RECONSTITUTION OF THE STEROID RECEPTOR-CENTER-DOT-HSP90 HETEROCOMPLEXASSEMBLY SYSTEM OF RABBIT RETICULOCYTE LYSATE

Rabbit reticulocyte lysate contains a multiprotein system that assembl es steroid receptors into a heterocomplex with hsp90. In the case of t he glucocorticoid receptor (GR), the receptor must be bound to hsp90 t o bind steroid, and assembly of the GR . hsp90 complex restores the ho rmone binding domain of the receptor to the steroid binding conformati on. Using both direct assay of heterocomplex assembly by Western blott ing and indirect assay of assembly by steroid binding, it has previous ly been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) ar e 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 s ystem has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into thr ee fractions by DEAE chromatography and then reconstitute the GR . hsp 90 heterocomplex assembly system in a manner that requires the presenc e of each fraction. Fraction A contains most of the hsp70 and all of t he p60 in lysate, and elimination of p60 by immunoadsorption inactivat es this fraction, with bioactivity being restored by the addition of b acterially 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 re placed by purified rabbit hsp90. Fraction C contains p23, and its acti vity is replaced in the recombined system by highly purified bacterial ly expressed human p23. A minimal GR . hsp90 heterocomplex assembly sy stem was reconstituted with purified rabbit hsp70 and hsp90 and bacter ially expressed human p23 and p60. This reports the first reconstituti on of this apparently ubiquitous protein folding/heterocomplex assembl y system.