A Novel RING Finger Protein, Vps8p, Functionally Interacts with the Small GTPase, Vps21p, to Facilitate Soluble Vacuolar Protein Localization

Bruce F Horazdovsky,Christopher R Cowles,Peg Mustol,Michael Holmes,Scott D Emr

doi:10.1074/jbc.271.52.33607

Abstract

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.

Highlights

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants that missort and secrete soluble vacuolar hydrolases
In wild-type cells, the fusion proteins were correctly delivered to the vacuole due to the vacuolar protein sorting information found in the carboxypeptidase Y (CPY) portion of the molecule
One mutant identified using this technique was vps8. vps8 mutant cells secreted the vast majority of CPYinvertase fusion proteins and this mislocalization phenotype was exploited to clone the wild-type VPS8 locus. vps8 mutant cells (SEY8 –3) carrying a CPY-invertase gene fusion were transformed with a yeast genomic library (CEN LEU2)

Summary

TABLE I Strains used in this study

MAT␣ leu112 ura his3-⌬200 trp1-⌬901 lys801 suc2-⌬9 MATa leu112 ura his3-⌬200 trp1-⌬90 ade101 suc2-⌬9 SEY6210 leu2–3, 112::pBHY11(CPY-Inv LEU2) SEY6211 leu2–3, 112::pBHY11(CPY-Inv LEU2) SEY6210, vps SEY6211, vps SEY8–30 leu2–3, 112::pBHY11(CPY-Inv LEU2) BHY11 VPS8-TRP1 SEY6210 vps8⌬1::HIS3 vps8⌬1::HIS3 sec vps8⌬1::HIS3 end MATa leu122 ura sec MATa end his ura bar. Many of the gene products affected in the class D mutants have been cloned and their gene products characterized [21,22,23,24,25,26,27,28,29,30] Included in this group are several gene products (Vps21p, Vps45p, Pep12p) that have been implicated in vesicle targeting and/or fusion events in the Golgi to endosome step of this transport pathway. A novel gene product affected in a class D mutant, Vps9p, has been implicated in this Golgi-to-endosome vesicle targeting/fusion reaction as a potential modulator of Vps21p function [28]. Overexpression of Vps21p can partially bypass Vps8p function, suggesting that Vps8p may be a positive modulator of Vps21p function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION