Complex I of the mitochondrial respiratory chain is a large multisubunit enzyme that assembles from nuclear and mtDNA-encoded components. Several complex I assembly factors have been identified, but their precise functions are not well understood. Here, we have investigated the function of one of these, NDUFAF7, a soluble matrix protein comprised of a DUF185 domain that harbors a methyltransferase motif. Knockdown of NDUFAF7 by siRNA in human fibroblasts produced a specific complex I assembly defect, as did morpholino-mediated knockdown of the zebrafish ortholog. Germline disruption of the murine ortholog was an early embryonic lethal. The complex I assembly defect was characterized by rapid, AFG3L2-dependent, turnover of newly synthesized ND1, the subunit that seeds the assembly pathway, and by decreased steady-state levels of several other structural subunits including NDUFS2, NDUFS1 and NDUFA9. Expression of an NDUFAF7 mutant (G124V), predicted to disrupt methyltransferase activity, impaired complex I assembly, suggesting an assembly factor or structural subunit as a substrate for methylation. To identify the NDUFAF7 substrate, we used an anti-ND1 antibody to immunoprecipitate complex I and its associated assembly factors, followed by mass spectrometry to detect posttranslational protein modifications. Analysis of an NDUFAF7 methyltransferase mutant showed a 10-fold reduction in an NDUFS2 peptide containing dimethylated Arg85, but a 5-fold reduction in three other NDUFS2 peptides. These results show that NDUFAF7 functions to methylate NDUFS2 after it assembles into a complex I, stabilizing an early intermediate in the assembly pathway, and that this function is essential for normal vertebrate development.
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