Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles.
Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete vacuolar hydrolases. A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble vacuolar proteins, yet maintain a near-normal vacuole structure. Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a protein family that includes the yeast proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms. These proteins are thought to participate in vesicle-mediated protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes. Significantly, this fraction 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 vesicles in the vacuolar protein sorting pathway.
Cowles
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CR
Division of Cellular and Molecular Medicine, University of California, San Diego, School of Medicine, La Jolla 92093-0668.
Emr
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Horazdovsky
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015415:Biomarkers; D002451:Cell Compartmentation / Q000502:physiology*; D002458:Cell Fractionation; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal / Q000235:genetics; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D016296:Mutagenesis; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins
D015415:Biomarkers; D005656:Fungal Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C497931:SEC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins