ALL OF THE FACTORS REQUIRED FOR ASSEMBLY OF THE GLUCOCORTICOID RECEPTOR INTO A FUNCTIONAL HETEROCOMPLEX WITH HEAT-SHOCK PROTEIN-90 ARE PREASSOCIATED IN A SELF-SUFFICIENT PROTEIN-FOLDING STRUCTURE, A FOLDOSOME

The hormone-binding domain of the glucocorticoid receptor must be boun d to heat shock protein (hsp) 90 for it to have a high-affinity steroi d binding conformation. We have recently demonstrated that hsp70 is re quired for cell-fi ee assembly of the receptor hsp90 complex and conco mitant activation of steroid binding activity (Hutchison, K.A, Dittmar , K. D., Czar, M. J., and Pratt, W. B. (1994) J. Biol. Chem. 269, 5043 -5049). hsp90 and hsp70 are known to exist together in a cytosolic com plex containing several other proteins, and in this work we ask if all of the factors required for proper receptor folding and heterocomplex assembly are preassociated in this multiprotein complex. The multipro tein complex was immunoadsorbed to protein A-agarose from rabbit retic ulocyte lysate using the 3G3 monoclonal IgM directed against hsp90. Wh en this immunopellet is mixed with immunadsorbed mouse glucocorticoid receptor and incubated at 30 degrees C with ATP/Mg2+ and KCl, the rece ptor is converted to the steroid binding conformation. When the immuno adsorbed multiprotein hsp90 complex is washed extensively, it loses a weakly bound protein (not hsp70 or hsp90) that is required for recepto r activation. This protein factor is contained in a Centricon C-100 fi ltrate of lysate which reconstitutes the receptor activating activity of the washed hsp90 complex. The hsp90 complex can be released from th e 3G3 antibody, and in the presence of the protein factor in the Centr icon C-100 filtrate it converts the receptor into a functional heteroc omplex with hsp90. The results support the proposal that the various c omponents of reticulocyte lysate that are required to refold the gluco corticoid receptor to the steroid binding state are preassociated with each other, acting as a self-sufficient protein folding machine.