The vacuolar membrane H+-ATPase (V-ATPase) of the yeast Saccharomyces cerevisiae is composed of peripheral catalytic (V1) and integral membrane (V0) domains. The 17-kDa proteolipid subunit (VMA3 gene product; Vma3p) is predicted to constitute at least part of the proton translocating pore of V0. Recently, two VMA3 homologues, VMA11 and VMA16 (PPA1), have been identified in yeast, and VMA11 has been shown to be required for the V-ATPase activity. Cells disrupted for the VMA16 gene displayed the same phenotypes as those lacking either Vma3p or Vma11p; the mutant cells lost V-ATPase activity and failed to assemble V-ATPase subunits onto the vacuolar membrane. Epitope-tagged Vma11p and Vma16p were detected on the vacuolar membrane by immunofluorescence microscopy. Density gradient fractionation of the solubilized vacuolar proteins demonstrated that the tagged proteins copurified with the V-ATPase complex. We conclude that Vma11p and Vma16p are essential subunits of the V-ATPase. Vma3p contains a conserved glutamic acid residue (Glu137) whose carboxyl side chain is predicted to be important for proton transport activity. Mutational analysis of Vma11p and Vma16p revealed that both proteins contain a glutamic acid residue (Vma11p Glu145 and Vma16p Glu108) functionally similar to Vma3p Glu137. These residues could only be functionally substituted by an aspartic acid residue, because other mutations we examined inactivated the enzyme activity. Assembly and vacuolar targeting of the enzyme complex was not inhibited by these mutations. These results suggest that the three proteolipid subunits have similar but not redundant functions, each of which is most likely involved in proton transport activity of the enzyme complex. Yeast cells contain V0 and V1 subcomplexes in the vacuolar membrane and in the cytosol, respectively, that can be assembled into the active V0V1 complex in vivo. Surprisingly, loss-of-function mutations of either Vma11p Glu145 or Vma16p Glu108 resulted in a higher degree of assembly of the V1 subunits onto the V0 subcomplex in the vacuolar membrane.