Dittmar K D, Hutchison K A, Owens-Grillo J K, Pratt W B
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1996 May 31;271(22):12833-9. doi: 10.1074/jbc.271.22.12833.
Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90. In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex. It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23. A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.
兔网织红细胞裂解物含有一个多蛋白系统，该系统可将类固醇受体与热休克蛋白90（hsp90）组装成异源复合物。就糖皮质激素受体（GR）而言，该受体必须与hsp90结合才能结合类固醇，并且GR-hsp90复合物的组装将受体的激素结合结构域恢复到类固醇结合构象。此前，通过蛋白质印迹法直接检测异源复合物组装以及通过类固醇结合间接检测组装，已确定组装系统既依赖ATP/Mg2+又依赖K+，并且热休克蛋白70（hsp70）和一种酸性23 kDa蛋白（p23）是形成功能性GR-hsp90复合物所必需的。也有人认为，孕酮受体-hsp90异源复合物组装可能需要一种60 kDa蛋白（p60），但从未从单个组分中重新构建出完整的异源复合物组装系统。在这项工作中，我们通过DEAE柱色谱法将兔网织红细胞裂解物中的蛋白质分离成三个组分，然后以需要每个组分存在的方式重新构建GR-hsp90异源复合物组装系统。组分A包含裂解物中大部分的hsp70和所有的p60，通过免疫吸附去除p60会使该组分失活，添加细菌表达的人p60可恢复其生物活性。组分A的活性可被高度纯化的兔hsp70和来自表达p60的细菌的裂解物的组合所替代。组分B包含hsp90，其活性可被纯化的兔hsp90所替代。组分C包含p23，在重组系统中其活性可被高度纯化的细菌表达的人p23所替代。用纯化的兔hsp70和hsp90以及细菌表达的人p23和p60重新构建了一个最小的GR-hsp90异源复合物组装系统。本文首次报道了这种明显普遍存在的蛋白质折叠/异源复合物组装系统的重新构建。
