The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

Ssal/2p, members of one of the yeast cytoso- lic hsp70 subfamilies, have been implicated in the trans- location of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunode- pleting Ssal/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post- translational translocation of prepro-ot-factor. Deple- tion of Ssal/2p had no effect on the efficiency of trans- location in this in vitro assay. The system was used to examine the effect of the absence of Ssal/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured lu- ciferase. Depletion of Ssal/2p had no effect on the abil- ity of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding ac- tivity of Ssal/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function spe- cific to Ssal/2p, apart from other cytosolic hsp70s. They also suggest that Ssal/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssal/2p activity on their proposed role in the process of translocation are discussed.