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The Small Heat-shock Protein IbpB from Escherichia coli Stabilizes Stress-denatured Proteins for Subsequent Refolding by a Multichaperone Network

1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês

10.1074/jbc.273.18.11032

1083-351X

Lea Veinger, Sophia Diamant, Johannes Büchner, Pierre Goloubinoff,

Enzyme Structure and Function

The role of small heat-shock proteins inEscherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins. The role of small heat-shock proteins inEscherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins. The small heat-shock proteins (sHSPs) 1The abbreviations used are: sHsp, small heat-shock protein(s); IbpB, inclusion body associated protein B; MDH, malate dehydrogenase; LDH, lactate dehydrogenase; S, GroES; L, GroEL; K, DnaK; J, DnaJ; E, GrpE. belong to a ubiquitous family of low molecular mass (15–30 kDa), stress-induced proteins in prokaryotes and eukaryotes. Whereas various sHSPs share weak sequence homologies (1Plesofsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar, 2Ehrnsperger M. Gaestel M. Buchner J. Fink A.L. Goto Y. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, New York1998: 533-575Google Scholar), many sHSPs appear to be functionally and structurally related. Many sHSPs assemble into large globular complexes, whose oligomeric structures may vary depending on the degree of subunit phosphorylation or the concentration of ions (3Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Ehrnsperger M. Gräber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar, 5Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar). The overexpression of sHSPs in plant, yeast, and in mammal cells correlates with increased levels of thermal resistance (6Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 147: 93-101Google Scholar, 7Knauf U. Bielka H. Gaestel M. FEBS Lett. 1992; 309: 297-302Crossref PubMed Scopus (75) Google Scholar, 8Schrimer E.C. Lindquist S. Vierling E. Plant Cell. 1994; 6: 1899-1909PubMed Google Scholar, 9van den Ijssel P. Overcamp P. Knauf U. Gaestel M. de Jong W.W. FEBS Lett. 1994; 355: 54-56Crossref PubMed Scopus (104) Google Scholar). In vitro, sHSPs specifically recognize, bind, and prevent the aggregation of non-native proteins during stress (3Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Ehrnsperger M. Gräber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar, 5Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar, 10Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar), suggesting that similarly to GroEL/GroES (LS) and Hsp70 (11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar, 12Freeman B.C. Toft D.O. Morimoto R.I. Science. 1996; 274: 1718-1720Crossref PubMed Scopus (289) Google Scholar), sHSPs can serve as an efficient binding reservoir for unstable protein-folding intermediates during stress. However, at variance with other molecular chaperones, such as Hsp70, Hsp60, Hsp104, and Hsp90 (for a review, see Ref. 13Buchner J. FASEB J. 1996; 10: 10-19Crossref PubMed Scopus (382) Google Scholar), small HSPs do not hydrolyze ATP and do not display a specific ability to promote the correct refolding of the bound stabilized proteins (3Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Ehrnsperger M. Gräber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar, 5Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar). The small heat shock proteins IbpA and IbpB from Escherichia coli are two sequence-related 14- and 16-kDa proteins, respectively, co-transcribed during stress by the bacterial heat-shock transcription factor ς32 (14Allen S.P. Polazzim J.O. Giersem J.K. Easton A.M. J. Bacteriol. 1992; 174: 6938-6947Crossref PubMed Google Scholar). IbpA and IbpB share a low sequence homology in their C-terminal region with sHSPs from yeast, plants, and mammals, including αB-crystallines (14Allen S.P. Polazzim J.O. Giersem J.K. Easton A.M. J. Bacteriol. 