The vacuolar ATPase is a large complex enzyme found in the endomembrane system of eucaryotic cells. It functions to acidify the interior of several organelles, e.g. lysosomes and secretory vesicles, and generates an electrochemical gradient to drive transport across the organellar membrane. We have characterized the ATPase in vacuolar membranes from Neurospora and have isolated the genes that encode three of the major subunits. The enzyme appears to have at least six additional subunits. The goals of this proposal are: (1) To isolate the other polypeptides which copurify with ATPase activity, obtain partial amino acid sequence, construct oligonucleotide probes, and isolate the corresponding genes. (2) To use genetics to determine the number of genes essential for synthesizing the vacuolar ATPase. (3) To examine the oligomeric structure of the ATPase by electron microscopy, using antibodies to map the location of several polypeptides. (4) To examine the chromosomal location, exon/intron structure, and the abundance and size of transcripts of vacuolar ATPase genes. Investigation of this enzyme is important because of the central role it plays in the function of many organelles. The uncoupling of ligands and receptors in endosomes, the packaging of neurotransmitters in secretory vesicles, and the hydrolytic action of enzymes in lysosomes all depend on the activity of the vacuolar ATPase. Furthermore, a vacuolar-type ATPase has been reported on the plasma membranes of osteoclasts, where it plays an important role in bone remodeling, and on the plasma membrane of some renal cells, where it plays a role in urinary acidification.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028703-15
Application #
2175253
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1981-02-01
Project End
1996-02-29
Budget Start
1995-03-01
Budget End
1996-02-29
Support Year
15
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California Santa Cruz
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Chavez, Christopher; Bowman, Emma Jean; Reidling, Jack C et al. (2006) Analysis of strains with mutations in six genes encoding subunits of the V-ATPase: eukaryotes differ in the composition of the V0 sector of the enzyme. J Biol Chem 281:27052-62
Shen, Ruichao; Lin, Cheng Ting; Bowman, Emma Jean et al. (2003) Lobatamide C: total synthesis, stereochemical assignment, preparation of simplified analogues, and V-ATPase inhibition studies. J Am Chem Soc 125:7889-901
Shen, Ruichao; Lin, Cheng Ting; Bowman, Emma Jean et al. (2002) Synthesis and V-ATPase inhibition of simplified lobatamide analogues. Org Lett 4:3103-6
Bowman, Barry J; Bowman, Emma Jean (2002) Mutations in subunit C of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site. J Biol Chem 277:3965-72
Boyd, M R; Farina, C; Belfiore, P et al. (2001) Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-atpases. J Pharmacol Exp Ther 297:114-20
Berg, O G; Gelb, M H; Tsai, M D et al. (2001) Interfacial enzymology: the secreted phospholipase A(2)-paradigm. Chem Rev 101:2613-54
Margolis-Clark, E; Hunt, I; Espinosa, S et al. (2001) Identification of the gene at the pmg locus, encoding system II, the general amino acid transporter in Neurospora crassa. Fungal Genet Biol 33:127-35
Tenney, K; Hunt, I; Sweigard, J et al. (2000) Hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores. Fungal Genet Biol 31:205-17
Bowman, E J; Bowman, B J (2000) Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A. J Exp Biol 203:97-106
Hunt, I E; Bowman, B J (1997) The intriguing evolution of the ""b"" and ""G"" subunits in F-type and V-type ATPases: isolation of the vma-10 gene from Neurospora crassa. J Bioenerg Biomembr 29:533-40

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