Analysis of the Interaction of Small Heat Shock Proteins with Unfolding Proteins
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m301640200
ISSN1083-351X
AutoresThusnelda Stromer, Monika Ehrnsperger, Matthias Gaestel, Johannes Büchner,
Tópico(s)Bee Products Chemical Analysis
ResumoThe ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, α-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals. The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, α-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals. heat shock proteins small heat shock proteins citrate synthase α-glucosidase size-exclusion chromatography N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine In response to environmental stress such as heat shock, which leads to the accumulation of nonnative proteins, cells increase the expression of several classes of proteins (1Lindquist S. Craig E.A. Annu. Rev Genet. 1988; 22: 631-677Crossref PubMed Scopus (4364) Google Scholar). The major conserved families of these heat shock proteins (Hsps)1 have been shown to be involved in protein folding as molecular chaperones (2Buchner J. FASEB J. 1996; 10: 10-19Crossref PubMed Scopus (380) Google Scholar).The most divergent of these chaperone classes are the small heat shock proteins (sHsps). sHsps have been found in almost all organisms investigated so far, with the number of members varying from species to species. They share conserved regions mostly in the C-terminal part of the protein, whereas the N-terminal part differs in sequence and length, leading to molecular masses of 16–42 kDa for sHsps in different organisms (3De Jong W.W. Caspers G.J. Leunissen J.A. Int. J. Biol. Macromol. 1998; 22: 151-162Crossref PubMed Scopus (434) Google Scholar). The conserved C-terminal domain of ∼100 amino acids shares sequence homology with the major eye lens protein αA-crystallin (4Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (673) Google Scholar). Almost all sHsps assemble into large oligomeric complexes of 9 to >30 subunits, and complexes in the range of 125 kDa to 2 MDa have been found (5Ehrnsperger M. Gaestel M. Buchner J. Methods Mol. Biol. 2000; 99: 421-429PubMed Google Scholar, 6Narberhaus F. Microbiol. Mol. Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (461) Google Scholar, 7Haslbeck M. Buchner J. Prog. Mol. Subcell. Biol. 2002; 28: 37-59Crossref PubMed Scopus (66) Google Scholar, 8van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (377) Google Scholar, 9Andreasi B.F. Arcovito G. De Spirito M. Mordente A. Martorana G.E. Biophys. J. 1995; 69: 2720-2727Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 10Spector A. Li L.-K. Augusteyn R.C. Schneider A. Freund T. Biochem. J. 1971; 124: 337-343Crossref PubMed Scopus (142) Google Scholar, 11Arrigo A.-P. Suhan J.P. Welch W.J. Mol. Cell. Biol. 1988; 8: 5059-5071Crossref PubMed Scopus (297) Google Scholar). Some sHsps such as those from plants form assemblies with well defined stoichiometries, whereas other sHsps, including the mammalian proteins, form a range of oligomeric sizes (12Haley D.A. Bova M.P. Huang Q.L. Mchaourab H.S. Stewart P.L. J. Mol. Biol. 2000; 298: 261-272Crossref PubMed Scopus (174) Google Scholar,13Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (563) Google Scholar). This polydispersity has limited the amount of structural information available. The crystal structure of an archaeal sHsp (14Kim K.K. Kim R. Kim S.H. Nature. 1998; 394: 595-599Crossref PubMed Scopus (783) Google Scholar) and the cryo-electron microscopy reconstruction of αB-crystallin (15Haley D.A. Horwitz J. Stewart P.L. J. Mol. Biol. 1998; 277: 27-35Crossref PubMed Scopus (260) Google Scholar) revealed that the overall organization is that of a hollow globular sphere. A variation of this scheme is the three-dimensional structure of wheat Hsp16.9, which assembles into a dodecameric double disk, where each disk is organized as a trimer of dimers (16van Montfort R.L. