Oligomeric forms of the 90-kDa heat shock protein.

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.