1992; 174: 6938-6947Crossref PubMed Google Scholar, 15Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (677) Google Scholar, 16Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar, 17Merck K.B. Groenen P.J Voorter C.E. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W.W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 18Boston R.S. Viitanen P.V. Vierling E. Plant Mol. Biol. 1996; 32: 191-222Crossref PubMed Scopus (510) Google Scholar, 19Waters E.R. Lee G.J. Vierling E. J. Exp. Bot. 1996; 47: 325-338Crossref Scopus (578) Google Scholar, 2Ehrnsperger M. Gaestel M. Buchner J. Fink A.L. Goto Y. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, New York1998: 533-575Google Scholar). Furthermore, they seem to be distantly related to other bacterial chaperones such as PapD and Caf1M. Based on the resolved x-ray structure of the PapD chaperone and on sequence homology, a model has been proposed where the three-dimensional structure of IbpB resembles that of immunoglobulins (20Zav'yalov V.P. Zav'yalova G.A. Denesyuk A.I. Gaestel M. Korpela T. FEMS Immunol. Med. Microbiol. 1995; 11: 265-272Crossref PubMed Google Scholar). In E. coli, IbpA and IbpB are found associated with endogenous proteins that aggregate intracellularly during heat-shock (21Laskowska E. Wawrzynow A. Taylor A. Biochimie. 1996; 78: 117-122Crossref PubMed Scopus (117) Google Scholar) and with non-native recombinant proteins in inclusion bodies (14Allen S.P. Polazzim J.O. Giersem J.K. Easton A.M. J. Bacteriol. 1992; 174: 6938-6947Crossref PubMed Google Scholar). They are implicated in the solubilization of protein aggregates after stress (21Laskowska E. Wawrzynow A. Taylor A. Biochimie. 1996; 78: 117-122Crossref PubMed Scopus (117) Google Scholar). In vitro, IbpB is a large globular complex, whose oligomeric state may vary at different temperatures and ionic concentrations. 2A. Azem, J. Buchner, and P. Goloubinoff, unpublished data. Similarly to other small HSPs, light-scattering measurements indicate that IbpB can specifically bind thermally or chemically denatured proteins such as MDH, LDH, and citrate synthase and thus prevent their irreversible aggregation (not shown). We show here that IbpB can bind and stabilize denatured proteins, and furthermore, deliver them to the DnaK/DnaJ/GrpE (KJE) chaperone system for subsequent active ATP-dependent refolding. KJE-mediated refolding of IbpB-stabilized proteins can be further activated by LS chaperonins, demonstrating cooperation between the different chaperone systems. IbpB can therefore function as a primordial protein-binding element of a multichaperone network, involved in stabilizing and active refolding of stress-denatured proteins. Wild type and E. coli B mutant T850 (22Takano T. Kakefuda T. Nat. New Biol. 1972; 239: 34-37Crossref PubMed Scopus (80) Google Scholar) were used for in vivocomplementation assays of T4 morphogenesis as described in Ref. 23Zeilstra-Ryalls J. Fayet O. Baird L. Georgopoulos C. J. Bacteriol. 1993; 175: 1134-1143Crossref PubMed Google Scholar. Plasmids pBF3-IbpA and pBF3-IbpB were from Dr. C. Hergersberg, Boehringer Mannheim GmbH, and pSE420 was from Invitrogen Inc. IbpB was purified as follows: late log phaseE. coli cells, containing plasmid pBF3-IbpB in LB medium and chloramphenicol (50 μg/ml), were incubated for an additional 12 h at 37 °C in the presence of isopropyl-1-thio-β-d-galactopyranoside (100 μg/ml), collected, and resuspended in 50 mm Tris-HCl, pH 7.5, 150 mm KCl, 20 mm MgAc2 (Buffer A), containing 5 μg/ml leupeptin. Cells were disrupted five times at 25 °C in a French Press at 900 p.s.i. Soluble proteins in the 30,000 × g supernatant were incubated with 6% polyethylene glycol 6000 (Merck) for 30 min at 4 °C. The protein pellet from 30,000 × g was resuspended in buffer A, filtered through a 0.45-μm filter (Schleicher & Schuell), and separated by gel filtration (0.5 ml/min) on a semi-preparative Superose 6B column (Pharmacia) in buffer A. The fractions collected between 6.0 and 7.5 ml (above 2.106 daltons), which were highly enriched with soluble oligomeric IbpB, were applied to a resource-Q column (Pharmacia), equilibrated with buffer