Assembly, super-assembly and impared assembly of the mitochondrial electron trasport chain: in situ validation of the plasticity model

Abstract

The oxidative phosphorylation system (OXPHOS) comprises three fundamental processes: electron transport, proton pumping and ATP synthesis. The OXPHOS system is organized as a branched chain of multi-protein complexes that can be assembled into supra-molecular structures (supercomplexes) to optimize the utilization of the different sources of electrons. We have proposed the plasticity model as a dynamic model where free and super-assembled RCs may coexist and be functional. This model is supported by the disruption of the mitochondrial membranes with mild detergents and visualization of supercomplexes (SCs) by blue native electrophoresis (BNGE) extracted from cell lines or tissues. In this thesis, we have developed an innovative and robust approach to visualize and quantitatively estimate the proximity of the mitochondrial complexes and SCs (I/III, III/IV, I/IV and I/III/IV) in intact cells, without the use of detergents. For that purpose, we have analyzed different combinations of mitochondrial endogenous subunits by Stimulated Emission Depletion super resolution microscopy (STED) using a variety of cellular tools: mtDNA depleted cells (º), complex III (CYTbM) and complex IV (Cox10KO) depleted cell lines, and their respective isogenic controls. Moreover, we have used different immunolabelling combinations to tag RCs and SCs (CI/CIII, CI/CIV, CIII/CIV and CI/CIII/CIV). Thus, STED imaging reveals the co-existence of free and superassembled complexes in intact cells demonstrating in situ that the cellular organization of the mitochondrial respiratory chain are correctly represented by the plasticity model. On the other hand, It is known that mutations in genes encoding subunits of the mitochondrial complexes may affect the stability of other complexes. Therefore, as a second main aim, we investigate the molecular mechanism that allows CIII mutants, to suppress the effect of mutations, which impede the assembly of respiratory complexes in normal circumstances.