Characterization of the protein-protein interactions determining the heat shock protein (hsp90.hsp70.hsp56) heterocomplex.

We have reported previously that the three heat shock proteins hsp56, hsp70, and hsp90 exist together in a heterocomplex in human lymphocyte cytosol (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). All three of these proteins also exist in the native glucocorticoid receptor heterocomplex isolated from WCL2 cell cytosol and we have recently shown that the three heat shock proteins are present when immunopurified mouse glucocorticoid receptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate (Hutchison, K. A., Scherrer, L. C., Czar, M. J., Ning, Y., Sanchez, E. R., Leach, K. L., Deibel, M. R., Jr., and Pratt, W. B. (1993) Biochemistry 32, 3953-3957). In this work, we show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp56, but hsp56 does not bind to purified mouse hsp70. In contrast to the equilibrium binding of hsp90 to hsp56, purified hsp90 binds poorly or not at all to purified hsp70 unless a third factor from reticulocyte lysate is present to permit complex formation. This hsp70.hsp90 complex-forming factor is heat-labile, and in the presence of this factor and ATP, a heat shock protein heterocomplex can be reconstituted from purified mouse hsp90 and hsp70 and rabbit hsp56 that is present in the factor preparation. Our data are consistent with a model in which hsp56 and hsp70 bind to different sites on hsp90 but do not interact with each other. The presence of hsp56 in the heat shock protein heterocomplex is not stabilized by molybdate but hsp56 is stabilized if the glucocorticoid receptor is present in addition to hsp90 and hsp70.