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Journal of Experimental Biology, Vol 203, Issue 1 97-106, Copyright © 2000 by Company of Biologists

JOURNAL ARTICLES

Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A

EJ Bowman and BJ Bowman
Department of Biology, University of California, Santa Cruz, CA 95064, USA. rbowman@biology.ucsc.edu

Vacuolar ATPases (V-ATPases) are large complex enzymes that are structural and mechanistic relatives of F(1)F(o)-ATPases. They hydrolyze ATP and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. The proton gradients generated are used to drive the movement of various compounds across membranes. V-ATPases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the plasma membranes of many specialized eukaryotic cells. They have been implicated in a wide variety of cellular processes and are associated with several diseases. Bafilomycin and concanamycin, specific inhibitors of V-ATPases, have been instrumental in implicating the V-ATPase in many of these roles. To understand further the mechanism of inhibition by these antibiotics and the physiological role of the enzyme in the cell, we have isolated mutants of the filamentous fungus Neurospora crassa that are resistant to concanamycin. Concanamycin has a dramatic effect on hyphal morphology at acid pH and is lethal at basic pH. In the resistant mutants, the cells can germinate and grow, although abnormally, in basic medium. Thus far, none of the mutants we have characterized is mutated in a gene encoding a subunit of the V-ATPase. Instead, the largest class of mutants is mutated in the gene encoding the plasma-membrane H(+)-ATPase. Mutations in at least four uncharacterized genes can also confer resistance. Inactivation of the V-ATPase by disruption of vma-1, which encodes the catalytic subunit (A) of the enzyme, causes a much more severe phenotype than inhibition by concanamycin. A strain lacking vma-1 is seriously impaired in rate of growth, differentiation and capacity to produce viable spores. It is also completely resistant to concanamycin, indicating that the inhibitory effects of concanamycin in vivo are due to inhibition of the V-ATPase. How the multiplicity of ATPases within a cell is regulated and how their activity is integrated with other metabolic reactions is poorly understood. Mutant analysis should help unravel this puzzle.




© The Company of Biologists Ltd 2000