Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.
vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.
Paravicini
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G
Division of Biology, California Institute of Technology, Pasadena 91125.
Horazdovsky
B F
BF
Emr
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SD
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis; D001483:Base Sequence; D002268:Carboxypeptidases / Q000096:biosynthesis; D002352:Carrier Proteins; D043402:Cathepsin A; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006867:Hydrolases / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000096:biosynthesis; D013347:Subcellular Fractions / Q000737:chemistry; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; C076106:VPS35 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D006867:Hydrolases; D000469:Alkaline Phosphatase; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D012697:Serine Endopeptidases; C023302:yeast proteinase B; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases