Differential steady-state tyrosine phosphorylation of two oligomeric forms of mitochondrial F0F1ATPsynthase: a structural proteomic analysis

Proteomics. 2006 Feb;6(3):921-6. doi: 10.1002/pmic.200500077.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Immunoblotting
  • Mitochondria, Heart / enzymology*
  • Mitochondrial Proton-Translocating ATPases / chemistry*
  • Mitochondrial Proton-Translocating ATPases / metabolism*
  • Phosphorylation
  • Phosphotyrosine / metabolism*
  • Proteomics*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Phosphotyrosine
  • F1F0-ATP synthase
  • Mitochondrial Proton-Translocating ATPases