Antiporter-like protein subunits of respiratory chain complex I

Complex I is the mitochondrial enzyme complex that oxidizes NADH produced in the citric acid cycle and reduces quinone in the membrane, coupled to proton pumping out of the mitochondrial matrix, creating a membrane potential. This process generates about 40% of the energy used by living organisms. High-resolution structures of complex I recently became available, but the molecular mechanism behind the coupling process remains unknown. This thesis is focused on better understanding of the three large membrane-spanning subunits that show primary sequence similarity to two proteins in Mrp-antiporters. The similarity suggests that these complex I subunits are somehow involved in transporting protons and/or other ions across the membrane, an essential element in the proton pumping/coupling functionality. First, a method of producing and purifying the NuoL, M, N and MrpA, D polypeptides in high amounts was developed. The C-terminal part of MrpA, that extends beyond the universally conserved 14 TM helices in the protein family, was further analysed and the transmembrane topology was determined. It was demonstrated that this domain in MrpA corresponds to the membrane parallel, so called lmp-helix in NuoL followed by the NuoJ subunit in complex I. Then, the purified antiporter-like polypeptides were reconstituted in liposomes, together with a novel pH sensitive probe. Glu3 is a dendritic porphyrin, which we have demonstrated can be enclosed inside lipid vesicles and used to accurately measure proton transport across membranes. Both the dye incorporation and protein insertion was successful, but the method requires further optimisation since the proteoliposomes were not tight enough to support build-up of a proton gradient. Subsequently, sodium interaction by the antiporter-like subunits was studied using Nuclear Magnetic Resonance (NMR), to investigate the putative interaction between sodium ions and the detergent-solubilised, purified protein molecules. No sodium binding was observed either for complex I subunits, nor for the control Mrp antiporter proteins. Then, the sodium translocation abilities were assessed in vivo in a Bacillus subtilis model system. The intracellular sodium content in a MrpA deletion strain, that exhibit a pH and sodium sensitive growth phenotype, was measured using 23Na-NMR and a membrane-impermeable shift agent. It was demonstrated that the growth defect was indeed caused by an inability to excrete sodium. Expression of Mrp proteins or homologous complex I subunits in the deletion strain was shown to influence the sodium homeostasis. A sequence motif in TM helix 8 that is present in MrpA/NuoL, but not in MrpD/NuoMN, that could possibly be responsible for the ion specificity of the respective subunits, was identified. The site-directed mutagenesis and subsequent functional analyses work of the motif is still ongoing, but some preliminary results are presented