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J. Biol. Chem., Vol. 283, Issue 6, 3329-3337, February 8, 2008

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Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V₁-ATPase Determined by Mass Spectrometry^*

Norton Kitagawa, Hortense Mazon^¶1, Albert J. R. Heck^¶1, and Stephan Wilkens²

From the Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, the Department of Cell, Molecular and Developmental Biology, University of California, Riverside, Riverside, California 92521, and the ^¶Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands

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-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 V₁-ATPase purified from yeast grown on ¹⁴N-containing medium 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 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 V₁-ATPase is A₃B₃DE₃FG₃H.

Received for publication, September 21, 2007 , and in revised form, November 16, 2007.

^* This work was supported by National Institutes of Health Grants GM58600 and CA100246 (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Netherlands Proteomics Centre and the Netherlands Research Council for Chemical Sciences.

² To whom correspondence should be addressed. Tel.: 315-464-8703; Fax: 315-464-8750; E-mail: wilkenss{at}upstate.edu.

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