A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization.

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn2+ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.

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

In the yeast Saccharomyces 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 cloned VPS9 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 degrees C. Electron microscopic examination of delta vps9 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 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 Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-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 binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.