YEAST VPS45P IS A SEC1P-LIKE PROTEIN REQUIRED FOR THE CONSUMPTION OF VACUOLE-TARGETED, POST-GOLGI TRANSPORT VESICLES

Over 45 VPS genes (vacuolar protein sorting) in Saccharomyces cerevisi ae are necessary for the correct sorting and delivery of vacuolar hydr olases. Yeast strains carrying mutations in a subset of these VPS gene s (class D vps mutants) are also defective in the segregation of vacuo lar material into the developing daughter cell and are morphologically characterized by having large central vacuoles. The class D VPS gene products, which include a Rab5 homologne (VPS21/YPT51) and a syntaxin homologue (PEP12/VPS6), have been proposed to function together at a p articular step along the vacuolar protein sorting pathway. We have clo ned another class D VPS gene, VPS45, which is homologous to a growing family of genes that encode Sec1p-like proteins. Vps45p is predicted t o be a hydrophilic protein of 577 amino acids with a molecular mass of 67 kDa. Fractionation studies show that Vps45p is a peripheral membra ne protein that cofractionates with Golgi-like membranes, consistent w ith Vps45p functioning in membrane traffic between the Golgi and the v acuole. Using a temperature sensitive allele of VPS45, we show that in activation of Vps45p causes the rapid accumulation of small (40-60 nm) vesicles and secretion of the vacuolar hydrolase carboxypeptidase Y. Because the entire yeast secretory pathway is functional after the tem perature induced inactivation of Vps45p, we conclude that the accumula ted vesicles represent transport intermediates between the Golgi and t he vacuole.