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

Abstract

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.

Catabolite Degradation of Fructose-1,6-bisphosphatase in the YeastSaccharomyces cerevisiae: A Genome-wide Screen Identifies Eight NovelGIDGenes and Indicates the Existence of Two Degradation Pathways

“…Our pulse-chase analysis of FBPase degradation under conditions allowing proteasome-dependent inactivation showed wild-type-like kinetics of FBPase breakdown in vid22⌬, vid27⌬, and cpr1⌬ cells (Figure 9). Also a deletion of Ssa2p, which was shown to affect vacuolar FBPase degradation (Brown et al, 2000), did not alter proteasome-dependent degradation of FBPase (our unpublished data). This suggests, that catabolite inactivation of FBPase might be mediated by two distinct pathways, which share some components such as Gid1p/ Vid30p, Gid4p/Vid24p, and Gid5p/Vid28p.…”

The Type 1 Phosphatase Reg1p-Glc7p Is Required for the Glucose-induced Degradation of Fructose-1,6-bisphosphatase in the Vacuole

“…Differential Centrifugation-Differential centrifugation experiments were performed as described (16,18).…”

“…To determine whether Reg1p plays a role in Vid vesicle formation, we performed a differential centrifugation analysis using Vid24p as a specific marker of Vid vesicles. Cells were starved and shifted to glucose-rich media for 30 min, and cell extracts were subjected to differential centrifugation as described previously (16,18). As is shown in Fig.…”

“…The formation of Vid vesicles requires the ubiquitin-conjugating enzyme Ubc1p (17). FBPase import into Vid vesicles is dependent on the heat shock protein Ssa2p (18), cyclophilin A (19), and Vid22p (20). Following FBPase import into Vid vesicles, this protein is then delivered to the vacuole for degradation via a Vid24p-, Ypt7p-, and SNARE-mediated process (21).…”

“…These include the molecular chaperone Ssa2p (18), the immunophilin cyclophilin A (19), and a plasma membrane protein Vid22p (20). Likewise, Vid24p (16) and various members of the SNARE and homotypic fusion vacuole protein sorting families of proteins (21) play roles in this process.…”