| In the secretory pathway, all newly synthesized proteins enter the endoplasmic reticulum, where they are properly folded. From here, correctly folded proteins are packaged into vesicles that transport them to their final destination. This thesis investigates aspects of both protein folding and transport. It examines the role of Vps33p and Vps16p in vacuolar and endosomal protein trafficking, and unravels the function of an essential protein, Pbn1p in ER protein folding and quality control in Saccharomyces cerevisiae.; The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here I report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. I also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other Class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. At the same time, I also demonstrate that the SM proteins Vps33p and Vps45p are not interchangeable. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.; My studies on Vps16p suggest that this protein acts at both the endosome and the vacuole of yeast, as opposed to the previously postulated role at the vacuole alone. I also demonstrate physical interaction between Vps16p and Vps8p, probably at the late endosome. The multiple roles of Vps16p explain the 'no vacuole' phenotype of vps16Delta cells.; PBN1 was isolated as a gene involved in Protease B (PrB) processing in Saccharomyces cerevisiae. It encodes for an ER localized, type I membrane protein and is essential for cell viability. (Abstract shortened by UMI.)... |
