Our laboratory is studying the 70-kDa heat shock proteins which act as molecular chaperones, that is, fold and unfold proteins in numerous processes in vivo. We have taken several approaches to understanding the mechanism of action of these proteins. First, we are carrying out a detailed investigation of the mechanism of action of hsp70 in uncoating bovine brain clathrin-coated vesicles and synthetic clathrin baskets. We previously discovered that a 100 kDa protein cofactor is required for the uncoating process to occur. We have now demonstrated that for clathrin baskets prepared with various types of assembly proteins, hsp70 always cause a distinctive time course of uncoating consisting of a rapid initial burst of uncoating followed by slow steady-state uncoating. We have also demonstrated that the initial burst of uncoating requires the presence of 100 kDa cofactor as does the initial burst of ATPase activity which accompanies the initial burst of uncoating. The activation of hsp70 by clathrin baskets at pH 6 where uncoating does not take place also requires the presence of cofactor. Therefore there is a highly specific interaction between hsp70, cofactor and clathrin baskets. Second, we have found that the new 20 kDa assembly protein which we discovered is myelin basic protein (MBP) and have shown that the ability of MBP to induce formation of clathrin baskets is specific and does not occur with other basic proteins. MBP is, by far, the smallest assembly protein yet discovered and therefore it may help us understand the role of assembly proteins in clathrin polymerization. Third, we are investigating the properties of polymerized hsp70; in ATP hsp70 is monomeric but in ADP it forms dimers and trimers. We have found that clathrin and peptide substrates bind only to monomeric hsp70 and not to polymerized hsp70. Therefore protein substrates shift the equilibrium between monomeric and polymerized hsp70 toward monomeric hsp70. On the other hand, we have found that catalytic amounts of dnaJ, a cofactor which interacts with hsp70 in E Coli, induces the polymerization of hsp70 into very large polymers. However, this effect only occurs in ATP and is reversed in ADP. We do not yet understand how this effect of dnaJ is related to reports that it presents various substrates to hsp70. Finally, we have succeeded in expressing large amounts of human stress hsp70 in E Coli, have found that the recombinant protein uncoats clathrin coated vesicles and are studying the effect of site-directed mutagenesis on the biochemical properties of this recombinant protein.