A. Elution was carried out with a linear gradient from 150 to 500 mm KCl in Buffer A. The fractions eluting at 230 ± 30 mm were collected and frozen as 34–45 μm (protomer) 95% pure IbpB stock solutions. GroEL14 and GroES7 were purified as described (24Török S. Horvath I. Goloubinoff P. Kovacs E. Glatz A. Balogh G. Vigh L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2192-2197Crossref PubMed Scopus (192) Google Scholar). Pig heart mitochondrial MDH and hog muscle LDH were from Boehringer Mannheim; hexokinase and pyruvate kinase from Sigma. Plasmid-encoded DnaK, DnaJ, and GrpE were overexpressed in E. coli and purified as described (25Schönfeld H.-J. Schmidt D. Schröder H. Bukau B. J. Biol. Chem. 1995; 270: 2183-2189Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 26Schönfeld H.-J. Schmidt D. Zulauf M. Colloid Polym. Sci. 1995; 99: 7-10Crossref Google Scholar). Protein concentrations were determined by the Bradford protein assay (Bio-Rad). In this study, all chaperone concentrations were expressed in terms of the individual protomers. Native MDH was labeled with NaB(3H)4 as described in Ref. 11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar. After purification by gel filtration (Superose 6B column, Pharmacia),3H-labeled MDH was found to be as active as unlabeled MDH. During heat-denaturation and chaperone-assisted refolding,3H-labeled MDH behaved indistinguishably from unlabeled MDH (not shown). MDH was heat-denatured for 30 min at 47 °C as in Ref. 11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar in the presence of various amounts of IbpB, DnaK, DnaJ, and/or GroEL, GroES as specified and subsequently refolded at 25 °C in the presence of supplemented DnaK/DnaJ/GrpE and/or GroEL/GroES (4, 4, 1, 4, and 6 μm, respectively) in 50 mm triethanolamine, pH 7.5, 20 mmMgAc2, 150 mm KCl, 5 mmdithiothreitol, 3 mm phosphoenol pyruvate, 7 μg/ml pyruvate kinase (Sigma), and 2 mm ATP (buffered with KOH), as indicated. The activity of MDH was measured as described in Ref. 27Diamant S. Azem A. Weiss C. Goloubinoff P. J. Biol. Chem. 1995; 270: 28387-28391Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, at 25 °C in 150 mm potassium phosphate buffer, pH 7.5, 10 mm dithiothreitol, 0.5 mm oxaloacetate, and 0.28 mm NADH (Sigma). The activity of LDH was measured as described in Ref. 28Badcoe I.G. Smith C.J. Wood S. Halsall D.J. Holbrook J.J. Lund P. Clarke A.R. Biochemistry. 1991; 30: 9195-9200Crossref PubMed Scopus (139) Google Scholar in 100 mm Tris, pH 7.5, 0.5 mm pyruvate, and 0.28 mm NADH. The time-dependent oxidation of NADH by MDH or LDH was monitored at 340 nm. Native MDH dimers remain stable and active in solutions above a concentration of 6 nm(protomers) (29Jaenicke R. Rudolph R. Heider I. Biochemistry. 1979; 18: 1217-1223Crossref PubMed Scopus (62) Google Scholar). The apparent rates of protein refolding were calculated from the time-dependent changes in the enzymatic activity of chaperonin solutions containing more than 20 nmenzyme. 3H-Labeled MDH (0.25 μm) was heat-denatured in the presence of IbpB (4 μm), DnaK + DnaJ (4, 0.8 μm) or GroEL (4 μm), and incubated for 1 h at 25 °C with ATP and ATP-regeneration systems, in the absence or presence of KJ, KJEL, or L (Fig. 5). A Superose 6B gel filtration column (Pharmacia) (at 0.5 ml min−1), run in the presence of the refolding buffer and 1 mm ATP was used for separation. 3H-Labeled samples were collected at the indicated elution volumes (Fig. 5), mixed with a 6-fold volume of LumaxTM, and counted. Wild type E. coli B cells are highly sensitive to T4 phages (23Zeilstra-Ryalls J. Fayet O. Baird L. Georgopoulos C. J. Bacteriol. 