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.
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.
Inoue
Takao
T
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Forgac
Michael
M
D000255:Adenosine Triphosphate / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001577:Benzophenones / Q000494:pharmacology; D015153:Blotting, Western; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D047468:Immunoprecipitation; D008027:Light; D008301:Maleimides / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology*
D000911:Antibodies, Monoclonal; D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D011522:Protons; D029701:Saccharomyces cerevisiae Proteins; C447616:VMA5 protein, S cerevisiae; D000255:Adenosine Triphosphate; C046109:benzophenone-4-maleimide; D025262:Vacuolar Proton-Translocating ATPases; D003545:Cysteine