Molecular characterization of the plasma membrane H+-ATPase, an antifungaltarget in Cryptococcus neoformans

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H+-ATPase , was isolated from a genomic DNA library of serotype A strain ATCC 6352, A n open reading frame of 3,380 nucleotides contains six Introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H+-ATPase wa s shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H+-ATPase, consistent with its predi cted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 op timum for ATP hydrolysis, K-m and V-max values of 0.5 mM and 3.1 mu mol mg( -1) min(-1), respectively, and an apparent K-i for vanadate inhibition of 1 .6 mu M. ATP hydrolysis in plasma membranes and medium acidification by who le cells were inhibited by ebselen, a nonspecific H+-ATPase antagonist whic h was also fungicidal, The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits mo re than 50% identity to PMA1 genes from plants, Collectively, this study pr ovides the basis for establishing the Cryptococcus H+-ATPase as a viable ta rget for antifungal drug discovery.