| Popis: |
In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly. |
