STRUCTURE AND FUNCTION OF THE VACUOLAR ATPASE

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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.