1993; 175: 1134-1143Crossref PubMed Google Scholar). In contrast, the mutant strain T850, which contains a chromosomal groEL with a R268C mutation, 3C. Weiss, S. Gross, and P. Goloubinoff, personal communication. do not support T4 growth (Fig. 1). We found that phage growth was specifically restored in T850 cells containing plasmid-encoded ibpA or ibpB genes (Fig. 1), however, only in the presence of a higher phage titer (108particles/ml) than in wild type cells. When exposed to 47 °C, the thermolabile enzyme MDH is inactivated at an apparent rate of 0.13 min−1 (11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar) and forms large insoluble protein aggregates that do not refold spontaneously after the heat shock (not shown). In contrast, when exposed to heat stress in the presence of a 4-fold molar excess of thermostable IbpB, MDH was inactivated at a slower rate (0.10 min−1) and formed a stable soluble IbpB·MDH complex.2 We found that the IbpB·MDH did not significantly reactivate, even after a 5-h incubation at room temperature (Fig. 2 A). However, when IbpB·MDH was supplemented with KJE chaperones and ATP, a significant amount (10%) of native MDH was slowly recovered, at a slow rate of ∼0.7 nm min−1. This KJE-mediated refolding of IbpB·MDH was a specific reaction, as it did not occur when IbpB was absent during the heat denaturation (Fig. 2,A and B), or when ATP or GrpE were absent during renaturation (not shown). When IbpB·MDH was supplemented with LS chaperonins and ATP, only insignificant amounts of MDH were reactivated (1.5%), in addition to the small amounts generated by spontaneous refolding (2.5% within 4–5 h, Fig. 2 A). Remarkably, when KJE and LS chaperones and ATP were concomitantly added to IbpB·MDH after the heat shock, a major fraction (40%) of the MDH was actively recovered at an apparent rate 5.5 times higher (3.7 nm min−1), than without LS (Fig. 2 A). Interestingly, a significant amount (10%) of MDH that was heat-inactivated without IbpB was also recovered at a slow rate of 0.8 nm min−1, but only in the presence of the two chaperone systems KJE and LS (Fig. 2 B). The strong dependence of the refolding rates and yields of MDH, on the presence of IbpB during denaturation, could be used as a first functional assay for a small heat-shock protein under stringent conditions. The yields of the reaction depended on the concentration of IbpB present during the heat-denaturation of MDH (Fig. 3 A). When denatured in the presence of a 20-fold molar excess of IbpB, up to 83% of the denatured MDH was recovered by KJE + LS after the stress. Half of the MDH molecules were recovered when denatured in the presence of a 5.7 molar excess of IbpB (Fig. 3 A). The refolding rate of IbpB·MDH was half-maximal when the LS concentration (fixed 1:1 ratio between L and S) was one-fifth that of DnaK (4, 4, and 0.5 μm of K, J, and E, respectively), or when the KJE concentration (with a fixed molar ratio of 4:4:0.5) was half that of the LS (4:4 μmeach) (Fig. 3 B). This suggests that at near equimolar DnaK and GroEL concentrations, as in the cell (30Neidhardt F.C. VanBogelen R.A. Neidhardt F.C. Escherichia coli and Salmonella typhymorium. 2. American Society for Microbiology, Washington, D. C.1987: 1334-1345Google Scholar), the KJE chaperones are more rate-limiting for this reaction than the LS chaperonins. The apparent refolding rate of heat-preformed KJE·MDH complexes was 8.6 times slower than the refolding rate of heat-preformed LS·MDH complexes (Table I). This difference was exploited to address, by kinetic measurements, the order of events during multichaperone IbpB/KJE/LS-mediated refolding reactions. The refolding rates of heat-preformed IbpB·MDH complexes (Fig. 4 A) was compared with heat-preformed KJ·MDH and GroEL·MDH complexes (Fig. 4 B), in the presence of various co-chaperones added either immediately or 53 min after the heat shock.Table IApparent rates of MDH refolding after heat denaturationChaperone present during heat shockChaperone and ATP added after heat shockApparent rate of refolding t = 0t = 53 minnm min −1IbpBATP + KJENo0.64IbpBATP + KJE0.67IbpBNoATP + KJE0.28IbpBATP + LSNo0.09IbpBATP + KJELSNo1.96IbpBATP + KJELS4.25IbpBNoATP + KJELS1.15IbpBATP + KJELS2.99KJATP + ENo1.17KJATP + IbpB + ENo0.40KJATP + ELSNo7.20KJATP + IbpB + ELSNo7.64LATP + SNo7.33 Open table in a new tab Significant refolding of IbpB·MDH was observed only when supplemented after the heat shock with the three chaperones KJE