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

Norton Kitagawa,Hortense Mazon,Albert J.R Heck,Stephan Wilkens

doi:10.1074/jbc.m707924200

Abstract

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.

Highlights

Vacuolar ATPases (V-ATPases,3 V1V0-ATPases) are ATP hydrolysis-driven proton pumps found in the endomembrane system of eukaryotic organisms, where they function to acidify the interior of subcellular organelles such as lysosomes, early and late endosomes, clathrin-coated vesicles, the Golgi, the plant tonoplast, and the yeast vacuole [1,2,3,4]
Much like the F-ATP synthase, the V-ATPase is a rotary molecular motor enzyme [5, 6]; ATP hydrolysis taking place on the A subunits of the A3B3 catalytic domain is coupled to proton translocation across the membrane domain via rotation of a central stalk made of subunits D, F, and d and a proteolipid ring
Using electron microscopy and single particle image analysis, we have previously shown that the C and H subunits are positioned in the interface connecting the V1 and V0, where they are connected to the A3B3 domain via elongated protein densities bound at the periphery of the B subunits (8 –10)

Summary

EXPERIMENTAL PROCEDURES

Yeast V1-ATPase Purification—V1-ATPase was purified from Saccharomyces cerevisiae strain SF838-5A␣ vma10⌬::kanMX expressing a FLAG-tagged VMA10 in plasmid pRS315 (CEN6, LEU2) as described previously [19]. Cells were harvested by low speed centrifugation, resuspended in Column Buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 7) up to a final volume of 25 ml, and frozen overnight at Ϫ20 °C. Isotope-labeled Subunit Titration and MALDI-Mass Spectrometry—Increasing amounts of 15N-labeled E and G subunits were mixed with a determined amount of yeast V1-ATPase, and the resulting protein mixture was separated on 12% SDS-PAGE gels. Bands containing both unlabeled and 15N-labeled E and G subunits were excised from the gels with a clean razor blade. A 1:1 peak ratio of 14N- and 15N-containing peptides was interpolated using the least squares linear regression analysis

RESULTS AND DISCUSSION

Name Composition

Analysis of isotope labeled subunit titrations