High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

Nikola Kovářová,Jiri Neuzil,Jakub Rohlena,Marek Vrbacký,Tomáš Mráček,Hana Nůsková,Kateřina Hejzlarová,Katarína Kľučková,Petr Pecina,Josef Houštěk,Eliška Holzerová

doi:10.1371/journal.pone.0071869

Abstract

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.

Highlights

The mitochondrial oxidative phosphorylation system (OXPHOS) is the main source of energy in mammals
Digitoninsolubilised CII from mitochondria of human fibroblasts was resolved by blue native electrophoresis (BNE) or hrCNE3 as a CII monomer of the expected mass of approximately 140 kDa (Figure 1A, B) which represented most of the CII signal
This was further demonstrated by 2D CNE/SDS PAGE analysis of the cells (Figure 2A), where the distributions of SDHA and SDHB in the second dimension gel indicate that the CIIhmw forms represent a complete and active CII, in accord with the profiles of CII activity in CNE

Summary

Introduction

The mitochondrial oxidative phosphorylation system (OXPHOS) is the main source of energy in mammals This metabolic pathway is localised in the inner mitochondrial membrane (IMM) and includes the respiratory chain complexes I, II, III and IV (CI, CII, CIII, CIV), ATP synthase (complex V, CV), plus the mobile electron transporters coenzyme Q (CoQ) and cytochrome c. CII (succinate: ubiquinone oxidoreductase; EC 1.3.5.1), catalyses electron transfer from succinate (via FADH2) to CoQ and represents important crossroads of cellular metabolism, interconnecting the tricarboxylic acid (TCA) cycle and the respiratory chain [1]. It consists of 4 nuclear encoded subunits. The SDHC and SDHD subunits form the hydrophobic membrane anchor and are the site of cytochrome b binding [2]

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Results

Conclusion