Subject Area

Biochemistry, Genetics, Life Sciences, Microbiology, Molecular Biology

Abstract

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, a complex E-F-G was the only soluble intermediate detected in Complex I assembly, but assembly was not optimal when they were expressed either first or last. Co-expression of subunits B and CD with E-F-G provided higher level of assembly, indicating that an integrated assembly of N- and Q-modules may provide a more efficient pathway. Mutations in the nuclearly-encoded subunits (peripheral arm subunits) have been regularly discovered in humans in recent years, and many lead to early death. We have identified mutations at 17 different sites and constructed 31 mutants in a bacterial model system. These mutations, found in NDUFS1, NDUFS2, NDUFS8, and NDUFV1, map to subunit interfaces, and we hypothesized that many 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. In general, there was a strong correlation between the pathogenicity of the human mutations, and the level of activity seen in the E. coli system. With compound heterozygotes, we could determine which mutation was more deleterious. In some cases we cold distinguish between phenotypes that were caused by loss of the original amino acid, and the introduction of the mutant residue.

Degree Date

Spring 5-14-2022

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

Advisor

Steven Vik

Number of Pages

133

Format

.pdf

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Download

Share

COinS