Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry

The stoichiometry of yeast V-1-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V-1-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V-1-ATPase purified from yeast grown on N-14-containing medium were titrated with defined amounts of N-15-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, N-14- and N-15-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V-1-ATPase is A(3)B(3)DE(3)FG(3)H.