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 (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-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.
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210; Department of Cell, Molecular and Developmental Biology, University of California, Riverside, Riverside, California 92521.
Norton
N
Kitagawa
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands.
Hortense
H
Mazon
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands.
Albert J R
AJR
Heck
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210. Electronic address: wilkenss@upstate.edu.
Stephan
S
Wilkens
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001671:Biochemistry / Q000379:methods; D003001:Cloning, Molecular; D004867:Equipment Design; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology*
D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases