Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE)

Abstract

Mitochondria are cellular organelles that produce energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in-gel activity of SCs and the individual complexes with a good resolution on blue native polyacrylamide gel electrophoresis (BN-PAGE).

COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation

“…The fact that respirasomes (but not SC III 2 +IV) are present in C57BL/6J mitochondria solubilized with a variety of detergent concentrations provides additional support for a role for COX7A2L to stabilize SC III 2 +IV. These data are in agreement with previous reports that show the presence of respirasomes in different tissues, including heart, liver and skeletal muscle, of C57BL/6 mice (Hatle et al, 2013; Ikeda et al, 2013; Jha et al, 2016; Milenkovic et al, 2013). In this regard, the previously reported absence of respirasomes in mouse strains with the truncated Cox7a2l allele (Lapuente-Brun et al, 2013) could have resulted from differences in the methodologies or reagents used for membrane solubilization.…”

“…Indeed, we observed some variations in the intensity of SC isoforms between C57BL/6J and CD1 mice at high detergent concentrations; however the pattern was not constant, as specific SCs were stabilized in C57BL/6J mice but not in CD1 mice and vice versa. Based on our data we cannot exclude the possibility that the mechanisms of SC assembly are regulated in a tissue-dependent manner as recently proposed (Jha et al, 2016). However, it is important to acknowledge that the observed changes may not necessarily be a consequence of genetic COX7A2L variation, but could well be explained by other types of genetic differences among mouse strains.…”

Improved Supra-Organization Is A Conformational And Functional Adaptation Of Respiratory Complexes

“…Probing with CI-subunit Ndufs1 revealed presence of CI in multiple species with different masses (Figure 1A). The band at ~800 kDa indicated the CIholocomplex (CI-HC) while the upper bands represented CI-supercomplexes (CI-SCs) (Figure1A) Jha et al, 2016). We also observed increase in SCs at 8 hr MG132 while CI-HC remained similar to control.…”

Cardiolipin is essential for early embryonic viability and mitochondrial integrity of neurons in mammals

“…Isolation of mitochondria and blue native-PAGE were performed essentially according to Jha et al 26 Mitochondrial protein concentration was quantified by Bradford protein assay (Nacalai tesque) using BSA as a standard. Isolation of mitochondria and blue native-PAGE were performed essentially according to Jha et al 26 Mitochondrial protein concentration was quantified by Bradford protein assay (Nacalai tesque) using BSA as a standard.…”