The yeast vacuole strongly resembles an animal cell lysosome, for it is an acid compartment, contains a set of hydrolases, and is the final destination of ligands taken up by fluid phase endocytosis and receptor-medicated endocytosis. Since yeast is amenable to genetical, biochemical, molecular and cell biological analyses, it affords an excellent model system for studies of the function and assembly of this organelle. The overall goals of this research are to determine how the lysosome-like vacuole of the yeast Saccharomyces is assembled, made functional, and its contained enzymatic activities activated.
Our specific aims for this period are 1) to genetically dissect the pathway of enzyme processing and localization for enzymes of the vacuole 2) to genetically dissect the mechanism of acidification of the vacuole and thereby determine whether acid pH acts to trigger autoactivation of proprotease A to initiate the proteolytic activation cycle and 3) to begin a genetic analysis of the mechanism of formation and assembly of the vacuole, per se. To achieve these goals we will isolate and characterize genes defined by pleiotropic mutations that eliminate vacuole hydrolases but not secreted proteins and try to discern from studies of the genes and gene products how the vacuolar enzymes are activated and targeted. We will isolate and characterize mutants defective in acidification of the vacuole using dyes whose fluorescence is pH-dependent. The genes defined by these mutations will be isolated to determine whether the vacuolar ATPase is the acidification agent and whether proteins other than the ATPase are involved. Mutants, especially conditional ones, that lack vacuoles will be isolated to facilitate identification of genes and gene products required for integrity, stability and formation of the vacuole. The corresponding genes will be isolated and studied.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029713-09
Application #
3277346
Study Section
Genetics Study Section (GEN)
Project Start
1981-07-01
Project End
1993-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Subramanian, Shoba; Woolford, Carol A; Drill, Emily et al. (2006) Pbn1p: an essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast. Proc Natl Acad Sci U S A 103:939-44
Subramanian, Shoba; Woolford, Carol A; Jones, Elizabeth W (2004) The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae. Mol Biol Cell 15:2593-605
Chang, Hak J; Jones, Elizabeth W; Henry, Susan A (2002) Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae. Genetics 162:29-43
Srivastava, A; Woolford, C A; Jones, E W (2000) Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae. Genetics 156:105-22
Coury, L A; Hiller, M; Mathai, J C et al. (1999) Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion. J Bacteriol 181:4437-40
Srivastava, A; Jones, E W (1998) Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases. Genetics 148:85-98
Woolford, C A; Bounoutas, G S; Frew, S E et al. (1998) Genetic interaction with vps8-200 allows partial suppression of the vestigial vacuole phenotype caused by a pep5 mutation in Saccharomyces cerevisiae. Genetics 148:71-83
Webb, G C; Zhang, J; Garlow, S J et al. (1997) Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome. Mol Biol Cell 8:871-95
Webb, G C; Hoedt, M; Poole, L J et al. (1997) Genetic interactions between a pep7 mutation and the PEP12 and VPS45 genes: evidence for a novel SNARE component in transport between the Saccharomyces cerevisiae Golgi complex and endosome. Genetics 147:467-78
Becherer, K A; Rieder, S E; Emr, S D et al. (1996) Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol Biol Cell 7:579-94

Showing the most recent 10 out of 33 publications