Mitochondrial supercomplexes molecular analysis Western-blot/Blue Native Page
Mitochondrial complexes and supercomplexes structure
Mitochondrial complexes are multi-subunits enzymes formed of up to ≈ 50 subunits (complexe I). Western blot analysis can be used to target specific subunits of interest, but complexes expression and or assembly are commobnly analyzed using Blue-Native electrophoresis. This method alos enable to study the composition of the super-structure (supercomplex) of the respiratory chain.
Native PAGE separations are run in non-denaturing conditions. Detergents are only used to lyselipid membranesin the cell. Complexes remain--for the most part--associated and folded as they would bein the cell. An additional separation method may then be used, SDS-PAGE or 2D BN-PAGE, which allows to analyse complexe subunits composition (BN/SDS-PAGE) or supercomplexe composition (BN/BN-PAGE).
Complex I from bovine heart mitochondria is associated with complexes III and IV. (A) BN-PAGE of bovine heart mitochondria after solubilization by digitonin. Most complex I and complex III was found assembled into two major supercomplexes a and b, and two minor supercomplexes c and d. The 200 kDa mass differences indicate the presence of varying copy numbers of monomeric complex IV. (B) Solubilization by DDM was used as a reference for quantitative solubilization of all OXPHOS complexes. Bovine complex I has binding sites for complex III and complex IV, and ATP synthase can be isolated as a dimer. (C) Complexes a–d and dimeric ATP synthase (VDim) separated by BN-PAGE were dissociated by 2D BN-PAGE using addition of DDM to the cathode buffer. Direct interaction of complexes I and III was apparent from the dissociation of complex a (I1III2) into monomeric complex I and dimeric complex III. Complexes b–d comprised complex IV in addition (silver staining required for c and d). Dimeric complex V dissociated into the monomeric form VMon.