Severe combined deficiency of the 2-oxoacid dehydrogenases, associated with a defect in lipoate synthesis and accompanied by defects in complexes I, II, and III of the mitochondrial respiratory chain, is a rare autosomal recessive syndrome with no obvious causative gene defect. A candidate locus for this syndrome was mapped to chromosomal region 2p14 by microcell-mediated chromosome transfer in two unrelated families. Unexpectedly, analysis of genes in this area identified mutations in two different genes, both of which are involved in [Fe-S] cluster biogenesis. A homozygous missense mutation, c.545G>A, near the splice donor of exon 6 in NFU1 predicting a p.Arg182Gln substitution was found in one of the families. The mutation results in abnormal mRNA splicing of exon 6, and no mature protein could be detected in fibroblast mitochondria. A single base-pair duplication c.123dupA was identified in BOLA3 in the second family, causing a frame shift that produces a premature stop codon (p.Glu42Argfs(∗)13). Transduction of fibroblast lines with retroviral vectors expressing the mitochondrial, but not the cytosolic isoform of NFU1 and with isoform 1, but not isoform 2 of BOLA3 restored both respiratory chain function and oxoacid dehydrogenase complexes. NFU1 was previously proposed to be an alternative scaffold to ISCU for the biogenesis of [Fe-S] centers in mitochondria, and the function of BOLA3 was previously unknown. Our results demonstrate that both play essential roles in the production of [Fe-S] centers for the normal maturation of lipoate-containing 2-oxoacid dehydrogenases, and for the assembly of the respiratory chain complexes.

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3) is the largest and the least understood of all the respiratory chain complexes. Mammalian complex I is composed of at least 46 subunits, which are encoded by both nuclear (39 subunits) and mitochondrial DNA (7 subunits) (1-3). The complex, which has an estimated molecular mass of almost 1 MDa, catalyzes the transfer of two electrons from NADH to ubiquinone coupled to translocation of four protons across the inner mitochondrial membrane.Low resolution structures based on the electron microscopy of the bovine (4), Escherichia coli (5, 6), and Neurospora crassa (7) complex I show that the complex has an L-shaped form with one arm in the membrane and a peripheral arm protruding into the mitochondrial matrix. A second, horseshoe-shaped conformation of the E. coli complex I has also recently been proposed (8). The complex can be dissociated by treatment with detergent into three subcomplexes: I␣, corresponding to the peripheral arm and composed of ϳ21 mostly hydrophilic subunits, and I␤ and I␥, both of which make up the membrane arm (9). Subcomplex I␣ contains the NADH binding site and most of the redox centers. All of the mtDNA-encoded 1 subunits and at least 16 of the nuclear-encoded subunits are found in the I␤ and I␥ fractions of the membrane arm.