Kanelakis K C, Morishima Y, Dittmar K D, Galigniana M D, Takayama S, Reed J C, Pratt W B
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1999 Nov 26;274(48):34134-40. doi: 10.1074/jbc.274.48.34134.
The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR).hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR.hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of approximately 0.03, and its elimination by immunoadsorption did not affect GR folding and GR. hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR.hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR.hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.
热休克蛋白hsp70/hsc70是由网织红细胞裂解液重构的形成糖皮质激素受体（GR）.hsp90异源复合物的五蛋白（hsp90、hsp70、Hop、hsp40和p23）最小伴侣系统的必需成分。BAG-1是一种辅因子，它与hsp70/hsc70的ATP酶结构域结合并调节其伴侣活性。由于已发现BAG-1与类固醇受体家族的多个成员相关联，我们研究了BAG-1对GR折叠和GR.hsp90异源复合物组装的影响。网织红细胞裂解液中BAG-1与hsp70/hsc70的摩尔比约为0.03，通过免疫吸附去除BAG-1并不影响GR折叠和GR.hsp90异源复合物组装。在低BAG-1:hsp70/hsc70比例下，BAG-1促进Hop从基于hsp90的伴侣系统中释放，而不抑制GR.hsp90异源复合物组装。然而，当摩尔比接近与hsp70化学计量时，BAG-1对GR折叠为类固醇结合形式产生浓度依赖性抑制，并相应地抑制由最小五蛋白伴侣系统进行的GR.hsp90异源复合物组装。此外，瞬时或稳定转染BAG-1的细胞中类固醇结合活性降低。这些观察结果表明，在生理浓度下，BAG-1通过促进Hop从组装复合物中释放来调节组装；但是，在接近转染细胞和一些转化细胞系中的浓度时，hsp70持续被BAG-1结合，异源复合物组装被阻断。