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (624) Google Scholar). Many sHsps have dynamic and variable quaternary structures with subunits that can freely and rapidly exchange between oligomers, such as αB-crystallin, Hsp25, Hsp27, and sHsps from Bradyrhizobium japonicum (8van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (377) Google Scholar,13Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (563) Google Scholar, 15Haley D.A. Horwitz J. Stewart P.L. J. Mol. Biol. 1998; 277: 27-35Crossref PubMed Scopus (260) Google Scholar, 17Ehrnsperger M. Lilie H. Gaestel M. Buchner J. J. Biol. Chem. 1999; 274: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18Studer S. Narberhaus F. J. Biol. Chem. 2000; 275: 37212-37218Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Changes in quaternary structure at temperatures that are physiologically relevant for sHsps are important for their chaperone activity (16van Montfort R.L. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (624) Google Scholar, 19Haslbeck 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 (380) Google Scholar, 20Giese K.C. Vierling E. J. Biol. Chem. 2002; 277: 46310-46318Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 21Bova M.P. Huang Q. Ding L. Horwitz J. J. Biol. Chem. 2002; 277: 38468-38475Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Van 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).In vitro, sHsps act as molecular chaperones in preventing unfolded proteins from irreversible aggregation and insolubilization (19Haslbeck 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 (380) Google Scholar, 23Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1735) Google Scholar, 24Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 25Ehrnsperger M. Buchner J. Gaestel M. Fink A.L. Goto Y. Structure and Function of Small Heat-Shock Proteins. Marcel Dekker, Inc., New York1998: 533-575Google Scholar, 26Lee G.J. Vierling E. Plant Physiol. 2000; 122: 189-198Crossref PubMed Scopus (365) Google Scholar, 27Haslbeck M. Cell. Mol. Life Sci. 2002; 59: 1649-1657Crossref PubMed Scopus (245) Google Scholar, 28Derham B.K. Harding J.J. Prog. Retin. Eye Res. 1999; 18: 463-509Crossref PubMed Scopus (273) Google Scholar). Because of their high binding capacity of up to one substrate molecule/sHsp subunit, sHsps are more efficient than other chaperones in this respect (19Haslbeck 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 (380) Google Scholar, 25Ehrnsperger M. Buchner J. Gaestel M. Fink A.L. Goto Y. Structure and Function of Small Heat-Shock Proteins. Marcel Dekker, Inc., New York1998: 533-575Google Scholar, 29Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar, 30Jaenicke R. Creighton T.E. Curr. Biol. 1993; 3: 234-235Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 31Wang K. Spector A. Investig. Ophthalmol. Vis. Sci. 1995; 36: 311-321PubMed Google Scholar, 32Farahbakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (185) Google Scholar). The nonnative proteins seem to be surface-exposed in the complexes because bound proteins and peptides are accessible to antibodies (33Ehrnsperger M. Hergersberg C. Wienhues U. Nichtl A. Buchner J. Anal. Biochem. 1998; 259: 218-225Crossref PubMed Scopus (38) Google Scholar). The range of substrates recognized covers peptides as well as oligomeric enzymes (25Ehrnsperger M. Buchner J. Gaestel M. Fink A.L. Goto Y. Structure and Function of Small Heat-Shock Proteins. Marcel Dekker, Inc., New York1998: 533-575Google Scholar, 33Ehrnsperger M. Hergersberg C. Wienhues U. Nichtl A. Buchner J. Anal. Biochem. 1998; 259: 218-225Crossref PubMed Scopus (38) Google Scholar, 34Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). No substrate specificity has been observed for sHsps so far, and complex formation with substrates has not been analyzed in detail. Here, we set out to address this question using Hsp25 from mouse and Hsp26 from yeast, two different well studied members of the sHsp family.Murine Hsp25 exists as a hexadecamer in solution. These oligomers are in a concentration-dependent equilibrium with tetramers, suggesting tetramers as the basic building block of the hexadecamer (17Ehrnsperger M. Lilie H. Gaestel M. Buchner J. J. Biol. Chem. 1999; 274: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). It was previously shown that Hsp25 is able to influence the inactivation and subsequent aggregation of the model substrate citrate synthase (CS). Hsp25 binds several nonnative CS molecules and protects them from irreversible aggregation. Under permissive folding conditions, the bound substrates can be released from Hsp25 and, in cooperation with other ATP-dependent chaperones, regain their native structures (34Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar).Under physiological conditions, yeast Hsp26 forms spherical 24-mers with particle diameters of ∼15 nm. Elevated temperatures induce the dissociation of the oligomeric complex into dimers. These dimers initiate the interaction with nonnative substrate proteins and reassemble into larger defined sHsp-substrate complexes (19Haslbeck 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 (380) Google Scholar).To gain insight into the general mode of the interaction of sHsps with nonnative proteins, we used four different model substrate proteins. We show that, for each substrate, morphologically distinct and defined complexes are formed. The stoichiometry of the complexes between sHsps and nonnative protein seems to be dependent on the substrate investigated. Furthermore, we demonstrate that Hsp25 and Hsp26 are able to form mixed complexes with CS and α-glucosidase (α-Gluc) and that the first substrate bound determines the morphology of the resulting sHsp-substrate complexes.EXPERIMENTAL PROCEDURESMaterialsRecombinant murine Hsp25 and yeast Hsp26 were expressed and purified as previously described (19Haslbeck 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 (380) Google Scholar, 24Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 35Gaestel M. Gross B. Benndorf R. Strauss M. Schunk W.H. Kraft R. Otto A. Bohm H. Stahl J. Drabsch H. Eur. J. Biochem. 1989; 179: 209-213Crossref PubMed Scopus (79) Google Scholar). Purified isoenzyme P1 of yeast α-Gluc (maltase, EC 3.2.1.20) (36Kopetzki E. Buckel P. Schumacher G. Yeast. 1989; 5: 11-24Crossref PubMed Scopus (24) Google Scholar) with a specific activity of >130 units/mg was a gift from Dr. A. Grossmann (Roche Diagnostics). Mitochondrial CS from pig heart (EC 4.1.3.7) was obtained from Roche Diagnostics. Bovine insulin and bovine rhodanese were from Sigma.CS was stored in 50 mm Tris-HCl and 2 mm EDTA, pH 8.0; α-Gluc was stored in 0.1 m potassium phosphate, pH 6.8; insulin was stored in 20 mm sodium phosphate and 0.1 m NaCl, pH 6.5; rhodanese was stored in 50 mm Tris-HCl, 20 mm dithioerythritol, 50 mm sodium thiosulfate, pH 7.7; Hsp25 was stored in 20 mm Tris-HCl, pH 7.6, 10 mm MgCl2, 20 mm NH4Cl, and 0.5 mmdithioerythritol; and Hsp26 was stored in 40 mm Hepes-KOH, 50 mm NaCl, 1 mm EDTA, and 1 mmdithioerythritol, pH 7.5. All protein concentrations refer to monomers.Analysis of Hsp25 and Hsp26 ActivitiesTo induce aggregation, CS and α-Gluc were diluted in 40 mm Hepes-KOH, pH 7.5, equilibrated at 43 and 46 °C, respectively (24Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 36Kopetzki E. Buckel P. Schumacher G. Yeast. 1989; 5: 11-24Crossref PubMed Scopus (24) Google Scholar, 37Höll-Neugebauer B. Rudolph R. Schmidt M. Buchner J. Biochemistry. 1991; : 11609-11614Crossref PubMed Scopus (90) Google Scholar). Rhodanese (30 μm) was diluted 1:100 in 40 mm sodium phosphate, pH 7.7, at 44 °C (38Mendoza J.A. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1992; 267: 17631-17634Abstract Full Text PDF PubMed Google Scholar). Assays were performed in the absence and presence of Hsp25 or Hsp26. Assays in the presence of IgG served as a control for unspecific protein effects (concentrations given in the figure legends). To monitor the kinetics of thermal aggregation in the presence of Hsp25 and Hsp26, light scattering was measured in a FluoroMax I fluorescence spectrophotometer in a stirred and thermostated quartz cell. During the measurements, both the excitation and emission wavelengths were set to 360 nm with a spectral bandwidth of 4 nm.The insulin aggregation assay in the presence of Hsp25 or Hsp26 was performed in 40 mm Hepes-KOH, pH 7.5. Insulin (45 μm) was equilibrated in the absence and presence of either Hsp25 or Hsp26 at 25 or 43 °C, respectively. Assays in the presence of IgG served as a control for unspecific protein effects. The aggregation reaction was started by the addition of dithioerythritol to a final concentration of 20 mm, and the resulting turbidity was monitored at 360 nm in an Amersham Biosciences Ultrospex 4060 UV-visible spectrophotometer equipped with a temperature control unit.Analysis of