Abstract

Respiratory Complex I, a multi-subunit, membrane-bound enzyme, oxidizes NADH in the electron transport chains of mammalian mitochondria, and many bacterial species. We have examined in vivo assembly of the membrane subunits of Complex I from E. coli. Complexes of J-K, L-M, M-N, and J-K-L-M-N were observed by both native gel electrophoresis and co-immunoprecipitation, when subsets of the genes were expressed. Subunit L (ND5 in humans), the most distal membrane subunit, with an unusual extended C-terminal segment, did not join with M-N, and but could join with J-K-M-N. When the genes were split between two plasmids, with L, M, and N subunits expressed in various combinations from one plasmid, the resulting enzyme activity in membrane vesicles dropped to 19–60% relative to expression from the whole operon encoded on one plasmid. When L was expressed after a time-delay, rather than simultaneously, the activity increased from 28% to 100%, indicating that it can efficiently join a pre-formed complex lacking L. In contrast, when larger groups of membrane subunits were expressed last, LMN or JKLMN, assembly was much less efficient. Clinical mutations located at the interfaces of membrane subunits of Complex I were also analyzed in this study. The results suggest E. coli model system can be effective in identifying deleterious human mutations, especially if appropriate assembly assays are carried out.

Degree Date

Fall 12-18-2021

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

Advisor

Steven Vik

Subject Area

Biochemistry, Molecular Biology

Number of Pages

125

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

Available for download on Wednesday, December 09, 2026

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