Differential steady‐state tyrosine phosphorylation of two oligomeric forms of mitochondrial F 0 F 1 ATPsynthase: A structural proteomic analysis

We investigated tyrosine phosphorylation of F 0 F 1 ATPsynthase using 3‐D blue native (BN)‐SDS‐PAGE, a refinement of the electrophoretic analysis of mitochondrial complexes. Bovine heart mitochondria were detergent‐solubilized and subjected to BN‐PAGE. Bands of ATPsynthase monomer (Vmon ) and dimer (Vdim) were excised and submitted to SDS‐PAGE and immunoblotting. One protein corresponding to F 1 γ subunit was detected by anti‐phosphotyrosine antibody in monomer but not in dimer. This was confirmed by MS peptide mapping. LC‐ESI/MS analysis after 3‐D SDS‐PAGE demonstrated phosphotyrosine in fragment 43–54. NetPhos scores predicted the phosphorylated residue to be Tyr52, in a solvent‐accessible loop at the foot of the F 1 central stalk.