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.
Biochemistry, Molecular Biology
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Zhang, Fang, "The Analysis of Assembly Pathway and Clinical Mutations of the Membrane Arm Subunits of Escherichia Coli Complex I" (2021). Biological Sciences Theses and Dissertations. 13.
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