Genetic, molecular and biochemical studies of vacuole biogenesis and maintenance in the yeast Saccaromyces cerevisiae

Citation

Robinson, Jane Suzanna (1991) Genetic, molecular and biochemical studies of vacuole biogenesis and maintenance in the yeast Saccaromyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/zfzc-0035. https://resolver.caltech.edu/CaltechTHESIS:04112011-112805564

Abstract

Using a selection for spontaneous mutants that mislocalize a vacuolar CPY-Inv fusion protein to the cell surface, 505 Saccharomyces cerevisiae mutants with defects in protein sorting were identified. Seventeen of these mutants were dominant; the others defined 25 new vps (for vacuolar protein sorting) complementation groups. Alleles of each vps complementation group exhibit defects in the targeting and final processing of soluble vacuolar enzymes (CPY, PrA and PrB). Two of the genes, VPS17 and VPS15, map to ChXV and ChII, respectively. The vpsll, vps16, vps18 and vps33 mutants exhibit morphological defects, their cells containing debris of a membranous nature and highly abnormal vacuole remnants. Intercrosses with other vacuole function-defective mutants (vpl, pep, sip and end), revealed genetic overlaps. It is evident that more than 50 gene products are involved in biogenesis and maintenance of the yeast vacuole. Alleles of 7 of the vps complementation groups are temperature-sensitive for vegetative cell growth at 37°C, and this recessive, Ts phenotype cosegregated with the vps defect in each case. This easily complemented phenotype has facilitated cloning of six of the genes. The VPS18 gene was chosen for further studies. A plasmid complementing the Ts growth defect of vps18-1 was isolated and shown by integrative mapping to carry DNA from the VPS18 locus. Yeast strains with a deletion of the entire VPS18 coding region (Δvps18), are viable and exhibit the same phenotypes as vps18-1. Δvps18, α strains have smaller α-factor halos on sst2,a lawns than do VPS18, α strains. Immunoprecipitation of α-factor indicated that it is secreted from the Δvps18 mutant in precursor form. Several of the other severely defective vps mutants also show this α-factor processing defect. DNA sequencing of VPs18 showed an open reading frame encoding a 918aa protein, hydrophilic in nature. The protein sequence revealed a zinc finger like, cysteine-rich motif in its C-terminal region. A synthetic mutant with a cysteine to serine alteration has a temperature-conditional CPY sorting defect with very rapid onset. Therefore, Vps18p may be a zinc-binding protein, directly necessary for correct vacuolar protein sorting and proper functioning of the Golgi compartment that contains Kex2p.

Thesis Files