Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H +-ATPase

Abstract

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 3B 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.