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Changes in Oligomerization Are Essential for the Chaperone Activity of a Small Heat Shock Protein in Vivo and in Vitro

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m208926200

1083-351X

Kim C. Giese, Elizabeth Vierling,

Bee Products Chemical Analysis

The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacteriumSynechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystisdepends on HSP16.6, a phenotype that is enhanced in a ΔClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restorein vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance. The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacteriumSynechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystisdepends on HSP16.6, a phenotype that is enhanced in a ΔClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restorein vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance. Molecular chaperones prevent irreversible damage to other proteins during heat stress. Most chaperones act to assist in protein folding, but small heat shock proteins (sHSPs) 1The abbreviations used for: sHSP, small heat shock protein; BSA, bovine serum albumin; SEC, size exclusion chromatography; luc, firefly luciferase; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. 1The abbreviations used for: sHSP, small heat shock protein; BSA, bovine serum albumin; SEC, size exclusion chromatography; luc, firefly luciferase; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. appear to be limited to maintaining the solubility of unfolding proteins, without catalyzing refolding (1Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1743) Google Scholar). The mechanism for this protection is not known, butin vitro studies with model substrates have identified stable, soluble complexes between sHSP oligomers (typically 9–30 or more monomers) and their substrates (for review, see Ref. 2Van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (382) Google Scholar). According to current models, de-oligomerization is an essential step in sHSP function (3Haslbeck M. Walke S. Stromer T. Ehrnsperger M. White H.E. Chen S. Saibil H.R. Buchner J. EMBO J. 1999; 18: 6744-6751Crossref PubMed Scopus (382) Google Scholar, 4van Boekel M.A. de Lange F. de Grip W.J. de Jong W.W. Biochim. Biophys. Acta. 1999; 1434: 114-123Crossref PubMed Scopus (51) Google Scholar, 5van Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (629) Google Scholar). Heat-induced destabilization of the sHSP oligomer may result in a smaller species that initiates the interaction with substrate, followed by re-assembly into a larger sHSP-substrate complex. Although sHSPs do not promote refolding of these model substrates themselves, sHSP-bound proteins have been refolded with ATP-dependent chaperones such as the HSP70 system or GroE (6Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (633) Google Scholar, 7Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (654) Google Scholar). How these biochemical activities relate to the action of sHSPsin vivo remains to be elucidated. The crystal structures of two sHSPs are known. HSP16.5, a spherical, 24-subunit oligomer from Methanococcus jannaschii was crystallized by Kim et al. (8Kim K.K. Kim R. Kim S.H. Nature. 1998; 394: 595-599Crossref PubMed Scopus (788) Google Scholar). Comparison with wheat TaHSP16.9, a dodecameric disk (5van Montfort R.L.M. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (629) Google Scholar), suggests that a dimer will be a common building block of many sHSP oligomers. The ∼100-amino acid α-crystallin domain, which is the region best conserved between sHSPs (9de Jong W.W. Caspers G.J. Leunissen J.A. Int. J. Biol. Macromol. 1998; 22: 151-162Crossref PubMed Scopus (437) Google Scholar), contains the dimer interface. This domain forms a β-sandwich in which a β-strand of each monomer is incorporated into a β-sheet of the other. The α-crystallin domain is flanked by a variable length, nonconserved N terminus and a short, flexible C-terminal arm. Both high resolution structures reveal inter-dimer interactions between hydrophobic residues in the C-terminal arm (β-strand 10) with a hydrophobic patch on the surface of the α-crystallin domain (largely β-strands 4, 5, and 8). Both groups of hydrophobic residues in this interaction are highly conserved in all sHSPs (9de Jong W.W. Caspers G.J. Leunissen J.A. Int. J. Biol. Macromol. 