Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes

Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a(1) and a(3) the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a(1) was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a(3) was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a(1) and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a(1) and a(3) isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype.