Dittmar K D, Banach M, Galigniana M D, Pratt W B
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1998 Mar 27;273(13):7358-66. doi: 10.1074/jbc.273.13.7358.
The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding. GR.hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60. We have shown that hsp90, p60, and hsp70 form an hsp90.p60. hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). The resulting GR.hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and Pratt, W. B. (1997) J. Biol. Chem. 272, 21213-21220). In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40. Elimination of the hsp40 from the purified GR.hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1. hsp40 is a component of the hsp90.p60.hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60. Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23. hsp90.p60.hsp70.hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins. The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70. Although hsp40 is not in the final GR.hsp90 heterocomplex isolated from L cell cytosol, it is in the GR.hsp90 heterocomplex assembled in reticulocyte lysate. We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR.hsp90 heterocomplex assembly.
糖皮质激素受体（GR）在无激素细胞中以与分子伴侣hsp90形成的异源复合物形式存在，hsp90对于产生类固醇结合所需的正确折叠状态是必需的。GR-hsp90异源复合物由一个似乎存在于所有真核细胞中的多蛋白系统形成。最近，我们用纯化的兔hsp90和hsp70以及细菌表达的人p23和p60重建了一个受体-hsp90异源复合物组装系统。我们已经表明，hsp90、p60和hsp70形成一个hsp90-p60-hsp70复合物，该复合物将GR从非类固醇结合形式转变为类固醇结合形式（迪特马尔，K.D.，和普拉特，W.B.（1997年）《生物化学杂志》272，13047 - 13054）。除非有p23存在以结合hsp90的ATP依赖构象并稳定其与受体的结合，否则形成的GR-hsp90异源复合物会迅速解离（迪特马尔，K.D.，德马迪，D.R.，斯坦卡托，L.F.，克里希纳，P.，和普拉特，W.B.（1997年）《生物化学杂志》272，21213 - 21220）。在当前的工作中，我们表明先前研究中使用的纯化兔hsp7被少量兔DnaJ同源物hsp40污染。从纯化的GR-hsp90组装系统中去除hsp40会降低组装活性，通过添加纯化的酵母DnaJ同源物YDJ-1可恢复该活性。hsp40是用抗p60抗体从网织红细胞裂解物中分离出的hsp90-p60-hsp70折叠体复合物的一个组分。在促进p23与hsp90结合的条件下（升高温度、ATP、诺乃洗涤剂P-40、钼酸盐），在网织红细胞裂解物中或由纯化蛋白形成了一个由伴侣蛋白组成的五元（p23-hsp90-p60-hsp70-hsp40）复合物。不含hsp40的纯化组装系统具有适度水平的组装活性，当YDJ-1以约hsp70浓度的二十分之一存在时，其组装活性得到最大程度增强。尽管hsp40不在从L细胞胞质溶胶中分离出的最终GR-hsp90异源复合物中，但它存在于在网织红细胞裂解物中组装的GR-hsp90异源复合物中。我们得出结论，hsp40是基于hsp90的多蛋白伴侣系统的一个组分，在该系统中它增强GR-hsp90异源复合物的组装。