1998; 22: 151-162Crossref PubMed Scopus (437) Google Scholar). This interaction appears to be important for oligomeric stability, but its role in the chaperone activity of sHSPs is unknown. sHSPs enhance stress tolerance in a variety of cell systems (10Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar, 11Soto A. Allona I. Collada C. Guevara M.A. Casado R. Rodriguez-Cerezo E. Aragoncillo C. Gomez L. Plant Physiol. 1999; 120: 521-528Crossref PubMed Scopus (142) Google Scholar), but are often nonessential for thermotolerance (12Petko L. Lindquist S. Cell. 1986; 45: 885-894Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 13Thomas J.G. Baneyx F. J. Bacteriol. 1998; 180: 5165-5172Crossref PubMed Google Scholar). Three organisms have been shown to become heat-sensitive in the absence of an sHSP gene: Neurospora crassa (14Plesofsky-Vig N. Brambl R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5032-5036Crossref PubMed Scopus (67) Google Scholar), Synechocystissp. strain PCC 6803 (15Lee S. Owen H.A. Prochaska D.J. Barnum S.R. Curr. Microbiol. 2000; 40: 283-287Crossref PubMed Scopus (63) Google Scholar) (referred to hereafter asSynechocystis), and recently Escherichia coli(16Kuczynska-Wisnik D. Kcdzierska S. Matuszewska E. Lund P. Taylor A. Lipinska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (85) Google Scholar). In these reports, the loss of viability of the sHSP deletions were mild, on the order of a 10-fold decrease compared with wild type, making these phenotypes difficult to exploit genetically. For this reason we undertook developing a more robust assay for sHSP activityin vivo that would allow selection for sHSP function and enable critical in vivo tests of the chaperone mechanism of sHSP action. Synechocystis has many advantages for molecular studies. In addition to having a fully sequenced genome (17Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2119) Google Scholar), it is easily transformed, and homologous recombination into the chromosome allows deletion and replacement of target genes (18Vermaas W.F. Methods Enzymol. 1998; 297: 293-310Crossref PubMed Scopus (40) Google Scholar). Therefore HSP16.6, the only sHSP in Synechocystis, can be deleted and replaced by mutant variants. In this study we describe a stress condition that demonstrates a strong requirement for functional HSP16.6, and allows the effects of point mutations on sHSP function in vivo to be assayed. Analysis of HSP16.6 in its homologous system may facilitate identification of mutants that disrupt in vivo function because of changes in essential, but as yet unrecognized activities of sHSPs. Synechocystis HSP16.6, which comprises relatively uniform, highly soluble oligomers, is also more readily studiedin vitro than the analogous sHSPs from E. coli, which aggregate on purification (16Kuczynska-Wisnik D. Kcdzierska S. Matuszewska E. Lund P. Taylor A. Lipinska B. Laskowska E. Microbiology. 2002; 148: 1757-1765Crossref PubMed Scopus (85) Google Scholar). Thus Synechocystispresents the opportunity to correlate in vivo and in vitro activities of an sHSP. We show here that mutations in HSP16.6 at Leu-66, a conserved residue in the hydrophobic patch on the α-crystallin domain, cause severe thermotolerance defects in Synechocystis. One of these mutant proteins, L66A, is also greatly impaired in both oligomerization and chaperone activity in vitro. In a novel selection for sHSP function, we randomly mutated hsp16.6 L66A and selected for intragenic suppressors that restore sHSP activity in vivo. This selection led to the identification of mutations that over-stabilize the HSP16.6 oligomer, and restore activity to the L66A mutant both in vivo and in vitro. The rate at which an sHSP-protected substrate is refolded by reticulocyte lysate is affected both by mutants with reduced oligomeric stability, which increase the rate, and strongly oligomerized mutants which slow it. This suggests a requirement for sHSP disassembly prior to substrate release. In total, these data demonstrate a correlation between sHSP function in vivo and chaperone activity in vitro, and support the hypothesis that dynamic changes in oligomerization are essential to both. pNaive (pAZ722) is a pUC118-based plasmid derived from pHK-2R, 2H. Kosaka and H. Fukuzawa, unpublished data. for integration at the hsp16.6 locus (open reading frame sll1514 (Ref. 17Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2119) Google Scholar)) via flanking sequence (500 bp each) from both ends of thehsp16.6 gene. Using unique restriction