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, 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.