and ATP, however, at a rate which was about half (Fig. 4 A; Table I, 0.64 nm min−1) that of GrpE-mediated refolding of KJ-MDH (Fig. 4 A; Table I, 1.17 nmmin−1). When the addition of ATP and KJE or KJELS was delayed for 53 min after the heat shock, the refolding rate of IbpB·MDH was significantly lower (Table I, 0.28 and 1.15 nm min−1, respectively), than without a delay (0.64 and 1.96 nm min−1, respectively). This suggests that the IbpB·MDH is a lose complex from which aggregation-prone MDH can dissociate. While LS and ATP alone did not directly assist the refolding of IbpB·MDH (Fig. 2 A; Table I, 0.09 nmmin−1), the presence of LS chaperonins tripled the rate (and yields, not shown) of KJE-mediated refolding of IbpB·MDH (Fig. 4 A, 1.96 nm min−1). Noticeably, the extent of LS activation of the KJE-mediated refolding of IbpB·MDH was yet 4 times slower than that of E + LS-mediated refolding of KJ-MDH (Fig. 4 B; Table I, 7.2 nmmin−1). Remarkably, delaying the addition of E + LS to IbpB·MDH which was incubated 53 min with ATP and KJ, increased the rate of refolding from 1.96 to 2.99 nm min−1 (Fig. 4 A; Table I). Likewise, delaying the addition of GroES to IbpB·MDH which was incubated 53 min with ATP and KJEL, more than doubled the refolding rates from 1.96 to 4.25 nm min−1 (Fig. 4 A; Table I). Thus, the rate-limiting dissociation of MDH from IbpB can be overcome by a delay during which it is allowed to transfer on KJ and then on GroEL. Whereas GrpE-mediated refolding of KJ-MDH was inhibited 3-fold in the presence of added free (equimolar) IbpB (Table I, 1.17 → 0.4 nm min−1), LS enhancement of KJE-MDH was not inhibited by added IbpB (Table I, 7.2 → 7.64 nm min−1). This indicates that, at this late stage of refolding, GroEL has a much higher affinity to the protein than KJE and IbpB. The refolding of urea-denatured LDH displayed a similar dependence on the various components of the IbpB/KJE/LS chaperone network. LDH is a thermostable enzyme, which therefore requires first to be denatured in 5 m urea (11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar) before being diluted into IbpB, LS, or KJ solutions and further incubated as MDH at 47 °C. Like MDH, LDH alone did not reactivate from the urea-heat denaturation, and no spontaneous refolding was observed even upon addition of KJE or ATP after the stress (not shown). Nor did urea-heat denatured LDH in the presence of IbpB (IbpB-LDH) refold after the stress in the presence of ATP alone, or with supplemented KJ or LS chaperones alone (not shown). Only in the presence of KJE or KJELS chaperones did IbpB-LDH actively refold in a strict ATP-dependent manner (TableII). KJE-mediated refolding of IbpB-LDH (0.73 nm min−1) was slower than GrpE-mediated refolding of KJ-LDH (0.89 nm min−1), and KJELS-mediated refolding of IbpB-LDH (0.93 nmmin−1) was slower than ELS-mediated refolding of KJ-LDH (1.27 nm min−1), showing that the release of LDH from IbpB was rate-limiting. In contrast to MDH refolding, however, LS chaperonins did not triple, but only activated 1.3-fold, the rate of KJE-mediated refolding of IbpB-LDH (Table II, 0.96 nmmin−1), suggesting that LS chaperonins are not as important for LDH refolding, as for MDH refolding.Table IIApparent rates of LDH refolding after urea-heat denaturationChaperone present during heat shockChaperone and ATP added after heat shock, t = 0Apparent rate of refoldingnm min −1IbpBATP + LS0.01IbpBATP + KJE0.73IbpBATP + KJELS0.96LATP + S0.00KJATP + E0.89KJATP + ELS1.27 Open table in a new tab Gel filtration of chaperone complexes with bound heat-denatured 3H-labeled MDH provided direct evidence for directional MDH transfer from IbpB to KJE, then to LS, during reactivation (Fig. 5). While native complexes of IbpB exceed 2 × 106 daltons2 and consequently eluted near the void volume of the column (6–7.5 ml), individual native complexes of GroEL, KJ, or MDH, independently resolved at 11–13, 13.5–15.5, and 15.5–18 ml, respectively (arrows shown instead of profiles, Fig. 5). The elution profile of [3H]MDH, which had been heat-denatured