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

Abstract

Mitochondria are cellular organelles that harvest 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 good resolution using blue native polyacrylamide gel electrophoresis (BN-PAGE).

Keywords: Cox7a2l; SCAFI; in-gel activity; mitochondria; oxidative phosphorylation; supercomplex.

Figure 1. Schematic representation of the oxidative phosphorylation (OXPHOS) system showing its individual complexes, supercomplexes and its nomenclature

(A) Mitochondrial electron transport chain (ETC) and ATP synthesis. In TCA cycle, NADH and succinate are electron donors, oxidized by NADH dehydrogenase (CI) and succinate dehydrogenase (CII), respectively. Ubiquinone (Q) is one of the electron acceptors which transfers the electrons to cytochrome c (c) by cytochrome c reductase (CIII). The electrons then pass to their terminal acceptor molecular oxygen (O₂) to release water by cytochrome c oxidase (CIV). This electron transfer is coupled with the transfer of protons (H⁺) across mitochondrial inner membrane, except complex II. The electrochemical proton gradient generated drives the generation of chemical energy in the form of adenosine triphosphate (ATP) through ATP synthase (CV). Note that in this figure, for simplicity, all components are shown as monomers, however in reality their stoichiometry is more complex e.g. complex III exists as dimers (as shown in the figure). Dimers and multiple aggregation of CIV are also present (not shown). (B and C) Representative stoichiometry and nomenclature of mitochondrial supercomplexes shown as a cartoon (B), and in a BN-PAGE (C). I, II, III, IV, V: complexes I to V; SC, supercomplex; M, matrix; IM, inner membrane; IMS, inner membrane space.

Figure 2. Schematic representation of the workflow

STEP 1 involves isolation and solubalization of mitochondria. In STEP 2, one can choose to either do BN-PAGE followed by immunoblotting (STEP 2(A)) or CN-PAGE (minor modification of BN-PAGE) followed by in-gel activity of the supercomplexes and complexes, with or without additional immunoblotting of a separate gel, run simultaneously (STEP 2(B)).

Figure 3. BN-PAGE showing mitochondrial complexes and supercomplexes

Liver mitochondria were isolated form DBA and C57BL6/ mice. 50 μg of mitochondrial protein (8g/g digitonin to protein ratio) was used for BN-PAGE for a run time of 30 min at 150 V and 1 h at 250 V followed by immunoblotting with an OXPHOS antiboty cocktail. The five SC stoichiometric combinations are indicated as numbers 1 to 5. Note, in C57BL/6 mice only 3 SC bands are observed (1, 2 and 5) in contrast to the 5 bands present in DBA mice.

Figure 4. BN-PAGE, followed by in gel activity assay and immunoblotting to define the SC bands in DBA and C57BL/6 (representing the 2 typical patterns of SCs in rodents)

Liver mitochondria were isolated and 50 μg of mitochondrial protein (4g/g digitonin to protein ratio) was used for BN-PAGE for a run time of 30 min at 150 V and 150 min at 250 V. In-gel activity assays and western blot was performed thereafter. (A) CI activity is shown in violet (20 min incubation). (B) CIV in brown (40 min incubation). (C) CIV+CI in brown and violet, respectively (40 min incubation in CIV substrate followed by 20 min in CI substrate). (D) CIII is shown by immunoblotting against the UQCRC2 subunit of CIII. (E) CII activity is shown in violet (40 min incubation). (F) CV activity is shown in red (16 h incubation). (G) Western blot developed with an OXPHOS antibody cocktail. Note, SCs 3, 4 and III₂+IV₁ are absent in C57BL/6 mice.

Figure 5. BN-PAGE, followed by Coomassie staining and destaining

Liver mitochondria were isolated and 150 μg of mitochondrial protein (4g/g digitonin to protein ratio) was used for BN-PAGE for a run time of 30min at 150V and 150min at 250V. The gel was stained and then destained using the colloidal blue staining kit.