Abstract
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.
Highlights
Ʈ To whom correspondence should be addressed: Dept. of Pharmacology, 1301 Medical Science Research Bldg
It should be mentioned that immunoblots of larger volumes of the DE52 pools than that analyzed in Fig. 2A show that pool B always contains some hsp70 and trace amounts of p23, as shown in Fig. 2A, the great majority of each of these proteins segregates to pools A and C, respectively
The components of the rabbit reticulocyte receptor1⁄7hsp90 heterocomplex assembly system were identified by analyzing the proteins that were bound to the chicken progesterone receptor (PR) at early and late stages of assembly of the PR1⁄7hsp90 heterocomplex [10, 12, 14, 19]
Summary
Ʈ To whom correspondence should be addressed: Dept. of Pharmacology, 1301 Medical Science Research Bldg. This multiprotein receptor heterocomplex can be assembled under cell-free conditions by incubating immunoadsorbed, hormone-free receptors (preincubated with salt to strip them free of associated proteins) with rabbit reticulocyte lysate [3, 4]. Steroid Receptor1⁄7hsp Heterocomplex Assembly [23], but a loosely associated component that is washed away is required to form a functional (i.e. steroid binding) GR1⁄7hsp complex [22]. This weakly bound component has been identified as p23 [15]
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