vps17 mutants missort and secrete several vacuolar hydrolases. To analyze the role of the VPS17 gene in vacuolar protein delivery, we have cloned this gene by complementation of the vacuolar protein sorting defects of a vps17-5 mutant. Disruption of the VPS17 gene had no effect on the viability of haploid yeast cells, although they show an obvious defect in vacuolar morphology. vps17-disrupted cells contain numerous small vacuole-like compartments and also exhibit a severe defect in the sorting of carboxypeptidase Y (CPY), a soluble vacuolar hydrolase. 95% of CPY is missorted and secreted from the mutant cells. Vacuolar sorting of two other soluble hydrolases, proteinase A and proteinase B, is also affected, but to a lesser extent. Delivery and maturation of the vacuolar membrane protein alkaline phosphatase does not appear to be affected in a delta vps17 strain. The DNA sequence of the VPS17 clone indicates that the gene encodes a 551-amino-acid protein with a calculated molecular mass of 63.1 kDa. The protein sequence is hydrophilic and contains no obvious N-terminal signal sequence or hydrophobic membrane-spanning domains, indicating that the Vps17p does not enter the secretory pathway. Using a Vps17p-specific polyclonal antiserum, we have demonstrated that the Vps17 protein is not modified with N-linked carbohydrates at any of its four potential N-linked glycosylation sites. The Vps17 protein, however, fractionates to a particulate fraction after centrifugation at 100,000 x g. Vps17p can be released from this particulate fraction by treatment with either Triton X-100 or urea, indicating that the Vps17p is peripherally associated with a crude membrane fraction. Based on these results, we propose that the Vps17p functions on the cytoplasmic surface of some intracellular organelle, possibly the Golgi complex or an intermediate in Golgi to vacuole transport, to facilitate the sorting and delivery of soluble vacuolar hydrolases. Vacuolar membrane protein traffic, however, appears to occur by a mechanism that is independent of Vps17p function.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|