A SORTING NEXIN-1 HOMOLOG, VPS5P, FORMS A COMPLEX WITH VPS17P AND IS REQUIRED FOR RECYCLING THE VACUOLAR PROTEIN-SORTING RECEPTOR

A number of the Saccharomyces cerevisiae vacuolar protein-sorting (vps ) mutants exhibit an altered vacuolar morphology. Unlike wild-type cel ls that contain 1-3 large vacuolar structures, the class B vps5 and vp s17 mutant cells contain 10-20 smaller vacuole-like compartments. To e xplore the role of these VPS gene products in vacuole biogenesis, we c loned and sequenced VPS5 and characterized its protein product. The VP S5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that shows significant sequence homology with mammalian sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic protein that is phosphorylated specifica lly on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and vacuolar membranes. This associati on was found to be dependent on the presence of another class B VPS ge ne product, Vps17p. Biochemical cross-linking studies demonstrated tha t Vps5p and Vps17p physically interact. Gene disruption experiments sh ow that the VPS5 gene product is not essential for cell viability; how ever, cells carrying the null allele contain fragmented vacuoles and e xhibit defects in vacuolar protein-sorting similar to vps17 null mutan ts. More than 95% of carboxypeptidase Y is secreted from these cells i n its Golgi-modified p2 precursor form. Additionally, the Vps10p vacuo lar protein-sorting receptor is mislocalized to the vacuole in vps5 mu tant cells. On the basis of these and other observations, we propose t hat the Vps5p/Vps17p protein complex may participate in the intracellu lar trafficking of the Vps10p-sorting receptor, as well as other late- Golgi proteins.