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Substrate-binding characteristics of proteins in the 90 kDa heat shock protein family.

ABSTRACT: In the present study we investigated the substrate-binding characteristics of three members of the 90 kDa heat shock protein (HSP90) family, namely the alpha isoform of human HSP90 (HSP90alpha), human GRP94 (94 kDa glucose-regulated protein, a form of HSP90 from endoplasmic reticulum), and HtpG (the Escherichia coli homologue of HSP90) and the domain responsible for these characteristics. The recombinant forms of HSP90alpha, GRP94 and HtpG existed as dimers and became oligomerized at higher temperatures. Among the three family members, HtpG required the highest temperature (65 degrees C) for its transition to oligomeric forms. The precipitation of the substrate protein, glutathione S-transferase, which occurred at 55 degrees C, was efficiently prevented by the simultaneous presence of a sufficient amount of HSP90alpha or GRP94, but not by HtpG, which was still present as a dimer at that temperature. However, precipitation was stopped completely at 65-70 degrees C, at which temperature HtpG was oligomerized. Thus the transition of HSP90-family proteins to a state with self-oligomerization ability is essential for preventing the precipitation of substrate proteins. We then investigated the domain responsible for the substrate binding of HtpG on the basis of the three domain structures. The self-oligomerizing and substrate-binding activities towards glutathione S-transferase and citrate synthase were both located in a single domain, the N-terminal domain (residues 1-336) of HtpG. We therefore propose that the primary peptide-binding site is located in the N-terminal domain of HSP90-family proteins.

SUBMITTER: Nemoto TK

PROVIDER: S-EPMC1221698 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

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Project description:Two isoforms of the 90-kDa heat shock protein, HSP90alpha and HSP90beta, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90alpha predominantly exists as a homodimer and that HSP90beta is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90alpha sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90beta oligomers sedimenting at 6-12S. All of the HSP90beta oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90beta dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90alpha oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90alpha migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.

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Substrate-binding characteristics of proteins in the 90 kDa heat shock protein family.

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