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Published online 10 July 2000. doi:10.1083/jcb.150.1.65
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© The Rockefeller University Press, 0021-9525/2000//65 $5.00
The Journal of Cell Biology, Volume 150, Number 1, , 2000 65-76

Original Article

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



C. Randell Browna, Jameson A. McCanna, and Hui-Ling Chianga

a Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033
Department of Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033.(717) 531-7667(717) 531-0859

crb13{at}psu.edu

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}ssa4 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.

Key Words: molecular chaperones • vesicle trafficking • • protein degradation • Ssa2p yeast vacuole

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AP1, aminopeptidase 1; CPY, carboxypeptidase Y; FBPase, fructose-1,6-bisphosphatase; hsc, heat shock cognate protein; Hsp, heat shock proteins; Vid, vacuole import and degradation; VPS, vacuolar protein sorting; WT, wild-type.


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