Analysis of the assembly pathway for membrane subunits of Complex I reveals that subunit L (ND5) can assemble last in E. coli.

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 preformed complex lacking L. In contrast, when larger groups of membrane subunits were expressed last, LMN or JKLMN, assembly was much less efficient. The two-plasmid expression system was used to re-analyze C-terminal mutations in subunit K (ND4L), which occur near the overlapping nuoK and L genes. These mutations were found to disrupt assembly, indicating the importance of the junction of L, N and K subunits. The results highlight the temporal and spatial aspects of gene expression that allow efficient assembly of the membrane subunits of Complex I.