Localization of subunits D, E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B.
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 Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) 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 F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.
Arata
Yoichiro
Y
Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Baleja
James D
JD
Forgac
Michael
M
D001577:Benzophenones / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics / Q000528:radiation effects; D017136:Ion Transport / Q000235:genetics / Q000528:radiation effects; D008301:Maleimides / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000528:radiation effects; D021122:Protein Subunits; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects
D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D021122:Protein Subunits; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; C046109:benzophenone-4-maleimide; D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine