A NOVEL SEC18P NSF-DEPENDENT COMPLEX REQUIRED FOR GOLGI-TO-ENDOSOME TRANSPORT IN YEAST/

The vacuolar protein-sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of proteins from the trans-Golgi to the vacuole via a prevacuolar endosome compartment. Mutations in class D vacuolar protein-sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of vacuolar proteins. Temperature-s ensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mu tations in VAC1, a gene implicated in vacuole inheritance and vacuolar protein sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway. The sequence of Vac1p co ntains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif. Site-directed mutations in the carboxyl-terminal RING motif strongly affected vacuolar protein sorting. Vac1p was foun d to be tightly associated with membranes as a monomer and in a large SDS-resistant complex. By using Pep12p affinity chromatography, we fou nd that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p. Consistent with a functi onal role for this complex in vacuolar protein sorting, double pep12(t sf)vac1(tsf) and pep12(tsf) vps45(tsf) mutants exhibited synthetic Vps (-) phenotypes, the tsf phenotype of the vac1(tsf) mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep1 2 allele in a sec18-1 strain resulted in a severe synthetic growth def ect that was rescued by deletion of PEP12 or VAC1, and subcellular fra ctionation of vac1 Delta cells revealed a striking change in the fract ionation of Pep12p and Vps21p, a rab family GTPase required for vacuol ar protein sorting. The functions of Pep12p, Vps45p, and Vps21p indica te that key aspects of Golgi-to-endosome trafficking are similar to ot her vesicle-mediated transport steps, although the role of Vac1p sugge sts that there are also novel components of the VPS pathway.