Five Entry Points of the Mitochondrially Encoded Subunits in Mammalian Complex I Assembly

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.Complex I (CI) (NADH-ubiquinone oxidoreductase; EC 1.6.5.3) is one of the main electron entry points in the mitochondrial respiratory electron transport chain catalyzing the oxidation of NADH to reduce ubiquinone to ubiquinol (31, 39, 40), contributing to the proton motive force to synthesize ATP by the process called oxidative phosphorylation (OXPHOS).CI assembly is a difficult problem to address due to the large size of the complex and its dual genomic nature, as 7 out of its 45 subunits are encoded by the mitochondrial DNA (mtDNA) (10, 11). Until very recently, mammalian CI assembly was explained using two different and apparently contradictory models. One model was proposed by following the time course of formation of CI intermediates in human cells in culture once mitochondrial protein synthesis had recovered after its inhibition by doxycycline (36). Based on these observations, human CI was proposed to be assembled through two different modules corresponding to the membrane and peripheral arms. The other model was proposed after analysis of a cohort of four CI-deficient patients in which seven putative assembly intermediates containing a combination of both peripheral- and membrane arm subunits were identified. Thus, an assembly pathway in which the peripheral- and membrane arm subassemblies came together before the completion of each of the arms was proposed (4). However, the most recent studies have refined the previous models and propose an overlapping view of the process. One study, by green fluorescent protein (GFP) tagging of the NDUFS3 subunit, identified six peripheral-arm intermediates. The second and third smaller NDUFS3-containing subassemblies were accumulated and could not advance into higher-molecular-mass species when mitochondrial protein synthesis was inhibited, thus determining the entry point of the mitochondrially encoded subunits in the CI assembly pathway (37). The most recent study analyzed the incorporation of the mitochondrial subunits in a time course to the fully assembled CI and, on the other hand, the incorporation of the nuclear subunits by importing them into isolated mitochondria (24). Although these two models differ in the order in which some subunits are incorporated, they agree on the general human CI assembly pathway, which takes place via evolutionarily conserved modular subassemblies (14, 25, 28, 37).However, the specific entry points of all the mtDNA-encoded CI subunits (ND subunits) in the CI assembly pathway and their roles in the stability of the complex remained to be clarified. Structural studies related to mutations in the ND subunits in pathological cases have given some hints as to the importance of each of them for CI assembly/stability. In this case, defects in specific ND subunits do not have the same effect: ND1, ND4, and ND6 seem to be fundamental to CI assembly, while ND3 and ND5 are important for its activity but not for assembly. On the other hand, mutations in ND2 alter CI assembly, with abnormal intermediate accumulation (19).In this article, we present new insights into the roles of the ND subunits by using mouse cells deficient for ND4, ND6, and a combination of ND6 and ND5. This study has allowed us to propose the five different entry points by which the mtDNA-encoded subunits are sequentially incorporated into the CI assembly pathway, completing the current view of the process. We conclude that ND4 and ND6 are required for the proper function and assembly of CI, although at different degrees due to their different entry points and roles in the CI assembly pathway.