sites (HpaI, found in the hsp16.6 promoter just upstream of the start codon, and an engineered ApaI site after the stop codon), hsp16.6 was cloned into pNaive to make pNaive.16 (pAZ768). The spectinomycin resistance gene,aadA, is 150 bp downstream of the hsp16.6 stop codon. The pBluescript (Stratagene)-based plasmids pClpB1-KO (pAZ804) and pClpB2-KO (pAZ805) are deletion constructs for clpB1 andclpB2, Synechocystis genes slr1641 andslr0156, respectively (17Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2119) Google Scholar). Each contains 500 bp of upstream and downstream flanking sequences from either clpB gene (generated by PCR on wild-type genomic DNA), separated by an erythromycin resistance gene from pRL425 (19Elhai J. Wolk C.P. Gene (Amst.). 1988; 68: 119-138Crossref PubMed Scopus (374) Google Scholar). pJC20/Hpa (pAZ877) was created from pJC20 (20Clos J. Brandau S. Protein Exp. Purif. 1994; 5: 133-137Crossref PubMed Scopus (148) Google Scholar) by adding anHpaI site to the polylinker. This allowed hsp16.6to be inserted using HpaI and ApaI to make pJC20/Hpa.hsp16 (pAZ730). All strains in this work were created by transforming pNaive into hsp16.6 deletion cells, to ensure that recombination occurs outside of the hsp16.6gene. The isogenic ΔHSP16.6 and +HSP16.6 strains were made by transforming pNaive and pNaive.16 into HK-1, a kanamycin-resistant,hsp16.6 deletion strain, provided by Drs. Kosaka and Fukuzawa of Kyoto University. Transformations were done as described by Williams (21Williams J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (850) Google Scholar), selecting for increasing spectinomycin resistance, at concentrations up to 250 μg/ml spectinomycin dihydrochloride. Initial ClpB deletion strains were made by transforming pClpB1-KO and pClpB2-KO into both +HSP16.6 and ΔHSP16.6, and selected for with up to 300 μg/ml erythromycin. pClpB1-KO was also transformed into HK-1 cells to create ΔClpB1/HK-1, which was used as the parental strain in most experiments. pNaive vectors carrying the appropriatehsp16.6 alleles were transformed to make +HSP16.6/ΔClpB1, ΔHSP16.6/ΔClpB1, and other mutant strains. Experiments were performed with at least two independent transformants for each strain. Cells were maintained in a lit 30 °C incubator on BG-11/agar (22Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) plates, buffered with 10 mm TES, pH 8.2, supplemented with 5 mm glucose, and either 50 μg/ml kanamycin sulfate, 100 μg/ml spectinomycin dihydrochloride, or 100 μg/ml erythromycin sulfate, as appropriate. Liquid media was BG-11, buffered with 5 mm HEPES, pH 7.8, supplemented with 5 mm glucose, and did not contain antibiotics. Suspension cultures were grown on a rotating wheel at 30 °C, resulting in doubling times of ∼8 h, and maximum cell densities of OD730 ∼2.5. Care was taken to ensure cells were in early log phase prior to stress treatments. Changes at thehsp16.6 and clpB1 loci did not affect cell growth rates or maximum densities prior to heat stress. Liquid cultures of logarithmically growing cells were diluted to an OD730 of 0.07 20 h before the stress. On the day of the experiment, densities were typically 0.3–0.6 OD730. Cultures were all diluted with fresh media to OD730 = 0.25, and serially diluted 1:10 four times. Spots (5 μl) were applied to 20.0 (± 0.2)-ml BG-11/glucose plates, with or without 140 mm MgSO4, as stated in the text. Plates were incubated either at 30 °C, or at 44 °C for up to 8 h in the dark in a Thelco Hi Performance incubator (Precision). Colonies typically appeared within 6 days. Survival was determined by comparing the number of colonies on heat-treated plates with unheated, BG-11/glucose-only plates. The hsp16.6 Leu-66 mutants were created with PCR using pJC20/Hpa.hsp16 as a template, and 5′-phosphorylated oligonucleotides designed to randomly mutate the Leu-66 codon. A pair of oligos was designed so that each annealed to opposite strands, and their 5′ ends annealed to adjacent nucleotides. PCR was performed with Pfu Turbo (Stratagene), and resulted in a linearized plasmid that could be circularized by ligating its blunt ends. These plasmids were amplified in E. coli. hsp16.6 was sequenced before being subcloned into pNaive. These plasmids were transformed into the HK-1/ΔClpB1 strain. This same procedure was used for all site-directed mutagenesis. Mutagenesis of hsp16.6 L66Awas done using error-prone PCR with Taq polymerase (Roche) in the presence of MnCl2, as described by Leung et al. (23Leung D.W. