ENDOSOME TO GOLGI RETRIEVAL OF THE VACUOLAR PROTEIN SORTING RECEPTOR,VPS10P, REQUIRES THE FUNCTION OF THE VPS29, VPS30, AND VPS35 GENE-PRODUCTS

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selecti ve protein sorting defect in which the vacuolar protein carboxypeptida se Y (CPY) is missorted and secreted from the cell, while other solubl e vacuolar hydrolases like proteinase A (PrA) are delivered to the vac uole. This phenotype is similar to that seen in cells with mutations i n the previously characterized VPS10 and VPS35 genes, Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for sol uble vacuolar hydrolases like CPY and PrA, while Vps35p is a periphera l membrane protein which cofractionates with membranes enriched in Vps 10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet giv e any clues to the functions of their products, Each is predicted to e ncode a hydrophilic protein with homologues in the human and C. elegan s genomes, Interestingly, mutations in the VPS29. VPS30, or VPS35 gene s change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The rout e that Vps10p takes to reach the vacuole in a vps35 mutant does not de pend upon Sec1p mediated arrival at the plasma membrane but does requi re the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A tempe rature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to misloca lize to a vacuolar membrane fraction at the nonpermissive temperature, Vps35p continues to cofractionate with Vps10p in vps29 mutants, sugge sting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the pre vacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydr olase sorting.