sHsp-Substrate ComplexesSize-exclusion Chromatography (SEC)To analyze complex formation between Hsp25 and the different substrates, SEC was performed using a TosoHaas TSK4000SW column (30 × 0.75 cm, separation range of 20–7000 kDa). Complex formation between Hsp26 and the different substrates was analyzed on a TosoHaas TSK4000PW column (30 × 0.75 cm; separation range of 10–1500 kDa). Hsp25 or Hsp26 in the absence and presence of substrate was incubated as described in the figure legends. All samples were centrifuged at 14,000 ×g for 5 min at 4 °C before application. Chromatography was carried out at 25 °C in running buffer (0.1 mHepes-KOH and 150 mm KCl, pH 7.5) at flow rates of 0.5 ml/min for samples with Hsp25 and 0.75 ml/min for those with Hsp26. The sample volume was 100 μl. Hsp25 and Hsp26 were detected by fluorescence at an excitation wavelength of 280 nm and emission wavelengths of 330 and 323 nm, respectively, using a Jasco FP920 fluorescence detector.Analysis of Binding StoichiometriesHsp25-substrate complexes were formed with 4.8 μm Hsp25 in the presence of increasing concentrations of CS (0.15–5 μm) for 30 min at 43 °C. During SEC (described above), the respective complex peaks were collected and precipitated with sodium deoxycholate/trichloroacetic acid. Precipitates were then resuspended in reducing SDS sample buffer (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar), separated by SDS-PAGE (10–20% precast Tricine gradient gels, Novex), and visualized by Coomassie Blue staining. Scanned gels were analyzed using ImageMaster-1D software (Amersham Biosciences). Protein amounts and ratios were calculated for monomers and corrected for different staining intensities due to differences in molecular mass.Electron MicroscopyComplex formation between Hsp25 or Hsp26 and the different substrates was performed following the protocol used for the SEC experiments. The samples were separated on a gel filtration column. The complex peak was collected; and immediately before application, samples were diluted to a protein concentration of 0.1 mg/ml in running buffer. As controls, substrates and Hsp25 or Hsp26 alone were also incubated under the respective experimental conditions. The samples were directly applied to glow-discharged carbon-coated copper grids and negatively stained with 3% uranyl acetate. Electron micrographs were recorded at a nominal magnification of ×60,000 using a Philips CM12 electron microscope operating at 120 kV or at a nominal magnification of ×33,000 using a Jeol 100CX electron microscope operating at 100 kV.DISCUSSIONThe overexpression of sHsps has been shown to convey thermotolerance in a number of organisms and cell types (20Giese K.C. Vierling E. J. Biol. Chem. 2002; 277: 46310-46318Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 40Sun W. Van Montagu M. Verbruggen N. Biochim. Biophys. Acta. 2002; 1577: 1-9Crossref PubMed Scopus (536) Google Scholar,46Aoyama A. Frohli E. Schafer R. Klemenz R. Mol. Cell. Biol. 1993; 13: 1824-1835Crossref PubMed Google Scholar, 47Landry J. Chretien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (579) Google Scholar, 48Mehlen P. Briolay J. Smith L. Diaz-Latoud C. Fabre N. Pauli D. Arrigo A.-P. Eur. J. Biochem. 1993; 215: 277-284Crossref PubMed Scopus (114) Google Scholar, 49Knauf U. Bielka H. Gaestel M. FEBS Lett. 1992; 309: 297-302Crossref PubMed Scopus (75) Google Scholar, 50van den Ijssel P.R. Overkamp P. Knauf U. Gaestel M. De Jong W.W. FEBS Lett. 1994; 355: 54-56Crossref PubMed Scopus (104) Google Scholar, 51Nakamoto H. Suzuki N. Roy S.K. FEBS Lett. 2000; 483: 169-174Crossref PubMed Scopus (88) Google Scholar), indicating a general thermoprotective function of the protein family. The mechanism of sHsp chaperone activity is poorly characterized. Previous work has clearly demonstrated that sHsps recognize misfolded proteins and maintain them in a soluble but inactive state. In contrast to other chaperones, the chaperone function of sHsps appears to be limited to binding and maintaining the solubility of unfolding proteins, without promoting refolding directly (8van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (377) Google Scholar, 19Haslbeck 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 (380) Google Scholar, 23Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1735) Google Scholar, 24Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 29Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar, 34Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar).Haslbeck et al. (19Haslbeck 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 (380) Google Scholar) suggested that, for yeast Hsp26, dissociation is a prerequisite of chaperone function and that the Hsp26 dimer may act as the primary substrate-binding species, followed by reassembly into a larger complex together with the substrate. This hypothesis was further supported by in vitro and in vivo studies (16van Montfort R.L. Basha E. Friedrich K.L. Slingsby C. Vierling E. Nat. Struct. Biol. 2001; 8: 1025-1030Crossref PubMed Scopus (624) Google Scholar, 20Giese K.C. Vierling E. J. Biol. Chem. 2002; 277: 46310-46318Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 26Lee G.J. Vierling E. Plant Physiol. 2000; 122: 189-198Crossref PubMed Scopus (365) Google Scholar) demonstrating that dynamic changes in oligomerization are a prerequisite for sHsp function in vivoand chaperone activity in vitro. For Hsp25, detectable dissociation of the oligomer does not precede the binding of nonnative protein, but an exchange of subunits has been suggested (34Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). We used these two members of the sHsp family, mouse Hsp25 and yeast Hsp26, which differ in their quaternary structures and in the mode of activation, to study the general principles of complex formation of sHsps with nonnative proteins.Formation of Large sHsp-Substrate ComplexesLittle is known about the structure of sHsp-substrate complexes. A striking feature of sHsps is that, upon substrate binding, they form very large complexes. For the four different model substrates used in this study, effective interactions with both Hsp25 and Hsp26 were observed. Both sHsps showed comparable promiscuity in substrate binding, and the morphologies of the sHsp-substrate complexes as visualized by negative-stain electron microscopy were similar. Hsp26 and Hsp25 exhibited globular structures with outer diameters of ∼15 and 20 nm, respectively. These structures increased in size upon substrate binding. Complexes formed with CS showed mean diameters of ∼50 and ∼70 nm for Hsp26 and Hsp25, respectively. Complexes formed with rhodanese were smaller, with mean diameters of ∼45 nm for both sHsps. The α-Gluc complexes were smaller (mean diameters of ∼30 nm) and more irregularly shaped than the CS and rhodanese complexes. The complexes formed with insulin revealed net-like structures with mean diameters of about the same size of the respective sHsp. The differences in complex sizes between Hsp26 and Hsp25 with CS might be due to different binding stoichiometries (see below).SEC experiments indicated that the process of complex formation of Hsp25 and Hsp26 with nonnative protein is influenced by the substrate used. After incubation of the sHsp with CS or rhodanese, full-sized sHsp-substrate complexes could be detected immediately. Only the amount of particles (and not their size) increased with prolonged incubation. With α-Gluc and insulin, intermediate-sized particles could be detected after incubation of substoichiometric amounts of substrate with sHsps.Surface binding of substrate has been shown for plant Hsp18.1 (29Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar), and the binding of peptides and proteins to Hsp25 (33Ehrnsperger M. Hergersberg C. Wienhues U. Nichtl A. Buchner J. Anal. Biochem. 1998; 259: 218-225Crossref PubMed Scopus (38) Google Scholar) also argues for accessibility of bound protein. In the case of Hsp26, the Hsp26-CS complex appears, however, to be a completely new assembly. The original Hsp26 shell is no longer detectable. Instead, an enlarged outer shell and an additional, internal shell of density become visible (19Haslbeck 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 (380) Google Scholar). As the bound substrates are still accessible to proteases, they are at least partially surface-exposed (data not shown). Due to the similarities in complex formation and morphology, it is reasonable to assume that, also in the case of Hsp25 and for sHsps in general, large rearrangements of