Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi-subunit complex composed of a catalytic domain (F(1)) and a membrane domain (F(0)) linked by two stalks. Subcomplexes of complex V containing the F(1) domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT-ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.

ABSTRACT:In the last decades, a large variety of oxidative phosphorylation (OXPHOS) defects have been reported, expressed as an increasing variety of clinical phenotypes. With the expanding number of genes and proteins involved, new screening techniques leading to more effective diagnostic routes are in ever-increasing demand. Cultured skin fibroblasts from a cohort of patients with various OXPHOS defects, previously recognized by enzyme activity studies and blue native PAGE, were investigated with an immunocytochemical technique. Cytospins of cultured fibroblasts were air dried, fixed, and stained with antibodies specifically directed against subunits of each OXPHOS complex. