Premium
Differential steady‐state tyrosine phosphorylation of two oligomeric forms of mitochondrial F 0 F 1 ATPsynthase: A structural proteomic analysis
Author(s) -
Di Pancrazio Francesca,
Bisetto Elena,
Alverdi Vera,
Mavelli Irene,
Esposito Gennaro,
Lippe Giovanna
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500077
Subject(s) - dimer , phosphorylation , tyrosine , monomer , tyrosine phosphorylation , protein subunit , mitochondrion , chemistry , peptide , biochemistry , polyacrylamide gel electrophoresis , residue (chemistry) , microbiology and biotechnology , biology , gene , organic chemistry , enzyme , polymer
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.