We are using three approaches to investigate the vacuolar ATPase, V-ATPase, from Neurospora crassa. (1) Examination in the electron microscope shows the enzyme has a 'ball and stalk' structure like the F-type ATPases. However, the vacuolar ATPase is significantly larger, has a prominent cleft in the head sector, and has extra components associated with the stalk and membrane sectors. (2) Genes encoding three of the major subunits of the vacuolar ATPase and the homologous subunits of the mitochondrial F-ATPase have been isolated. The exon/intron structures of the genes have been analyzed and the chromosomal locations have been determined. Two of the vacuolar ATPase genes map very close to each other, suggesting the possibility of a cluster of ATPase genes. (3) The function of the ATPase is being investigated by isolating strains with altered or inactivated ATPase. We are characterizing strains that are resistant to bafilomycin A1, a potent and specific inhibitor of the vacuolar ATPase. Initial attempts to inactivate a vacuolar ATPase gene indicate that the enzyme may be essential for growth.

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