly(A) tails, both terminating at identical positions after a run of seven T residues. Since the template R N A was selected by binding to oligo(dT)-cellulose, the poly(A) tails must have been lost during the cDNA cloning procedure, either after priming of first strand synthesis with oligo(dT), or possibly as a result of self-priming through annealing of the poly(A) tail to the U tract. The D N A sequence upstream of the open reading frame (ORF) has many features typical of yeast promoters (Struhl 1986, 1987). Two TATA boxes are present at positions - 1 4 5 and - 7 7 , and a pyrimidine-rich tract (CT block), found in many yeast promoters (Dobson et al. 1982), spans nucleotides - 9 8 to - 8 0 . The 5' untranslated leader is typically A-rich. The sequence surrounding the initiation codon is characteristic of highly expressed yeast genes, showing an A at position - 3 and a T at + 6 (Dobson et al. 1982), and corresponds in 9 out of 10 positions to the consensus for yeast m R N A identified by Cigan and Donahue (1987). Sequences upstream of the essential promoter region include two putative heat shock elements (HSE), in acreement with the observation that the gene is heat inducible (see below). The proximal one at position - 1 8 0 consisting of a 5/6 match to the central consensus G A A N N T T C , and the distal one at - 4 5 9 matches the extended consensus C N N G A A N N T T C N N G in 7 out of 8 positions. However, the motif TTCTAGAA, re-

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Fig. 3A and B. Structure of the predicted hsp12 polypeptide. A Hydropathy plot, using the UWGCG program, based on values according to Kyte and Doolittle (1982). Moving window average: 11 residues. B Alignment of a portion of the predicted hsp12 polypeptide (1) with those of soybean hsp17 (2) (Raschke et al. 1988) and fruitfly hsp26 (3) (Ingolia and Craig 1982) ported to be present in all previously identified yeast heat shock genes (Tuite et al. 1988), is not found in the HSP12 promoter. An additional, striking feature of the upstream region is the repeated hexanucleotide G G A A A A , which occurs in an almost perfect tandem repeat of five copies as well as in two separate copies. The D N A sequence 3' to the coding region contains two motifs related to the polyadenylation signal of higher eukaryotes, A A T A A A (Proudfoot and Brownlee 1976), although it is not clear whether this element functions as polyadenylation signal in yeast (Zaret and Sherman 1982).

Analysis of the coding region The longest ORF, as specified by the first initiation codon following the transcript initiation sites, consists of 109 codons potentially encoding a protein of Mr 11680 dalton. It has a codon bias index of 0.82, characteristic of very highly expressed yeast genes (Bennetzen and Hall 1982; Sharp et al. 1986). A comparison of the deduced amino acid sequence with polypeptides in the Swissprot and N B R F databases revealed no significant homologies. The predicted polypeptide sequence shows a remarkable excess of negatively charged (20.2%) and positively charged (18.4%) residues. This is reflected in a negative hydropathy plot (Fig. 3 A) characteristic of other small hsps (Lindquist and Craig 1988). Figure 3B represents the best alignment achieved of hsp12 with other small hsps, showing limited homology of up to 33% between

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Fig. 5. HSP12 expression of mutants affected in cAMP-dependent protein phosphorylation. Northern blot of total RNA (10 gg) hybridised to HSP12 cDNA. Cultures were grown in YPD medium and harvested in exponential phase (Expon.) or stationary phase (St.). Genotypes are wild type (wt), bcyi (bcy), cyrl-2 (cyr), and bcyl/cyrI-1 (bcy/cyr)

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Fig. 4A and B. Hspl2 gene expression during batch growth in YPD. A Northern blot of RNA (5 gg per lane) extracted at various stages from a batch culture growing at 30° C. Hybridisation probes were Hspl2 and HSP26 cDNA and actin DNA (hybridisation to the actin probe served as control for RNA loading, although actin RNA was absent from stationary phase cells). Numbers 1-7 refer to sampling points indicated in B. HS, RNA from a culture growing exponentially at 25°C (OD6so=2), after a heat shock of 40 rain at 37° C. B Growth curve with histogram of transcript levels of HSPI2 (solid bars) and HSP26 (open bars). After autoradiography, individual spots were cut out from the blot and radioactivity measured by scintillation spectrometry. Values are expressed as a percentage of the highest levels reached, i.e. in sample 5

a portion of hspl 2 with hsp 17 of soybean (Raschke et al. 1988) and hsp26 of Drosophila (Ingolia and Craig 1982). However, this region o f homology does not correspond to the regions of greatest conservation between most hsps. Surprisingly, the hsp12 sequence shows no homology at all with hsp26 of yeast, nor with e-crystallin, which is related to most small hsps (Lindquist and Craig 1988).

