The yeast vacuole strongly resembles an animal cell lysosome, for it is an acid compartment, contains a set of hydrolases, is the final destination of ligands taken up by fluid phase endocytosis and receptormediated endocytosis. Since yeast is amenable to gentical, biochemical and molecular analyses, it affords an excellent model system for studies of the function and assembly of this important organelle. The overall goals of this research are to understand the role of the set of vacuolar hydrolases in the metabolism and differentiation of yeast, to understand how these enzyme activities are generated, to understand how the activities of these enzymes are regulated and integrated into cellular function and to understand the regulatory circuits that govern the genes for these enzymes.
Our specific aims for this period are 1) to genetically dissect the role that protease A plays in effecting processing of hydrolase precursors 2) to delineate the processing pathway for the protease B precursor and test whether our proposed pathway is correct and 3) to analyze the regulatory circuit that effects carbon catabolite repression and expression of the set of vacuolar proteases. To achieve these goals we will isolate and characterize mutants obtained by in vitro mutagenesis of the cloned protease. A structural gene and relate the phenotypic changes to the sequenced mutations. We will delineate the protease B processing pathway by kinetic experiments using antibodies and by engineering derivatives of the structural gene that should be exempt from a processing requirement. To analyze regulation we will place PRB1 upstream regulatory sequences upstream of the SUC2 and lacZ structural genes and use these hybrid genes to isolate regulatory mutations and to define cis regulatory sequences of PRB1.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018090-18
Application #
3225937
Study Section
Genetics Study Section (GEN)
Project Start
1977-08-01
Project End
1992-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
18
Fiscal Year
1991
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
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Naik, R R; Nebes, V; Jones, E W (1997) Regulation of the proteinase B structural gene PRB1 in Saccharomyces cerevisiae. J Bacteriol 179:1469-74
Manolson, M F; Wu, B; Proteau, D et al. (1994) STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. J Biol Chem 269:14064-74
Bachhawat, A K; Manolson, M F; Murdock, D G et al. (1993) The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H(+)-ATPase. Yeast 9:175-84
Woolford, C A; Noble, J A; Garman, J D et al. (1993) Phenotypic analysis of proteinase A mutants. Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae. J Biol Chem 268:8990-8
Manolson, M F; Proteau, D; Jones, E W (1992) Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases. J Exp Biol 172:105-12
Nebes, V L; Jones, E W (1992) N-linked glycosylation of proteinase B precursors of the yeast Saccharomyces cerevisiae is not required for proper targeting or processing of the enzyme. Yeast 8:353-9
Preston, R A; Reinagel, P S; Jones, E W (1992) Genes required for vacuolar acidity in Saccharomyces cerevisiae. Genetics 131:551-8
Manolson, M F; Proteau, D; Preston, R A et al. (1992) The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase. J Biol Chem 267:14294-303
Preston, R A; Manolson, M F; Becherer, K et al. (1991) Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae. Mol Cell Biol 11:5801-12

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