A YEAST PROTEIN RELATED TO A MAMMALIAN RAS-BINDING PROTEIN, VPS9P, ISREQUIRED FOR LOCALIZATION OF VACUOLAR PROTEINS

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein s orting (WS) genes result in secretion of proteins normally localized t o the vacuole, Characterization of the VPS pathway has provided consid erable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of th e vacuolar protein sorting defect of vps9 cells, characterized its gen e product, and investigated its role in vacuolar protein sorting. Cell s with a vps9 disruption exhibit severe vacuolar protein sorting defec ts and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of Delta vps9 cells revealed the appearance o f novel reticular membrane structures as well as an accumulation of 40 - to 50-nm-diameter vesicles, suggesting that Vps9p may be required fo r the consumption of transport vesicles containing vacuolar protein pr ecursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature , newly synthesized carboxypeptidase Y was secreted, indicating that V ps9p function is directly required in the VPS pathway. Antibodies rais ed against Vps9p immunoprecipitate a rare 52-kDa protein that fraction ates with cytosolic proteins following cell lysis and centrifugation, Analysis of the VPS9 DNA sequence predicts that Vps9p is related to hu man proteins that bind Ras and negatively regulate Res-mediated signal ing. We term the related regions of Vps9p and these Ras-binding protei ns a GTPase binding homology domain and suggest that it defines a fami ly of proteins that bind monomeric GTPases. Vps9p may bind and serve a s an effector of a rab GTPase, like Vps21p, required for vacuolar prot ein sorting.