hsp90 is a ubiquitous heat shock protein and a major cytoplasmic molecular chaperone. It has been described mainly in relation to its association with steroid receptors and some other regulatory proteins where it appears to assist in their folding to a native state. The main objectives of this proposal are to describe the multi-protein chaperoning pathway involving hsp90 and the mechanisms of action of hsp90 and its co-chaperones. Three specific aims are proposed. In Aim 1, we will continue to develop and study an hsp70 and hsp90 dependent chaperoning system for the renaturation of denatured proteins. This system involves the cooperation of at least four proteins and an optimal system will probably include a few additional proteins. The individual and cooperative functions of these proteins will then be examine din detail. In Aim 2, we will continue to describe the structural and functional domains of hsp90. Emphasis will be on the importance of the nucleotide binding domain in hsp90 activity and the descriptions of domains that interact with protein substrates and with the co-chaperone, p23. Our main approach will be the characterization of a wide variety of hsp90 mutants. A novel hsp90 homolog, TRAP-1, will be studied in Aim 3. Several basic properties of this protein must first be described. It will then be compared with hsp90 in a variety of ways. This will provide new information on the general features of proteins in the hsp90 family and should reveal whether TRAP-1 has very distinct functions that overlap with hsp90. These studies should provide important nw information on cytoplasmic chaperoning in general plus a much clearer definition of the functions of hsp90.