Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis

Citation

Banta, Lois Margaret (1990) Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/TZHF-3J73. https://resolver.caltech.edu/CaltechETD:etd-06112007-104332

Abstract

The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment containing a number of hydrolytic glycoproteins including carboxypeptidase Y (CPY), proteinase A (PrA) and proteinase B (PrB). A gene fusion-based selection scheme was utilized to isolate ~600 mutants defective in the localization and processing of vacuolar proteins. These vacuolar protein sorting (vps) mutants define >33 complementation groups and exhibit hybrid protein-independent defects in the sorting of CPY, PrA, and PrB. Light and electron microscopic analyses of the vacuole morphology revealed three distinct classes of vps mutants. The class A mutants (26 complementation groups) contain 1-3 large vacuoles that resemble those of the parental strain. One class A mutant is sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a role for vacuolar pH in protein sorting, perturbation of vacuole acidification resulted in the missorting and secretion of CPY and PrA in wild-type cells. Mutants in the three class B complementation groups exhibit a fragmented vacuole morphology. The class C vps mutants (four complementation groups) lack any compartment resembling a wild-type vacuole, but accumulate vesicles and other membranous structures. Many class C strains exhibit genetically linked defects including temperature-sensitivity and sensitivity to osmotic stress. Unlike other vps mutants, these mutants secrete up to 50% of a vacuolar membrane marker enzyme. The gene defined by one class C mutant, vps33, has been cloned. The predicted VPS33 gene product is hydrophilic and shares sequence similarity with a family of ATP-binding proteins. Disruption of VPS33 is not lethal but results in temperature-sensitive growth. Vps33p-specific antisera recognize a cytosolic protein of ~75 kD. One temperature-sensitive vps33 mutant carrying a missense mutation contains apparently normal vacuoles at the permissive temperature, but lacks vacuoles specifically in the bud at the nonpermissive temperature. We propose that the abnormalities in vacuole morphology and inheritance in vps33 mutants are a consequence of a primary defect in Golgi-to-vacuole protein delivery. A second VPS gene, VPS28, has also ben cloned. Our data suggest that the VPS28 gene product only indirectly affects vacuole protein sorting, but may function in a late protein modification process.

Thesis Files