in the presence of IbpB alone and then incubated 1 h with ATP alone prior to injection, distributed in two peaks; one together with IbpB, and the other in the low molecular weight region corresponding to free MDH (Fig. 5, profile 1). The elution profile of heat-denatured IbpB-bound MDH, incubated 1 h with KJ and ATP but without GrpE, showed 3H label associated to the broad KJ fraction, at the expense of the IbpB containing and the free MDH fractions (Fig. 5, profile 2). Thus, a significant amount of the non-native MDH was transferred after the heat shock from IbpB and from the free state to the KJ chaperones. In contrast, when IbpB-MDH was incubated with ATP, KJ, and in addition with E + L (without S), the majority of the3H label eluted associated to the GroEL fraction, at the expense of the IbpB-, KJ-, and free MDH-containing fractions (Fig. 5, profile 3). Thus, a significant amount of IbpB bound and free MDH, that was initially transferred to KJ, was further transferred to the GroEL. When KJ chaperones were omitted and IbpB-MDH was incubated directly with GroEL and ATP, less 3H label co-eluted with the GroEL fraction, and correspondingly, more MDH eluted with the IbpB-containing and free MDH fractions (Fig. 5, profile 4). When S and ATP were subsequently added, MDH activity was fully recovered from the GroEL fractions from profile 3 (+KJE + L) but not from profile 4 (+L) (data not shown), showing that direct binding and IbpB-released MDH to GroEL is non-productive. When, in a control experiment, [3H]MDH (0.35 μm) was heat-denatured in the presence of an equimolar excess all three types of chaperones together (IbpB, KJ, and L, 10 μm each), gel filtration showed that over 90% of the3H label co-eluted with GroEL (not shown). When GroEL was omitted during the denaturation, most of the 3H label co-eluted with KJ. Only when GroEL and KJ were both omitted during denaturation was a majority (60%) of the 3H label co-eluted with the IbpB (Fig. 5, track 1). This suggests that changes of affinity can define the sequence of transfer of folding intermediates between the various components of a chaperone network. Whereas small Hsps share with other molecular chaperones the ability to recognize, bind, and prevent the aggregation of non-native proteins under stress, the classification of small Hsps as true chaperones remains an open question because of their poor performance at specifically promoting the correct refolding of the sHsps-stabilized proteins. A first indication that sHsps may collaborate with other chaperones in the refolding of bound denatured proteins came from the observation that the refolding of heat-denatured citrate synthase bound to mammalian Hsp25, can be activated by Hsp70, even without ATP (4Ehrnsperger M. Gräber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar). We observed here that similarly to other small Hsps from eukaryotes (3Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Ehrnsperger M. Gräber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar, 5Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar, 10Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 16Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar), IbpB from E. coli can stabilize denatured proteins such as citrate synthase, α-glucosidase, and MDH in a soluble non-aggregated form.2 Remarkably, whereas IbpB alone did not promote the active refolding of bound proteins after the stress, IbpB-bound MDH or IbpB-LDH were specifically reactivated by KJE chaperones and ATP, but not directly by LS chaperonins. Hence, the apparent rate of refolding of IbpB-MDH was 50 times higher in the presence of KJE + LS chaperones and ATP, then without KJE + LS and the yield of the reaction was about one recovered MDH for 10 IbpB subunits present during denaturation. Similarly, ATP- and KJE-assisted refolding of IbpB-LDH was 70–90 times faster than without ATP or KJE. The kinetic and gel filtration data strongly suggested that MDH, but not necessarily LDH or glucose-6-phosphate dehydrogenase, was refolded in a