HSP12 gene expression A Northern blot analysis of R N A from various stages of a batch culture (in YPD) shows that the HSPI2 transcript was undetectable in exponentially growing cultures, but in stationary phase was induced several hundred-fold (Fig. 4). By comparison, HSP26 m R N A was similarly absent in cultures of low density, but began to accumulate at an earlier stage than HSP12 m R N A . Maximal transcript levels were found at the same stage in both cases, immediately after the cessation of exponential growth. During the course of stationary phase,

both HSP12 and HSP26 transcript levels decreased gradually, that of HSP26 at a slightly faster rate. HSPI2 was also dramatically induced by heat shock (Fig. 4A). Incubation at 37 ° C for 40 rain of a culture previously growing at 25 ° C, resulted in HSP12 transcript levels approaching those of stationary phase cultures, and comparable with heat-induced levels o f the HSP26 transcript.

Regulation o f liSP12 by cAMP Since it was found that the expression of two yeast heat shock genes, UBI4 and SSA3 (a member of the HSP70 gene family), is induced by low cAMP levels, we investigated HSP12 expression in three mutants affected in cAMP-dependent protein phosphorylation. The temperature - sensitive cyrl-2 mutant has low levels of cAMP at permissive temperatures, and the bcyl mutation results in constitutive activation of cAMP-dependent protein kinase (Matsumoto et al. 1985). Cultures were grown in rich medium and harvested during exponential phase, and HSP12 expression was analysed by Northern blotting (Fig. 5). The results show that HSPI2 m R N A was absent from both the bcyl mutant and cyrl-2, bcyI double mutant, but present in the mutant containing low cAMP levels (cyrl-2) at a level approaching that of wild-type cells in stationary phase. Thus, it appears that HSP12 expression is regulated by cAMP in addition to heat shock. The suppression of the C Y R - phenotype by the bcyl mutation suggests that the effect of c A M P upon HSPI2 expression is mediated via the activity of cAMP-dependent protein kinase.

Effects of disrupting the HSP12 gene A diploid yeast strain (842) was transformed to uracil prototrophy with linearised HSPI2 c D N A which contained a copy of the URA3 gene inserted at the unique StyI site. Tetrad analysis showed that all four spores were viable, and colonies derived from these showed a 2:2 segregation o f uracil prototrophy, as expected for

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Fig. 6A a n d B. HSP12 gene disruption. A Southern blot of DNA from various strains, digested with BglII and hybridised to an HSPI2 cDNA probe. Lanes are: 1, hspl2: : URA3, ura3 haploid spore a; 2, HSP12, ura3 haploid spore b; 3, hsp12: : URA3/HSP12, ura3/ura3 diploid parent, URA + transformant of 842; 4, HSP12/ HSP12, ura3/ura3 diploid 842; 5, HSP12 haploid strain $288C. B SDS-polyacrylamide gel of proteins from stationary phase cultures stained with Coomassie blue. Lanes 1-4, spores a (Ura÷), b (Ura-), c (Ura +) and d (Ura-) derived from the diploid Ura + transformant in lane 5; lane 6, diploid untransformed strain 842; lane 7, haploid strain $288C; M, low molecular weight peptide markers (Sigma), and trypsinogen. Arrow indicates the 14.4 kDa protein absent from two of the spores (lanes 1, 3)

