Abstract
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.
Highlights
From the $Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109 and the §Department of Cell Biology and Biochemistry, The Upjohn Company, Kalamazoo, Michigan 49001
The steroidreceptorcomplex isolated from WCL2 cell cytosol and we have hsp heterocomplex can be formed under cell-free conditions by recently shown that the three heatshock proteins are present when immunopurified mouse glucocorticoriedceptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate
We show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp66, but hsp56 does not bind to purified mouse hsp70
Summary
Cell Culture and Fractionation"L929 mouse fibroblasts (L cells) weregrown in monolayer in Dulbecco'smodifiedEagle's medium passing the fraction pool through ATP-agarose to remove hsp7O. Deatment of Reticulocyte Lysate-To prepare hsp90- and hsp7O-free supplemented with 10% bovineserum. The WCLB line of Chinese hamster ovary cellsoverexpressingthe mouse GR was established by Hirst et al [32], and the cells were grown in monolayer culture using Dulbecco'smodifiedEagle'smedium plus 3 p~methotrexate, 40 pdml proline, and 10%iron-supplemented calf serum. The drop-throughmaterial and all fractions that did notcontain hsp were combinedand appliedto a 25-ml column ofATP-agaroseA.ll of the material that was not adsorbedto the matrix was passed through a second columnof ATP-agaroseto remove the last traces of hsp7O. Cell ho- 100m~ KC1 and 5 m~ dithiothreitol, concentrated to one-halfthe origimogenateswere centrifugedfor 1h at 100.000 xg, and the supernatant nal volume of lysate, flash frozen, and stored in small aliquots. Zmrnunoadsorption-The GR heterocomplex was immunoadsorbed from replicate (100-200 pl) aliquots of L cellor WCLB cellcytosol
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