the complex occur upon binding of nonnative protein.Taken together, our data suggest that the formation of large defined sHsp-substrate complexes is a general feature of sHsps. The morphology of these complexes is substrate-dependent, but independent of the sHsp used.Cooperativity of Substrate Protein BindingThe large complex structures observed by SEC and electron microscopy indicate binding of several substrate molecules to Hsp25 or Hsp26 oligomers. A maximum binding capacity of one protein/sHsp subunit has repeatedly been suggested (29Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar, 31Wang K. Spector A. Investig. Ophthalmol. Vis. Sci. 1995; 36: 311-321PubMed Google Scholar, 43Farahbakhsh Z.T. Huang Q.L. Ding L.L. Altenbach C. Steinhoff H.J. Horwitz J. Hubbell W.L. Biochemistry. 1995; 34: 509-516Crossref PubMed Scopus (201) Google Scholar). Especially in the case of CS and rhodanese, the particles formed with Hsp25 or Hsp26 showed little variance in size and shape. Only spherical, regularly shaped particles appeared. Partially loaded and therefore smaller sHsp complexes were not detected. This observation suggests coordinated binding of substrate molecules to sHsps. For CS and Hsp26 (19Haslbeck 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 (380) Google Scholar) and for CS and Hsp25 (Fig. 4), this process was found to be highly cooperative.The simultaneous binding of several nonnative proteins seems to be a prerequisite for efficient and stable complex formation. Interestingly, for all proteins studied, except insulin, binding could be saturated at a defined ratio of nonnative protein to sHsp. The cooperativity and morphology of the complexes indicate that the substrates direct the process of complex formation.Incorporation of Different Substrate Proteins into sHsp ComplexesThe formation of mixed substrate complexes is important for the function of sHsps in the crowded environment of the cell. Here, upon stress, different polypeptides unfold and have to be bound simultaneously. We were interested in whether sHsps are able to bind different substrates in one complex. Interestingly, two different substrates could be incorporated into the sHsp-substrate complex, leading to the formation of morphologically uniform mixed complexes. The analysis of the mixed complexes by electron microscopy revealed significant differences in complex morphologies depending on the order of addition. If either Hsp25 or Hsp26 was first incubated with CS, the complexes showed marked similarity to the structures that were observed upon incubation of only CS and Hsp25 or Hsp26. Preincubation of sHsp with α-Gluc led to typical complexes between sHsp and α-Gluc. When all three proteins were incubated together, the resulting complexes showed morphologies similar to the sHsp-CS complexes, but they were significantly smaller. Thus, the first substrate bound seems to determine the morphology of the complex.In conclusion, the analysis of the interaction of different substrate proteins with sHsps provides new insights into the mode of complex formation between sHsps and nonnative substrate proteins. sHsps bind substrates in a range of at least 3–100 kDa. The degree of cooperativity seems to be substrate-dependent. Taken together, our results show that the formation of large, morphologically distinct complexes with nonnative proteins is a conserved feature of the sHsp family of chaperones. In response to environmental stress such as heat shock, which leads to the accumulation of nonnative proteins, cells increase the expression of several classes of proteins (1Lindquist S. Craig E.A. Annu. Rev Genet. 1988; 22: 631-677Crossref PubMed Scopus (4364) Google Scholar). The major conserved families of these heat shock proteins (Hsps)1 have been shown to be involved in protein folding as molecular chaperones (2Buchner J. FASEB J. 1996; 10: 10-19Crossref PubMed Scopus (380) Google Scholar). The most divergent of these chaperone classes are the small heat shock proteins (sHsps). sHsps have been found in almost all organisms investigated so far, with the number of members varying from species to species. They share conserved regions mostly in the C-terminal part of the protein, whereas the N
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