The heat shock protein Ssa2p is required for import of fructose-1,6-bisphosphatase into Vid vesicles

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Delta ssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Delta ssa1, Delta ssa3, or Delta ssn4 strains. Likewise, in vitro import was impaired for the Delta ssa2 strain, but not for the other Delta ssa strains. The cytosol was identified as the site of the Delta ssa2 defect; Delta ssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Delta ssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Delta ssa2 Vid vesicles, providing Delta ssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.