Localization of Subunits D, E, and G in the Yeast V-ATPase Complex Using Cysteine-Mediated Cross-Linking to Subunit B

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Abstract
Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H+)-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu106 and Asp199 led to cross-linking of subunits B and E, at positions Asp341 and Ala424 to cross-linking of subunits B and D, and at positions Ala15 and Lys45 to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F1-ATPase, and energy minimization, Glu106, Asp199, Ala15, and Lys45 are all predicted to be located on the outer surface of the complex, with Ala15 and Lys45 located near the top of the complex furthest from the membrane. By contrast, Asp341 and Ala424 are predicted to face the interior of the A3B3 hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V1 and V0 domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the γ subunit of F1, which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.