Analysis of compound heterozygous and homozygous mutations found in peripheral subunits of human respiratory Complex I, NDUFS1, NDUFS2, NDUFS8 and NDUFV1, by modeling in the E. coli enzyme.

Respiratory Complex I (NADH:ubiquinone oxidoreductase) is composed of 45 subunits, seven mitochondrially-encoded and 38 imported. Mutations in the nuclearly-encoded subunits have been regularly discovered in humans in recent years, and many lead to cardiomyopathy, Leigh Syndrome, and early death. From the literature, we have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. Many of these mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that they would disrupt assembly of Complex I. The mutations were constructed in the homologous E. coli genes, nuoG, nuoCD, nuoI and nuoF, respectively, and expressed from a plasmid containing all Complex I genes. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity measured, which indicated a range of reduced activity. Some mutants were also analyzed using recently developed assays of assembly, time-delayed expression, and co-immunoprecipitation, which showed that assembly was disrupted. With compound heterozygotes, we determined which mutation was more deleterious. Construction of alanine mutations allowed us to distinguish between phenotypes that were caused by loss of the original amino acid or introduction of the mutant residue.

Analysis of Human Clinical Mutations of Mitochondrial ND1 in a Bacterial Model System for Complex I.

The most common causes of mitochondrial dysfunction and disease include mutations in subunits and assembly factors of Complex I. Numerous mutations in the mitochondrial gene ND1 have been identified in humans. Currently, a bacterial model system provides the only method for rapid construction and analysis of mutations in homologs of human ND1. In this report, we have identified nine mutations in human ND1 that are reported to be pathogenic and are located at subunit interfaces. Our hypothesis was that these mutations would disrupt Complex I assembly. Seventeen mutations were constructed in the homologous nuoH gene in an E. coli model system. In addition to the clinical mutations, alanine substitutions were constructed in order to distinguish between a deleterious effect from the introduction of the mutant residue and the loss of the original residue. The mutations were moved to an expression vector containing all thirteen genes of the E. coli nuo operon coding for Complex I. Membrane vesicles were prepared and rates of deamino-NADH oxidase activity and proton translocation were measured. Samples were also tested for assembly by native gel electrophoresis and for expression of NuoH by immunoblotting. A range of outcomes was observed: Mutations at four of the sites allow normal assembly with moderate activity (50−76% of wild type). Mutations at the other sites disrupt assembly and/or activity, and in some cases the outcomes depend upon the amino acid introduced. In general, the outcomes are consistent with the proposed pathogenicity in humans.

Subunits E-F-G of E. coli Complex I can form an active complex when expressed alone, but in time-delayed assembly co-expression of B-CD-E-F-G is optimal.

Respiratory Complex I from E. coli is a proto-type of the mitochondrial enzyme, consisting of a 6-subunit peripheral arm (B-CD-E-F-G-I) and a 7-subunit membrane arm. When subunits E-F-G (N-module), were expressed alone they formed an active complex as determined by co-immunoprecipitation and native gel electrophoresis. When co-expressed with subunits B and CD, only a complex of E-F-G was found. When these five subunits were co-expressed with subunit I and two membrane subunits, A and H, a complex of B-CD-E-F-G-I was membrane-bound, constituting the N- and Q-modules. Assembly of Complex I was also followed by splitting the genes between two plasmids, in three different groupings, and expressing them simultaneously, or with time-delay of expression from one plasmid. When the B-CD-E-F-G genes were co-expressed after a time-delay, assembly was over 90 % of that when the whole operon was expressed together. In summary, E-F-G was the only soluble subcomplex detected in these studies, but assembly was not optimal when these subunits were expressed either first or last. Co-expression of subunits B and CD with E-F-G provided a higher level of assembly, indicating that integrated assembly of N- and Q-modules provides a more efficient pathway.

Analysis of Human Mutations in the Supernumerary Subunits of Complex I.

Complex I is the largest member of the electron transport chain in human mitochondria. It comprises 45 subunits and requires at least 15 assembly factors. The subunits can be divided into 14 "core" subunits that carry out oxidation-reduction reactions and proton translocation, as well as 31 additional supernumerary (or accessory) subunits whose functions are less well known. Diminished levels of complex I activity are seen in many mitochondrial disease states. This review seeks to tabulate mutations in the supernumerary subunits of humans that appear to cause disease. Mutations in 20 of the supernumerary subunits have been identified. The mutations were analyzed in light of the tertiary and quaternary structure of human complex I (PDB id = 5xtd). Mutations were found that might disrupt the folding of that subunit or that would weaken binding to another subunit. In some cases, it appeared that no protein was made or, at least, could not be detected. A very common outcome is the lack of assembly of complex I when supernumerary subunits are mutated or missing. We suggest that poor assembly is the result of disrupting the large network of subunit interactions that the supernumerary subunits typically engage in.