Fig. 7A and B. Comparison of protein and RNA synthesised by the hspl2: : URA3 disrupted haploid spore a (D) and the HSPI2 wild-type haploid spore b (wt), during exponential growth (Ex), after a 40 min heat shock at 37° C (HS) and in stationary phase (St). Cultures were grown in minimal medium. A SDS-polyacrylamide gel of in vivo labelled proteins. The arrow on the right marks the position of the 14.4 kDa marker. Arrows on the left indicate positions of polypeptides present in exponentially growing disrupted, but not wild-type cells. B Northern blot of RNA (10 gg) probed with HSPI2 and HSP26 cDNA

a single copy locus. The disruption o f the H S P t 2 locus in the U R A ÷ transformants was confirmed by Southern blotting (Fig. 6A). An analysis of stationary phase proteins by SDSpolyacrylamide gel electrophoresis, using high percentage polyacrylamide gels, showed that disruption of H S P 1 2 led to the loss of a protein co-migrating with the 14.4 kDA peptide marker. In the wild-type haploid and diploid cells this protein represented an abundant polypeptide easily visible by Coomassie blue staining (Fig. 6B). This 14.4 kDa protein was shown by in vivo labelling to be synthesised after heat shock and during stationary phase by the wild type but not the h s p I 2 : ." U R A 3 mutant haploid (Fig. 7 A). Thus the synthesis of the 14.4 k D a protein correlates with the induction o f H S P t 2 transcription. Since the predicted hspl2 polypeptide contains only one internal methionine, the amino acid used for in vivo labelling, the intensity of the labelled 14.4 kDa band indicates this protein to be one of the most abundant proteins synthesised after heat shock and in stationary phase. A Northern blot analysis of R N A from wild-type

and h s p t 2 : . U R A 3 mutant cells, showed that no normal H S P 1 2 transcript was synthesised by the mutant strain, whereas the wild-type control showed high levels of normal H S P 1 2 expression after heat shock and on entry into stationary phase (Fig. 7B). Instead, the hsp12.': U R A 3 disruption mutant contained low levels of a truncated H S P 1 2 transcript after heat shock. For this experiment, cultures were grown in minimal medium to facilitate in vivo protein labelling, and it can be seen that, in contrast cultures grown in YPD, considerable levels o f H S P t 2 m R N A were present in exponential phase cells. We noted too that under these conditions the h s p i 2 : : U R A 3 mutant contained detectable levels of an H S P 2 6 transcript which was not seen in the wild-type cells (Fig. 7B). This was in addition to low levels of the truncated H S P I 2 transcript which were visible after prolonged exposure of the autoradiography (not shown). Interestingly, in vivo labelling of proteins in exponentially growing h s p l 2 : : U R A 3 mutants revealed the synthesis of several additional proteins when compared with the wild-type strain, as indicated in Fig. 7A. These co-migrated with some of the polypeptides induced by heat

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Fig. 8. Effect of HSP12 disruption on the acquisition of thermotolerance. Cultures of a haploid hspl2: ."URA3 disruption mutant (triangles) and wild-type HSP12 haploid (circles) were incubated

at 51° C for the times indicated,after a 1 h heat treatment at 37° C (solid symbols), or without a prior heat treatment (open symbols)

shock and in stationary phase. Thus it appears that disruption of the HSP12 gene led to an induction of the heat shock response in growth conditions which are associated with low-level HSP12 expression in wild-type cells. Role of HSP12 during growth at different temperatures and in thermotolerance In order to determine whether disrupting the HSP12 locus had an effect on growth at different temperatures, exponentially growing cultures derived from all four spores of a tetrad were plated on YPD plates at equal densities and incubated at 20 °, 30° and 37° C, respectively. The hspi2: : URA3 mutant did not differ from the wild type in its ability to form colonies at these temperatures, and there was no apparent difference in the rate of colony formation. In an experiment designed to test the effect of HSP12 inactivation on induction of thermotolerance, isogenic hspl2: : URA3 mutant and wild-type cells were incubated at 51 ° C with or without a prior heat shock at 37° C. Viability was assessed at 30° C after various incubation times as shown in Fig. 8. The results show that the ability of the hspl2: : URA3 mutant to acquire thermotolerance after prior heat treatment at 37°C was not affected. Furthermore, in the absence of a 37° C heat shock, incubation at 51 ° C resulted in equally rapid loss of viability for both mutant and wild-type cells. This suggests that the product of the H S P I 2 gene is unlikely to be the sole agent responsible for induced thermotolerance.

