The role of DnaJ-like proteins in glucocorticoid receptor•hsp90 heterocomplex assembly by the reconstituted hsp90•p60•hsp70 foldosome complex
The role of DnaJ-like proteins in glucocorticoid receptor•hsp90 heterocomplex assembly by the reconstituted hsp90•p60•hsp70 foldosome complex
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DOI：
10.1074/jbc.273.13.7358
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发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Pratt, WB
中科院分区：
文献类型：
--
作者：
Dittmar, KD;Banach, M;Pratt, WB
糖皮质激素受体（GR）是从无激素细胞中与分子伴侣hsp90的异复合体中回收的，这是产生适当折叠状态以进行类固醇结合所必需的。GR.hsp90异位复合物是由一个多蛋白系统形成的，似乎存在于所有真核细胞中，最近我们重组了一个受体。用纯化兔Hsp90和hsp70和细菌表达人p23和p60的Hsp90杂合体组装系统。我们已经证明hsp90、p60和hsp70组成hsp90.p60。将GR从非类固醇结合形式转化为类固醇结合形式的hsp70复合物(Dittmar， K. D, and Pratt， W. B.（1997）。化学，272,13047-13054)。产生的GR.hsp90异复合体迅速分解，除非p23存在于hsp90的atp依赖构象上并稳定其与受体的联系(Dittmar, k.d, Demady， D， R., Stancato, l.f ., Krishna， P.，和Pratt， w.b. (1997) J. Biol。化学，272,21213-21220)。在目前的工作中，我们发现先前研究中使用的纯化兔hsp70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可以恢复组装活性。Hsp40是hsp90.p60的组成部分。从网织红细胞裂解液中分离Hsp70折叠体复合物，具有抗p60抗体。在促进p23与hsp90结合的条件下（高温、ATP、Nonidet P-40、钼酸盐），网状细胞裂解液或纯化蛋白质形成一个五元（p23.hsp90.p60.hsp70.hsp40）的伴侣蛋白复合物。不含hsp40的纯化组装系统具有中等水平的组装活性，当YDJ-1存在于约1 / 20 hsp70浓度时，其最大程度地增强了组装活性。虽然hsp40不存在于最终从L细胞质中分离出来的GR.hsp90异复合体中，但它存在于网状细胞裂解液中组装的GR.hsp90异复合体中。我们得出结论，hsp40是基于hsp90的多蛋白伴侣系统的一个组成部分，它增强了GR.hsp90异质复合物的组装。
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.