Our current work on a vacuolar membrane proton ATPase in the yeast Saccharomyces cerevisiae has revealed that it is a third type of H+-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F0F1-type ATP synthase and the plasma membrane E1E2-type H+-ATPase. The vacuolar membrane H+-ATPase is composed of three major subunits, subunit a (Mr = 67 kDa), b (57 kDa), and c (20 kDa). Subunit a is the catalytic site and subunit c functions as a channel for proton translocation in the enzyme complex. The function of subunit b has not yet been identified. The functional molecular masses of the H+-ATPase under two kinetic conditions have been determined to be 0.9-1.1 x 10(5) daltons for single-cycle hydrolysis of ATP and 4.1-5.3 x 10(5) daltons for multicycle hydrolysis of ATP, respectively. N,N'-Dicyclohexyl-carbodiimide2 does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-sensitive catalytic site. Cloning of structural genes for the three subunits of the H+-ATPase (VMA1, VMA2, and VMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.