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar). pNaive.16.L66A (pAZ697) was used as a template. The oligos anneal on either side of the hsp16.6 gene, amplifying the entire gene. Buffer conditions were as directed by Roche forTaq polymerase, except that there was 0.1 mmMnCl2, 4.9 mm MgCl2, and 80 μm dNTPs. 30 cycles of amplification were performed. Under these conditions, we estimated an average of ∼1.5 base pair changes/gene, and found a range from 0 to 6. Resulting PCR fragments were digested with HpaI and ApaI and cloned into pNaive as described above. Pools of plasmids were amplified in E. coli before transforming into Synechocystis. Liquid cultures of logarithmically growing cells were incubated in a 42 °C water bath for 2 h, and then pelleted at 4 °C before being resuspended in SDS sample buffer. The protein concentration of the cell lysates was measured with Coomassie Blue binding (24Ghosh S. Gepstein S. Heikkila J.J. Dumbroff E.B. Anal. Biochem. 1988; 169: 227-233Crossref PubMed Scopus (159) Google Scholar). 0.5 μg of protein/lane was loaded on 15% SDS-PAGE gels. Western blot analysis was performed with anti-HSP16.6 rabbit antiserum, created against purified recombinant HSP16.6. 3G. J. Lee and E. Vierling, unpublished data. Pools of plasmids containing randomly mutagenized hsp16.6 L66A were transformed into HK-1/ΔClpB1. ∼3000 mutagenized genes from 10 independent PCR reactions were transformed as described above, except that cells were replica-plated to 250 μg/ml spectinomycin plates, and then 7 days later to drug-free plates. Four days later they were again replica-plated to 20 ml, 140 mm MgSO4BG-11/glucose plates, and heated at 44 °C for 8 h. Plates were moved to 30 °C, and allowed to grow for 8–10 days. By this time, large patches of cells were observed from surviving colonies. Thehsp16.6 genes were amplified out of potential suppressor strains and sequenced. To ensure that the observed phenotype washsp16.6-dependent, the genes were then re-transformed into Synechocystis, and cells were re-assayed for their heat stress sensitivity. HSP16.6 and its mutant versions were purified as previously described (25Lee G.J. Vierling E. Methods Enzymol. 1998; 290: 350-365Crossref PubMed Scopus (34) Google Scholar). Proteins were expressed from pJC20/Hpa plasmids in the E. coli strain BL21 (Stratagene). Unlike the wild-type HSP16.6, L66A and L66A/D80V were in the insoluble fraction of the lysate and were resolubilized with 6 murea. When the urea was dialyzed away, the sHSPs remained soluble. Similar treatment of wild-type protein had no effect on its activity or oligomerization. L66A and L66A/D80V were insoluble in low concentrations of ammonium sulfate; therefore, this step of the purification was omitted for them. The 0.2–0.85 m sucrose gradient, and the ion exchange on DEAE in 3 m urea were the same for all samples. Proteins were stored in 20 mmNaPO4, 20 mm NaCl, pH 7.3, 1 mmdithiothreitol. Protein concentration of HSP16.6 was determined using an extinction coefficient of ε280 = 5960 m−1cm−1, based on the aromatic amino acid content, as described by Pace et al. (26Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3436) Google Scholar). Mutant proteins were assayed by Bradford assay (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar), using HSP16.6 as a standard. Proteins were run on a Bio-Sil SEC 400 (Bio-Rad), equilibrated with 20 mmNaPO4, 20 mm NaCl, pH 7.3, at a flow-rate of 1 ml/min. Unless otherwise stated, both buffer and column were at room temperature. For high temperature experiments, both column and buffer were heated to 38 °C, and samples were incubated at appropriate temperature for at least 20 min before being injected onto column. Proteins were diluted into the same buffer and, when appropriate, heated at 42 °C for 7.5 min before centrifuging at 16,000 ×g for 15 min at 4 °C. 100 μl of sample were injected onto the column. Protection of firefly luciferase (luc) from thermal aggregation by sHSPs was assayed basically as described in Lee and Vierling (25Lee G.J. Vierling E. Methods Enzymol. 