We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR.hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src.hsp90 and Raf.hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor.hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.

We have recently shown that hsp56, the FK506-binding immunophilin component of both the heat shock protein (hsp90.hsp70.hsp56) heterocomplex and the untransformed glucocorticoid receptor heterocomplex, is bound directly to hsp90 (Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Deibel, M. R., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161). In this work, we show that both untransformed glucocorticoid receptor and hsp90 heterocomplexes contain CyP-40, a 40-kDa immunophilin of the cyclosporin A-binding class. CyP-40 is present in both native glucocorticoid receptor heterocomplexes and receptor heterocomplexes reconstituted with rabbit reticulocyte lysate, and the presence of CyP-40 in the receptor heterocomplex is stabilized by molybdate. Immunoadsorption of hsp90 from cell lysate yields coimmunoadsorption of both hsp56 and CyP-40, showing that both immunophilins are in native heterocomplex with hsp90. However, immunoadsorption of hsp56 does not yield coimmunoadsorption of CyP-40; thus, the two immunophilins do not exist in the same heterocomplex with hsp90. Both purified CyP-40 and hsp56 bind directly to purified hsp90, and excess CyP-40 blocks the binding of hsp56, consistent with the presence of a common immunophilin binding site on hsp90. Our data also suggest that there are at least two types of untransformed glucocorticoid receptor-hsp90 heterocomplexes, one that contains hsp56 and another that contains CyP-40. The role played by the immunophilins in steroid receptor action is unknown, but it is clear that the peptidylprolyl isomerase activity of immunophilins is not required for glucocorticoid receptor-hsp90 heterocomplex assembly and proper folding of the hormone binding domain by the hsp90-associated protein folding system of reticulocyte lysate.

In animal cell lysates, multiprotein complexes containing hsp90, hsp70, p60, p23, and several immunophilins can assemble steroid receptors and oncogenic protein kinases, such as v-Src and v-Raf, into heterocomplexes that contain hsp90 and either immunophilins or, in the case of protein kinases, p50. The complexes with hsp90 are required for the proper functioning of these signal transduction systems. Wheat germ lysate contains a similar protein folding activity that forms functional steroid receptor complexes with hsp90, but not all the components of this system have been identified. The plant chaperone system has conserved interactions with animal chaperones in that wheat hsp70 functions in the rabbit reticulocyte lysate heterocomplex assembly system and human p23 functions in the wheat germ lysate. Here, we ask if wheat germ lysate also contains immunophilins of the FK506-binding class (FKBPs) that bind to the hsp90 component of the chaperone complex via tetratricopeptide repeat (TPR) domains. To demonstrate the plant heterocomplex, we add purified mammalian p23, preadsorbed with the JJ3 antibody to protein A-Sepharose, to wheat germ lysate and allow ATP-dependent formation of an animal p23. plant hsp90 complex. The complex is then washed and incubated with the radiolabeled immunosuppressant drug [3H]FK506, which binds in a specific manner to a coimmunoadsorbed plant FKBP. Binding of the plant FKBP to plant hsp90 is prevented by adding to wheat germ lysate a purified fragment containing the TPR domains of human cyclophilin-40. Geldanamycin, a benzoquinone ansamycin that binds to animal hsp90s and prevents their chaperone activity, binds in a temperature-dependent manner to wheat hsp90 to block formation of the p23.hsp90.FKBP heterocomplex. These data show that immunophilin binding to hsp90 via TPR domains is conserved in the plant kingdom as well as in the animal kingdom and that geldanamycin will be an important tool for the study of hsp90-mediated protein chaperoning in plant cells.