Evidence for a universal TPR binding domain onhsp90.

Abstract

Prior to their hormone-dependent activation, steroid receptors are recovered in the cytosolic fraction of target cells in multimeric protein complexes containing both heat shock protein (hsp) and immunophilin chaperones. It is the goal of this thesis to examine in detail the interactions between hsp90 and immunophilins, and to show that these complexes are ubiquitously conserved in eukaryotes. All members of the immunophilin protein family have peptidyl-prolyl isomerase activity, and like hsp90, they are thought to play major roles in protein folding and trafficking in the cell. We have previously shown that FKBP52/hsp56, an immunophilin of the FK506-binding class, is bound directly to hsp90 in both the hsp (hsp90$\cdot$hsp70$\cdot$hsp56) heterocomplex that exists independent of receptors and the glucocorticoid receptor (GR) heterocomplex formed by this chaperone machinery. I show here that the 40 kDa cyclosporin A-binding immunophilin CyP-40 is also present in both the hsp and GR heterocomplexes. Both FKBP51/hsp56 and CyP-40 bind directly to hsp90 via their tetratricopeptide repeat (TPR) domains, and excess CyP-40 blocks FKBP52/hsp56 binding, suggesting that hsp90 may contain a common immunophilin binding site. Unlike other proteins recovered in steroid receptor heterocomplexes, the immunophilins are not required for receptor heterocomplex assembly. Since FKBP52/hsp56 has been implicated in targeted protein trafficking, I examined the hsp90 binding of four TPR-containing proteins all thought to be involved in protein movement. FKBP52/hsp56 and CyP-40 each contain 3 TPR domains and are weakly bound to hsp90. Mas70p, a mitochondrial membrane receptor with 7 TPR domains, and p50, a component of hsp90$\cdot$oncogenic protein kinase complexes, both bind very tightly to hsp90. Their binding is not blocked by the TPR domain segment of CyP-40, but the binding of all four proteins is blocked by bacterially-expressed p60, an hsp90-binding TPR protein required for GR-hsp90 complex assembly. The data are consistent with the notion that hsp90 has a region that acts as a general TPR domain acceptor. The thesis proposes a model for protein movement in which proteins that are chaperoned by hsp90 move as dynamic complexes to cellular sites of action with the TPR-containing protein targeting movement of the complexes.