Discussion

We have demonstrated the presence in yeast of a new small heat shock gene, HSP12. Our results show that

the protein encoded by HSP12 may be one of the most abundant proteins synthesised after heat shock and on entry into stationary phase. The fact that it has escaped detection until now is probably due primarily to its small size, 12 kDa, requiring non-standard conditions for resolution on protein gels. The polypeptide sequence encoded by HSP12 is structurally related to other small hsps, showing a similar hydrophilic profile caused mainly by an excess of charged amino acid side chains (Lindquist and Craig 1988). In addition, a region of the hspl2 polypeptide has limited sequence homology with small hsps from other organisms, notably those from soybean and fruitfly. However, this region does not correspond to the region of greatest conservation between other small hsps. Thus, while suggesting common evolutionary ancestry, this observation seems to point to an early divergence of hspl2. Although it has been noted previously that small hsps are more related by their structures than their primary sequences, the absence of any homology with hsp26 from yeast is somewhat surprising. Although initially isolated as a result of its high level expression on entry into stationary phase, HSP12 was found to be strongly heat-inducible. As expected for a gene with this pattern of expression, the promoter contains two elements which could act as binding sites for the yeast HSF. Other features of the gene, including TATA box, pyrimidine-rich tract, A-rich untranslated leader, translation initiation environment, and very high codon bias index, are typical of highly expressed yeast genes and thus constitute the basis for very efficient expression at both the transcriptional and the translational level. Our results show that the HSP12 transcript is induced several hundred-fold upon heat shock and on entry into stationary phase. In agreement with our prediction of efficient expression, an abundant protein of apparent Mr 14.4 kDa was synthesised under inducing conditions by wild-type HSP12, but not hspl2: : URA3 mutant cells. The absence of this protein from the hspl2: : URA3 mutant, its regulated synthesis paralleling that of HSP12 mRNA synthesis, and its mobility on SDS-polyacrylamide gels, which is close to that predicted from the nucleic acid sequence, strongly suggest that it is indeed the HSP12 gene product. As yet it is not clear what exactly constitutes the trigger for the heat shock response (Pelham 1989). Results from work with hsp70 of Drosophila suggested that the synthesis of hsps was self-regulating, and yeast mutants defective in some of the hsp70 genes constitutively synthesised hsps, thereby supporting a model involving feedback regulation (DiDomenico et al. 1982; Craig and Jacobsen 1984). Alternatively, the heat shock response may be elicited by the presence of denatured or aberrant proteins, as supported by work with Xenopus and by the recent finding that mistranslation induces the heat shock response in yeast (Ananthan et al. 1986; Grant et al. 1989). Our results show that growth conditions which result in detectable levels of HSP12 transcript in wild-type cells even during exponential growth, appear to trigger a mild heat shock response in hspl2: ."URA3 mutants, as characterised by the presence of HSP26

