Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it ex-erts its function remains unknown. Herein, we demonstrate that theNeurospora crassaNDUFAF2 homologue, the 13.4L pro-tein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the con-necting part but lacking the Nmodule of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. Mitochondrial complex I (NADH:ubiquinone oxidoreduc-tase, EC 1.6.5.3) is an L-shaped structure embedded in the inner mitochondrial membrane that transfers electrons from NADH to ubiquinone coupled to the translocation of protons to the intermembrane space (1). In mammals, it is composed of 45 dissimilar polypeptides encoded by both the mitochondrial and the nuclear DNA (2) These proteins, together with an FMNmol