EXPLORING AN ANTIFUNGAL TARGET IN THE PLASMA-MEMBRANE H(+)ATPASE OF FUNGI

The plasma membrane H+-ATPase is a promising new antifungal target tha t is readily probed with the sulfhydryl-reactive reagent omeprazole. I nhibition of the H+-ATPase by omeprazole is closely linked to cell kil ling, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M i and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81-90). In this study, the molecular nature of this interaction was examined by s creening a series of 26 well-characterized pmal mutations residing in the first two transmembrane segments of the H+-ATPase from Saccharomyc es cerevisiae. Only two pmal mutants, A135G and G158D,G156C, were foun d to significantly decrease the sensitivity of cells for omeprazole. I n contrast, enhanced sensitivity was observed at a number of positions , with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggestin g that this region was not a direct site of covalent modification. Rat her, its conformation influences omeprazole binding at some other locu s. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co -reconstituted with the H+-ATPase and the light-driven proton pump bac teriorhodopsin. Omeprazole was found to completely inhibit proton tran sport by the H+-ATPase at 50 mu M in this system. An asymmetrically-di stributed chemical trap system involving glutathione was used to demon strate that this inhibition appears localized to the extracellular por tion of the enzyme. This work indicates that omeprazole can inhibit th e Hf-ATPase from its extracellular face, and this inhibition is influe nced by changes in the M1, M2 region of the protein.