104 mRNA in addition to truncated HSP12 transcript, and by the synthesis of several proteins which in wild-type cells are associated with heat shock or stationary phase. In this situation it seems possible, therefore, that the heat shock response may have been induced by abnormal protein synthesised from the truncated mRNA, rather than as a result of a lack of functional hspl2 protein. This possibility applies equally to the hsp70 mutants of yeast, as these consisted of insertions into the coding regions of the SSAI and SSA2 genes (Craig and Jacobsen 1984). Since expression of these two genes is constitutive, it is possible that in the disruption mutants the heat shock response was triggered by abnormal polypeptide, and not as a result of a lack of functional hsp70 protein. Although the regulation of heat shock gene expression by heat shock factor is fairly well understood, it is not clear how the same genes are switched on during stationary phase. The expression of two other stationary phase-inducible heat shock genes, UBI4 and SSA3, was found to be stimulated by low levels of cAMP, and in the case of UBI4 this was shown to be the result of a lower activity of cAMP-dependent protein kinase (cAPK; Werner-Washburne etal. 1989; Tanaka etal. 1988). Our results which showed high level transcription of HSPI2 during exponential phase in a cyrl-2 mutant containing low cAMP levels, but not in a cyrl-2, bcy-I double mutant whose protein kinase is constitutively active, suggest that HSPI2 gene expression is similarly regulated by cAMP via the activity of cAPK. As has been suggested previously, cAPK could directly regulate positive or negative transcription factors by phosphorylation (Tanaka et al. 1988). Since cAMP levels are low in cultures approaching stationary phase and under starvation conditions (Matsumoto et al. 1985), both conditions known to be associated with the synthesis of hsps (Iida and Yahara 1984), it seems likely that the developmental induction of HSPI2 and other hsp genes during stationary phase is the result of a decline in cAMP levels. Such a model would also account for the observed low level induction of HSP12 expression during exponential growth in minimal medium, if the limited supply of nutrients, resulting in a much reduced growth rate, also leads to lower cAMP levels. The regulation of UBI4 by cAMP, the developmental induction of several Drosophila heat shock genes, and the regulation by serum of a human hsp70 gene, all involve promoter elements separate from those operating during heat shock (Tanaka et al. 1988; Riddihough and Pelham 1986; Wti et al. 1986). Deletion analysis of the HSP12 promoter will show whether induction of this gene during stationary phase also involves separate elements. An immediate candidate for such a study is the repeated hexanucleotide GGAAAA, as this is clearly related to the sequence A G A A G G G A A A A G G conferring serum inducibility on the human HSPTO (Wu et al. 1986) and to the repeated motif GAAAATPu found within the ecdysone-responsive element of the Drosophila HSP27 promoter (Riddihough and Pelham 1986). We have noted that the UBI4 promoter also has several sequence elements related to this motif (Oezkaynak et al.

1987). Furthermore, the similarity with the heptanucleotide A/CA/GGAAAT involved in SUC2 expression is interesting as this gene is also expressed on entry into stationary phase (Sarokin and Carlson 1986). Curiously, the HSP12 hexanucleotide also perfectly matches the consensus sequence AAA/GT/GGA repeated seven times in the regulatory region required for viral activation of the human interferon-fl gene (Fujita et al. 1985). So far no single hsp has been shown to be indispensable for the acquisition of thermotolerance. In order to examine whether HSP12 is involved in the protection against thermal stress, we compared the performance of an HSP12 mutant with that of wild-type cells under a variety of conditions. The mutation appeared to have no effect on viability nor on growth at normal temperature. This is perhaps not surprising since the absence of HSP12 transcript during exponential growth in rich medium suggests that hsp12 protein is not required during this phase. In addition, the hspl2: ."URA3 mutant was indistinguishable from wild type in its performance during growth at various temperatures and it retained the ability to acquire thermotolerance to acute heat stress. Since the same has been found to be true for hsp26 (Petko and Lindquist 1986), it seemed possible that these two proteins perform similar functions during heat stress and can compensate for each other. Preliminary results with an hspl2, hsp26 double mutant suggest that even this is not compromised in its ability to survive acute heat stress. Therefore, if the small hsps are important in protection against heat damage, there must exist yet another protein which can compensate for the lack of hsp12 and hsp26. As discussed above, UBI4 is required for survival of cells at a temperature just above maximal for continued growth, although it was dispensable during acute heat stress. Thus it seems possible that the two small hsps and ubiquitin, which is inducible above a considerable basal level by heat shock, can all compensate for each other during acute heat stress. Hsps are synthesised at very high levels during heat shock, and while this is likely to reflect their importance during this kind of stress, the amounts synthesised may be in excess of cellular requirements, thereby allowing for the apparent dispensability of individual members. Similar studies on a hspl2, hsp26, ubi4 triple mutant may reveal whether other, as yet unidentified, factors are also involved in the acquisition of thermotolerance.

Acknowledgements. We thank Steve Crowe for constructing the hspl2::URA3 disruption mutant, Debbie Seaman for technical assistance, and Sarah Gilbert and Christine Fleming for helpful discussions. We should also like to thank Dr. A. Wheals for providing the cAMP mutant strains. This work was supported by a MAFF contract CSA 938 to P.A.M.

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C o m m u n i c a t e d b y C.P. H o l l e n b e r g

HSP12, a new small heat shock gene of Saccharomyces cerevisiae: analysis of structure, regulation and function.

We have isolated a new small heat shock gene, HSP12, from Saccharomyces cerevisiae. It encodes a polypeptide of predicted Mr 12 kDa, with structural s...
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