Differential steady-state tyrosine phosphorylation of two oligomeric forms of mitochondrial F0F1ATPsynthase: 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)gamma 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.
Di Pancrazio
Francesca
F
Department of Biomedical Sciences and Technologies, University of Udine, MATI Center of Excellence, Udine, Italy.
Bisetto
Elena
E
Alverdi
Vera
V
Mavelli
Irene
I
Esposito
Gennaro
G
Lippe
Giovanna
G
D000818:Animals; D002417:Cattle; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019000:Phosphotyrosine; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases