Identification and reconstitution of an isoform of the 116-kDa subunit of the vacuolar proton translocating ATPase

We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a₂, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a₁) we described (Peng, S.-B., Crider, B.P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262-17266). Sequence comparison indicates that the a₂ isoform is the bovine homologue of a 116- kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.- K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137-1144). The bovine a₁ and a₂ isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane regions. Northern blot analysis demonstrates that isoform a₂ is highly expressed in lung, kidney, and spleen. To determine the possible role of the a₂ isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (V(O)) containing isoform a₂. This V(O) conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V₁) of vacuolar-type proton translocating ATPase (V- ATPase) and sub-58-kDa doublet, a 50-57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a₂ isoform of the 116- kDa polypeptide functions as part of the proton channel of V-ATPases.