1998; 290: 350-365Crossref PubMed Scopus (34) Google Scholar). Heating reactions were prepared in 25 mm HEPES, pH 7.5, 15 mm KCl, 5 mm MgCl2, 2 mm dithiothreitol (D buffer) to a final volume of 50 μl. Reactions had 24 or 96 μm sHSP and 1 μm luc. Samples were heated at 42 °C for 7.5 min, cooled on ice, and centrifuged at 16,000 × g for 20 min at 4 °C. Equal volumes of remaining soluble protein were run on a 14% SDS-PAGE and compared with unheated luc. The ability of sHSPs to maintain luc in a re-foldable state was assayed in D buffer. Samples were heated at 42 °C, 7.5 min, then cooled on ice. Reactions were diluted into refolding buffer (60% rabbit reticulocyte lysate (Green Hectares, Oregon, WI) in D buffer with 2 mm ATP added). All samples were diluted to a final concentration of 30 nm sHSP in the refolding step, independent of the concentration during heat inactivation. To achieve this, samples were first diluted to 1.2 μm sHSP in D buffer with 6% reticulocyte lysate to protect the luc activity. In the absence of ATP, this mixture does not promote refolding. In the refolding reaction, heated samples were incubated at 31 °C for up to 2 h. Luciferase activity, relative to activity before heating, was determined by adding 2.5 μl of reaction to 50 μl of luc assay system (Promega) and measuring in a luminometer. We sought conditions that require functional HSP16.6 for survival in a simple plating assay. A variety of stress conditions were tested, and a combination of MgSO4 and 44 °C heat stress was determined to best demonstrate sHSP-dependent survival. Fig.1 A shows the isogenic strains +HSP16.6, a wild-type HSP16.6-expressing strain, and ΔHSP16.6, anhsp16.6 deletion strain, plated onto standard agar plates or plates supplemented with 140 mm MgSO4. In the absence of heat stress, there is no loss of viability by either strain on MgSO4. When heated for 8 h at 44 °C on MgSO4, less than 0.1% of ΔHSP16.6 survive compared with greater than 10% of +HSP16.6. Thus, the deletion of the sHSP causes more than 100-fold loss of viability. The ClpB/HSP100 proteins are a family of chaperones that have the ability to resolubilize aggregated protein (28Parsell D.A. Kowal A.S. Singer M.A. Lindquist S. Nature. 1994; 372: 475-478Crossref PubMed Scopus (736) Google Scholar, 29Glover J.R. Lindquist S. Cell. 1998; 94: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, 30Zolkiewski M. J. Biol. Chem. 1999; 274: 28083-28086Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The loss of sHSP function, which might lead to increased protein aggregation, could be compensated for by the action of ClpB. A search of the Synechocystisdata base, CyanoBase (www.kazusa.or.jp/cyano), identified twoclpB genes (slr1641 and slr0156) that we have named clpB1 and clpB2, respectively, based on the similarity of the former to the heat-inducedclpB1 in Synechococcus sp. strain PCC 7942 (31Eriksson M.J. Clarke A.K. J. Bacteriol. 1996; 178: 4839-4846Crossref PubMed Google Scholar).clpB1 deletions were readily obtained in both +HSP16.6 and ΔHSP16.6 backgrounds with no effect on cell growth at 30 °C. Parallel attempts to delete clpB2 were unsuccessful in both strains, suggesting that, as was found in Synechococcus(32Eriksson M.J. Schelin J. Miskiewicz E. Clarke A.K. J. Bacteriol. 2001; 183: 7392-7396Crossref PubMed Scopus (18) Google Scholar), this gene is essential under standard growth conditions. As shown in Fig. 1, there are not significant differences in the survival of the +HSP16.6 and +HSP16.6/ΔClpB1 strains after heat shock. However, in the ΔClpB1 background, the hsp16.6deletion, ΔHSP16.6/ΔClpB1, is >10,000-fold less viable than +HSP16.6/ΔClpB1. Our data are suggestive, but do not prove, that there is a genetic interaction between these proteins. Nevertheless, because of the strong dependence of Synechocystisthermotolerance on HSP16.6 in the absence of ClpB1, all selections and subsequent analyses were performed in ΔClpB1 cells. As described in the Introduction, several conserved hydrophobic amino acids form a patch on the surface of sHSPs that may be an important oligomerization site. We wished to test the importance of sHSP oligomerization on in vivo function by mutating one of these conserved residues in HSP16.6, and examining the effect on thermotolerance inSynechocystis. Leu-66, on β-strand 4, was chosen because mutagenesis of a homologous residue, Val-76 in Pisum sativumHSP18.1, was found to destabilize the sHSP oligomer in vitro. 4D. S. Kim and E. Vierling, unpublished data. Transformation of Leu-66 mutant alleles into a ΔHSP16.6/ΔClpB1 background (described under “Experimental Procedures”) results in expression of these mutants by the endogenous hsp16.6promoter in the absence of wild-type HSP16.6. As shown in Fig.2 A, mutations of Leu-66 have varied effects. L66T has little effect on thermotolerance, whereas L66E and L66K are so deleterious that cells expressing these mutants are less viable than the deletion strain. Even cells carrying the conservative mutation L66A are nearly as defective as ΔHSP16.6/ΔClpB1, demonstrating that small changes at Leu-66 can greatly impair HSP16.6 function in vivo. The accumulation of HSP16.6 was measured by Western blot after a nonlethal incubation at 42 °C (Fig. 2 B). The levels of L66A, L66E, and L66K mutant proteins are greatly reduced relative to wild-type HSP16.6, suggesting either that they are unstable or that they are degraded because their presence is deleterious to the cell. Even L66T-expressing cells, which are wild type in survival, do not accumulate wild-type levels of sHSP, indicating that cells with reduced levels of sHSP can remain thermotolerant. Intragenic suppressor analysis was undertaken to identify regions of HSP16.6 that share their function with Leu-66. Selection for sHSP function was attempted with multiple cycles of heat shock and recovery. However, it became apparent that cells can become resistant to heat stress, even in the absence of an sHSP. After as few as two rounds of heat stress, a population of hsp16.6 deletion cells became nearly as resistant to heat stress as +HSP16.6 and stayed resistant for many generations without further selection. Resistance also occurs in the ΔHSP16.6/ΔClpB1 strain. Based on the high frequency at which this occurs, it appears that sHSP-independent thermotolerance can be achieved by many different mechanisms, but this has not been pursued. As a result of this observation, only a single round of heat shock has been used to select for sHSP function. The severe reduction of thermotolerance of cells carrying hsp16.6 L66K made this mutation appear to be an excellent tool to isolate suppressors that would restore sHSP function in vivo. However, multiple attempts to identify suppressors of L66K failed, suggesting that it may be too severe to suppress in the manner tried. In contrast, suppressors of the weaker mutant, L66A, were readily obtained. Intragenic suppressors were generated by random mutagenesis ofhsp16.6 L66A by error-prone PCR and transformed into aSynechocystis Δhsp16.6/ΔclpB1 strain. Thehsp16.6 genes of colonies that survived 44 °C 8 h were recovered and sequenced. Mutant genes were re-transformed intoSynechocystis to verify that thermotolerance was sHSP-dependent. Eight suppressors were isolated (TableI), representing single amino acid changes at five residues, and one double mutant (P8L/K137E) out of ∼3000 colonies screened. Three changes at Asp-80 (to Val, His, or Asn) all suppress the L66A defect. L66A/N40Y has been independently isolated three times, suggesting that this selection is approaching saturation. The back mutation, Ala-66 to Leu, was not recovered, but this mutation is unlikely as it would require two base changes (GCG to either CTG or TTG). Ala-66 to Thr (ACG), which can substitute for Leu-66 (Fig. 2 A), was recovered.Table ISuppressors of L66ASuppressor mutant(s)Independent isolates (no.)Increase in hydrophobicityaAs calculated by Radzicka and Wolfenden (38) for side-chain analogs, in kcal/mol. The hydrophobicity of proline was not determined.Thermotolerance of L66A with suppressorbScale is “+++” = wild-type thermotolerance, “−” = L66A.Thermotolerance of suppressor alonebScale is “+++” = wild-type thermotolerance, “−” = L66A.N40Y36.5++++++T76I17.5+++++D80V112.8+++++D80H14.1++NDcND, not determined.D80N12.1++NDV108L10.9++++++V133A2−2.2++++P8L/K137E1ND+++++P8LND++++K137E−1.3++++a As calculated by Radzicka and Wolfenden (38Radzicka A. Wolfenden R. Biochemistry. 1988; 27: 1664-1670Crossref Scopus (550) Google Scholar) for side-chain analogs, in kcal/mol. The hydrophobicity of proline was not determined.b Scale is “+++” = wild-type thermotolerance, “−” = L66A.c ND, not determined. Open table in a new tab The ability of the suppressors to restore thermotolerance is shown in Fig. 3 A. Some suppressors, such as N40Y and V108L, are strong enough to rescue L66A to nearly wild-type levels of survival, whereas L66A/V133A is just 10-fold better than L66A alone. Suppression by P8L and K137E individually has also been tested. Each mutation can at least slightly suppress L66A, although neither does as well as P8L/K137E. Some of the suppressor mutations improve HSP16.6 accumulation. Fig.3 B shows HSP16.6 levels in cells expressing the suppressor mutants relative to wild-type and L66A-expressing strains. None of the suppressors is able to ful