title: Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.
abstract: The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.
authors.affiliation: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: T
authors.initials: T
authors.lastname: Xu
authors.forename: E
authors.initials: E
authors.lastname: Vasilyeva
authors.forename: M
authors.initials: M
authors.lastname: Forgac
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001577:Benzophenones / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002966:Clathrin / Q000737:chemistry*; D018699:Coated Vesicles / Q000201:enzymology*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003553:Cystine / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D011193:Potassium Iodide / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D014357:Trypsin; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D000596:Amino Acids; D001577:Benzophenones; D002966:Clathrin; D003432:Cross-Linking Reagents; D008301:Maleimides; D013388:Succinimides; C096464:disuccinimidyl glutarate; D011193:Potassium Iodide; D003553:Cystine; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; C046109:benzophenone-4-maleimide; D014357:Trypsin; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005022:Ethyldimethylaminopropyl Carbodiimide