A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeastSaccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have clonedVPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38&C. Electron microscopic examination of Dvps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele ofvps9was 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 Vps9p functionisdirectlyrequiredintheVPSpathway.AntibodiesraisedagainstVps9pimmunoprecipitatearare52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase bindinghomologydomainandsuggestthatitdefinesafamilyofproteinsthatbindmonomericGTPases.Vps9p may bind and serve as an effector of a rab GTPase, like Vps21p, required for vacuolar protein sorting. In eukaryotic cells, the identities and functions of organelles are determined largely by their unique repertoire of resident proteins. Proper intracellular organization, therefore, requires sorting and transport of proteins from their common site of synthesis to the appropriate resident organelle. Studies of vacuolar protein biosynthesis in Saccharomyces cerevisiae have provided an extremely useful experimental system for studying protein localization. Genetic selections for mutants that mislocalize vacuolar proteins (vacuolar protein sorting [vps] mutants) have led to the identification of more than 40 genes required in this process (2, 34‐36). Ongoing analysis of VPS genes and vps mutants continues to provide considerable insight into mechanisms of protein sorting and intracellular trafficking of macromolecules. The vacuole ofS. cerevisiaeis an acidic organelle containing large amounts of degradative enzymes and is therefore analogous to the lysosome of animal cells (21). Of all vacuolar proteins, the biosynthesis of carboxypeptidase Y (CPY) is best understood, and it serves as a prototype in models of vacuolar protein sorting. CPY is an abundant, soluble, vacuolar protease whose trafficking status can be easily assessed by identification of electrophoretically distinct forms generated during its biosynthesis (44). CPY enters the secretory pathway at the endoplasmic reticulum (ER), where core glycosyl chains are added (forming p1CPY [67 kDa]), and then transits through the Golgi apparatus, where it is further modified by glycosylation (forming p2CPY [69 kDa]). The vacuolar targeting signal of CPY, a short stretch of amino acids found in the pro seg