Reference: Kitagawa N, et al. (2008) Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry. J Biol Chem 283(6):3329-37

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Abstract


The stoichiometry of yeast V1-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 V1-ATPase complex, and each of the individual protein components, using native electrospray ionization-mass spectrometry (ESI-MS). The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 Da and 428,300 Da, respectively. Second, defined amounts of V1-ATPase purified from yeast grown on 14N containing media were titrated with defined amounts of 15N labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14N and 15N containing peptide pairs were used for quantification of subunit stoichiometry using MALDI-TOF MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V1-ATPase is A3B3DE3FG3H.

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Journal Article
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Kitagawa N, Mazon H, Heck AJ, Wilkens S
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