sequential, multichaperone reaction. When native MDH was heat denatured in the presence of IbpB alone, an IbpB·MDH complex was formed which was in equilibrium after the heat shock with a free inactive MDH species. The IbpB-released MDH tended to convert into a kinetically trapped inactive species, unless allowed to bind KJE, but not LS chaperones. The slow dissociation of MDH from the IbpB·MDH complex was the rate-limiting step. Delays in the addition of the various chaperone components showed that MDH was first transferred to KJE chaperones, where it accumulated, unless allowed to partially fold and then interact with LS chaperonins. In contrast with the IbpB-released species (MDH1), which was characterized by a high affinity for the KJE chaperones and a low (non-productive) affinity for LS chaperonins, the KJE-released species (MDH2) had a low affinity for IbpB and a high affinity for LS chaperonins. The increased affinity of IbpB-released MDH1 for KJE, and of KJE-released MDH2 for LS provides a preferential pathway for the sequential MDH refolding in the multichaperone network, as follows: IbpB-MDH1 + KJE + LS ⇒ IbpB + KJE-MDH1 + LS ⇒ IbpB + KJE + LS-MDH2 ⇒ IbpB + KJE + LS + MDHnat. Binding to IbpB during the heat shock was also a prerequisite for a strict KJE- and ATP-dependent refolding of urea/heat-denatured LDH. However, KJE-released LDH species proceeded to the native state almost as rapidly without as with LS chaperonins, suggesting that LS was optional. Interestingly, glucose-6-phosphate dehydrogenase displayed a yet different behavior. Fluorescence and light scattering indicated that heat-inactivated glucose-6-phosphate dehydrogenase was neither aggregated nor bound to IbpB or LS. Whereas denatured glucose-6-phosphate dehydrogenase did not refold spontaneously, it was specifically refolded by KJE + ATP, but not by IbpB and/or by LS + ATP (data not shown). Hence, IbpB can serve as a specific high capacity scavenger for aggregation-prone folding intermediates, but it does not necessarily interact with all types of denatured proteins. It should be noted that the sequential IbpB → KJE → LS mediated refolding of MDH was observed only when MDH was heat denatured in the presence of IbpB alone. When saturative amounts of LS and KJE were also present during the denaturation, IbpB displayed the lowest affinity for denatured MDH. This implies that in the cell, various protein intermediates can bind and distribute according to the relative amounts, binding capacities of each of the chaperones present, and according to the specific affinity of the folding intermediates for the chaperones present during the stress. We propose a model that describes the various protein-folding pathways that may exist in the multichaperon network (Fig. 6). Upon denaturation, native proteins form an unstable folding intermediate that can either aggregate irreversibly, refold spontaneously (pathway 1), or distribute between several components of the chaperone network. Whereas acid-denatured barnase refolds spontaneously without chaperones (31Corrales F.J. Fersht A.R. Folding & Design. 1996; 1: 265-273Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), heat-denatured MDH can bind all three components, depending on their relative abundance during denaturation. IbpB-MDH sequentially transfers to KJE, then to LS (pathway 4 → 5 → 6). KJE-MDH and LS-MDH preferentially used pathways 3 → 6 and2, respectively. Whereas, LDH denatured in urea for 30 s is preferentially refolded by LS (pathway 2) (11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar), urea-heat denatured LDH was refolded by IbpB and KJE, but not by LS (pathway 4 → 5). In contrast, the refolding of firefly luciferase denatured by guanidinium chloride and heat first required LS, then KJE (pathway 2 → 6) (32Buchberger A. Schröder H. Hesterkamp T. Bukau B. J. Mol. Biol. 1996; 261: 328-333Crossref PubMed Scopus (135) Google Scholar). In a yet different pathway, heat-denatured glucose-6-phosphate dehydrogenase was refolded by KJE and not by IbpB or LS (pathway 3). Plasmid-encoded ibpA or ibpB partially suppressed a chromosomal groEL mutation leading to phage-growth deficiency. Because LS chaperonins are involved in protein-folding, prevention of protein aggregation during stress, and correct refolding after heat shock (11Goloubinoff P. Diamant S. Weiss C. Azem A FEBS Lett. 1997; 407: 215-219Crossref PubMed Scopus (37) Google Scholar), this suppression is evidence that either IbpA or IbpB can replace, yet with low efficiency, some of the cellular functions carried on by the LS chaperonin. In eukaryotes, Hsp27, Hsp25, and αB-crystallines are found in protein granules of neurodegenerative diseases and after heat shock (33Mydlarski M.B. Schipper H.M. Brain Res. 1993; 627: 113-121Crossref PubMed Scopus (28) Google Scholar, 34Schipper H.M. Cisse S. Glia. 1995; 14: 55-64Crossref PubMed Scopus (55) Google Scholar, 35Head M.W. Corbin E. Goldman J.E. Am. J. Pathol. 1993; 143: 1743-1753PubMed Google Scholar). In Drosophila, heat-induced granules dissolve after stress (36Feder J.H. Rossi J.M. Solomon J. Solomon N. Lindquist S. Genes Dev. 1992; 6: 1402-1413Crossref PubMed Scopus (331) Google Scholar). Similarly, IbpA/B proteins were found in E. coliassociated with inclusion bodies and with heat-induced intracellular proteins aggregates. IbpA/B were found to be involved in the re-solubilization of protein aggregates after stress (21Laskowska E. Wawrzynow A. Taylor A. Biochimie. 1996; 78: 117-122Crossref PubMed Scopus (117) Google Scholar). We demonstrated here in vitro that IbpB-bound proteins are stabilized in a conformation that can be subsequently released and specifically refolded by the KJE chaperones and ATP. It is tempting to speculate that stress-denatured proteins that accumulate together with IbpA/B proteins in the E. coli cell, can be solubilized and refolded, or degraded after stress, in processes involving the KJE chaperones, ATP hydrolysis, and possibly other chaperones. Moreover, in eukaryotes where heat-shock granules and amyloid plaques often contain sHSPs (37Yokoyama N. Iwaki T. Goldman J.E. Tateishi J. Fukui M. Acta Neuropathol. 1993; 85: 248-255Crossref PubMed Scopus (23) Google Scholar, 35Head M.W. Corbin E. Goldman J.E. Am. J. Pathol. 1993; 143: 1743-1753PubMed Google Scholar), a similar solubilization mechanism of sHSP-bound protein aggregates, involving Hsp70 and other ATP-hydrolyzing chaperones such as Hsp104, is therefore also conceivable as a defense mechanism against neurodegenerative and prion diseases (38Patino M.M. Liu J.-J. Glover J.R. Lindquist S. Science. 1996; 273: 622-625Crossref PubMed Scopus (572) Google Scholar). There are major differences in the expression level of the various families of Hsps between organisms. While heat shock induces a number of distinct Hsp families in E. coli and yeast (39Lorimer G.H. FASEB J. 1996; 10: 5-9Crossref PubMed Scopus (198) Google Scholar), Hsp70 is by far the major heat-induced protein in Drosophila (38Patino M.M. Liu J.-J. Glover J.R. Lindquist S. Science. 1996; 273: 622-625Crossref PubMed Scopus (572) Google Scholar), and small heat-shock proteins Hsp18.1 and Hsp17.7 are the major heat-induced proteins in higher plants (3Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 5Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (656) Google Scholar). We find that small heat-shock proteins, while possibly binding non-native proteins with relatively low affinity, may yet serve as a primary protein-binding matrix for subsequent refolding by a highly cooperative multichaperone machinery. Despite their high binding efficiency, LS chaperonins may not be present in sufficient amounts to bind all non-native protein during cellular stress (39Lorimer G.H. FASEB J. 1996; 10: 5-9Crossref PubMed Scopus (198) Google Scholar). Hence, auxiliary chaperone systems such as sHsps may become essential in vivo for the stress response. We thank C. Hergersberg for plasmids pBF3-IbpA and pBF3-IbpB, H.-J Schönfeld for purified DnaK, DnaJ, and GrpE; B. Bukau for DnaK and DnaJ producing plasmids, and G. Lorimer for discussions.