COOPERATIVE ACTION OF HSP70, HSP90, AND DNAJ PROTEINS IN PROTEIN RENATURATION

The proteins required for the repair of damaged proteins in the eukary otic cytoplasm remain largely uncharacterized. The renaturation of the rmally denatured firefly luciferase readily occurs in rabbit reticuloc yte lysate by an ATP-dependent process. Earlier studies had shown that this chaperoning activity could be reconstituted, in part, using puri fied preparations of hsp70 and hsp90. We have extended the description of this system by clarifying the importance of hsp70 and hsp90 and ha ve tested for additional factors that enhance renaturation. Using muta nt hsp70 proteins, we have shown that hsp70 is required for luciferase renaturation. We have also found that hsp70 and hsp90 preparations pu rified by common procedures were contaminated with low levels of DnaJ proteins that are essential for the renaturing activity. When hsp70 an d hsp90 preparations free of DnaJ proteins are used, the system must b e supplemented with a DnaJ protein to obtain renaturation activity. Th e yeast DnaJ protein, YDJ-1, was found to be very effective for this p urpose. Although significant renaturation can occur with only hsp70 an d DnaJ proteins, hsp90 also contributes to the renaturation process, b oth in the complex environment of reticulocyte lysate and in a purifie d system. However, using highly purified hsp90 and geldanamycin, a spe cific inhibitor of hsp90 function, we have determined that hsp90 is no t an essential component of the renaturation system. The contribution of hsp90 to renaturation is only partially blocked by geldanamycin, su ggesting that this protein may influence activity in more than one way . This study indicates that hsp70, hsp90, and DnaJ proteins function c ooperatively to renature damaged proteins in the eukaryotic cytoplasm and provides a framework by which additional components can be identif ied and individual chaperone contributions can be investigated.