Nemoto T, Sato N
Department of Biochemistry, Iwate Medical University School of Dentistry, 19-1 Uchimaru, Morioka 020-8505, Japan.
Biochem J. 1998 Mar 1;330 ( Pt 2)(Pt 2):989-95. doi: 10.1042/bj3300989.
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.
90 kDa热休克蛋白的两种同工型，即HSP90α和HSP90β，存在于哺乳动物细胞的胞质溶胶中。在非变性条件下通过聚丙烯酰胺凝胶电泳（天然PAGE）分析表明，HSP90α主要以同二聚体形式存在，而HSP90β主要以单体形式存在[Minami，Kawasaki，Miyata，Suzuki和Yahara（1991）J. Biol. Chem. 266，10099 - 10103]。然而，在其他分析条件下，如梯度离心，仅观察到二聚体形式。因此，在本研究中，我们使用本实验室最近开发的同工型特异性单克隆抗体，通过免疫化学技术研究了HSP90的天然形式。在生理盐浓度下进行甘油梯度离心以及对大鼠肝脏胞质溶胶进行天然PAGE分析，结果显示HSP90α的寡聚体形式以8 - 10S沉降为主。另一方面，甘油梯度离心显示HSP90β寡聚体有多种形式，沉降在6 - 12S。然而，所有的HSP90β寡聚体在天然PAGE上均迁移至100 kDa单体和190 kDa二聚体位置。一种新型的二维双天然PAGE显示，该实体在电泳过程中从HSP90β二聚体转变为单体。相同的PAGE进一步显示，HSP90α寡聚体在电泳过程中也解离为二聚体。细菌表达的人HSP90α全长形式以二聚体形式迁移，但在天然PAGE条件下，相当一部分未穿透凝胶，表明存在寡聚体形式。对HSP90缺失突变体的电泳研究表明，C末端的200个氨基酸能够形成寡聚体。综上所述，我们得出结论，即使在未受应激的条件下，两种HSP90同工型在胞质溶胶中也主要以寡聚体形式存在，但在进行天然PAGE时它们会人为解离为较小的形式。
