title: Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F(0)F(1)ATP synthase.
abstract: The role of the integral inner membrane subunit e in self-association of F(0)F(1)ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F(0)F(1)ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, gamma and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits gamma and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F(0)F(1)ATP synthase.
authors.affiliation: Department of Biomedical Sciences and Technologies and M.A.T.I. Centre of Excellence, University of Udine, Udine, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Bisetto
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Valentina
authors.initials: V
authors.lastname: Giorgio
authors.forename: Vera
authors.initials: V
authors.lastname: Alverdi
authors.forename: Irene
authors.initials: I
authors.lastname: Mavelli
authors.forename: Giovanna
authors.initials: G
authors.lastname: Lippe
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D008929:Mitochondria, Heart / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D021122:Protein Subunits; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases