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.

Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90. In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex. It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23. A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.

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.

The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified "foldosome" directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K.A., Dittmar, K.D., and Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directing folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.

The hormone-binding domain of the glucocorticoid receptor must be bound to heat shock protein (hsp) 90 for it to have a high-affinity steroid-binding conformation. Cell-free assembly of a glucocorticoid receptor-hsp90 heterocomplex is brought about in reticulocyte lysate by a preformed protein-folding complex containing hsp90, hsp70, and other proteins [Hutchison, K.A., Dittmar, K. D., & Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899]. In this "foldosome" system, hsp70 is required for assembly of the receptor-hsp90 complex and concomitant activation of steroid-binding activity [Hutchison, K.A., Dittmar, K.D., Czar, M.J., & Pratt, W.B. (1994) J. Biol. Chem. 269, 22157-22161]. All previous experiments involving cell-free assembly of both receptor-hsp90 and protein kinase-hsp90 heterocomplexes have been carried out with the protein-folding system in rabbit reticulocyte lysate. In this work, we show that concentrated lysates of receptor-free mouse (L cells) and insect (Sf9) cells and also a plant (wheat germ) lysate fold the immunopurified glucocorticoid receptor into a functional (i.e., steroid binding) heterocomplex with hsp90. Receptor heterocomplex formation in animal lysates and in the plant lysate are not identical in that the dynamics of complex assembly are different, but both systems produce a functional complex that binds steroid. Also, in contrast to animal and insect complexes, receptor-plant hsp90 complexes are not stabilized by molybdate. When added to the other lysate, purified plant and animal hsp90s show partial complementarity, in that a receptor-hsp90 complex is formed but the receptor is not converted to the steroid-binding conformation. When added to rabbit reticulocyte lysate that has been depleted of endogenous hsp70, purified wheat germ and mouse hsp70's are equally active in promoting both assembly of receptor-hsp90 heterocomplexes and conversion of receptor to the steroid-binding conformation. Thus, hsp70 from the plant kingdom has conserved the ability to interact functionally with chaperone proteins of the animal kingdom to cooperate in protein folding as evidenced by formation of a functional receptor-hsp90 heterocomplex.