MUTATIONS IN THE VPS45 GENE, A SEC1 HOMOLOG, RESULT IN VACUOLAR PROTEIN SORTING DEFECTS AND ACCUMULATION OF MEMBRANE-VESICLES

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisi ae have uncovered a large number of mutants (vps) that missort and sec rete vacuolar hydrolases, A small subset of vps mutants exhibit a temp erature-conditional growth phenotype and show a severe defect in the l ocalization of soluble vacuolar proteins, yet maintain a near-normal v acuole structure, Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been fo und to encode a member of a protein family that includes the yeast pro teins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from o ther eukaryotic organisms, These proteins are thought to participate i n vesicle-mediated protein transport events, Polyclonal antiserum rais ed against a TrpE-Vps45 fusion protein specifically detects a stable 6 7 kDa protein in labeled yeast cell extracts, Subcellular fractionatio n studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport ve sicles and, potentially, endosomal membranes, Significantly, this frac tion lacks ER, vacuole and plasma membranes, Overexpression of Vps45p saturates the sites with which Vps45p associates, A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters, This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicl es in the vacuolar protein sorting pathway.