Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain

J Biol Chem. 2005 Jul 29;280(30):27896-903. doi: 10.1074/jbc.M504890200. Epub 2005 Jun 10.


The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Antibodies, Monoclonal / chemistry
  • Benzophenones / pharmacology
  • Blotting, Western
  • Cloning, Molecular
  • Cross-Linking Reagents / pharmacology
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / pharmacology*
  • Electrophoresis, Polyacrylamide Gel
  • Hydrolysis
  • Immunoprecipitation
  • Light
  • Maleimides / pharmacology
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Phenotype
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Protons
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / physiology*
  • Time Factors
  • Vacuolar Proton-Translocating ATPases / chemistry*
  • Vacuolar Proton-Translocating ATPases / physiology*


  • Antibodies, Monoclonal
  • Benzophenones
  • Cross-Linking Reagents
  • Maleimides
  • Protons
  • Saccharomyces cerevisiae Proteins
  • VMA5 protein, S cerevisiae
  • Adenosine Triphosphate
  • benzophenone-4-maleimide
  • Vacuolar Proton-Translocating ATPases
  • Cysteine