hsp70 interacting protein hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of BAG-1

Reticulocyte lysate contains a chaperone system that assembles glucocortico id receptor (GR). hspBO heterocomplexes. Using purified proteins, we have p repared a five-protein heterocomplex assembly system consisting of two prot eins essential for heterocomplex assembly-hsp90 and hsp70-and three protein s that act as co-chaperones to enhance assembly-Hop, hsp40, p23 [Morishima, Y., Kanelakis, K. C., Silverstein, A. M,, Dittmar, K, D., Estrada, L,, and Pratt, W. B, (2000) J. Biol. Chem. 275, 6894-6900]. The hsp70 co-chaperone Hip has been recovered in receptor . hsp90 heterocomplexes at an intermedi ate stage of assembly in reticulocyte lysate, and Hip is also thought to be an intrinsic component of the assembly machinery. Here we show that immuno depletion of Hip from reticulocyte lysate or addition of high levels of Hip to the purified five-protein system does not affect GR . hsp90 heterocompl ex assembly or the activation of steroid binding activity that occurs with assembly, Despite the fact that Hip does not affect assembly, it is recover ed in GR . hsp90 heterocomplexes assembled by both systems. In the five-pro tein system, Hip prevents inhibition of assembly by the hsp70 co-chaperone BAG-1, and cotransfection of Hip with BAG-1 opposes BAG-I reduction of ster oid binding activity in COS cells. We conclude that Hip is not a component of the assembly machinery but that it could play a regulatory role in oppos ition to BAG-1.