Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate

Rabbit reticulocyte lysate contains a multiprotein chaperone system that as sembles the glucocorticoid receptor (GR) into a complex with hsp90 and conv erts the hormone binding domain of the receptor to its high affinity steroi d binding state. This system has been resolved into five proteins, with hsp 90 and hsp70 being essential and Hop, hsp40, and p23 acting as co-chaperone s that optimize assembly. Hop binds independently to hsp70 and hsp90 to for m an hsp90(.)Hop(.)hsp70 complex that acts as a machinery to open up the GR steroid binding site. Because purified hsp90 and hsp70 are sufficient for some activation of GR steroid binding activity, some investigators have rej ected any role for Hop in GR(.)hsp90 heterocomplex assembly. Here, we count er that impression by showing that all of the Hop in reticulocyte lysate is present in an hsp90(.)Hop(.)hsp70 complex with a stoichiometry of 2:1.1. T he complex accounts for similar to 30% of the hsp90 and similar to9% of the hsp70 in lysate, and upon Sephacryl S-300 chromatography the GR(.)hsp90 as sembly activity resides in the peak containing Hop-bound hsp90. Consistent with the notion that the two essential chaperones cooperate with each other to open up the steroid binding site, we also show that purified hsp90 and hsp70 interact directly with each other to form weak hsp90-hsp70 complexes with a stoichiometry of 2:1.