Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H+-ATPase.
We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H(+)-ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. Photoactivation resulted in cross-linking of the unique sulfhydryl groups on subunit B with other subunits in the complex. Four of the eight mutants constructed containing a unique cysteine residue at Ala(15), Lys(45), Glu(494), or Thr(501) resulted in the formation of cross-linked products, which were recognized by Western blot analysis using antibodies against both subunits B and E. These products had a molecular mass of 84 kDa, consistent with a cross-linked product of subunits B and E. Molecular modeling of subunit B places Ala(15) and Lys(45) near the top of the V(1) structure (i.e. farthest from the membrane), whereas Glu(494) and Thr(501) are predicted to reside near the bottom of V(1), with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A(3)B(3) hexamer that forms the core of the V(1) domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V(1) and V(0) domains.
Arata
Yoichiro
Y
Departments of Physiology and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Baleja
James D
JD
Forgac
Michael
M
D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine; D007074:Immunoglobulin G; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D021122:Protein Subunits; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine