The Heat Shock Protein Ssa2p Is Required for Import of Fructose-1,6-Bisphosphatase into Vid Vesicles

• Published in: The Journal of cell biology 2000-07, Vol.150 (1), p.65-76
• Main Authors: Brown, C. Randell, McCann, Jameson A., Chiang, Hui-Ling
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Antibodies; Biological Transport; Carrier proteins; Carrier Proteins - metabolism; Cell-Free System - enzymology; Cells; Centrifugation; Cytosol; Cytosol - enzymology; Fructose-Bisphosphatase - metabolism; Fungal Proteins - metabolism; Heat; HSP70 Heat-Shock Proteins - metabolism; Imports; Molecules; Organelles; Original; Ports; Proteins; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vacuoles; Vacuoles - metabolism; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.150.1.65

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 Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.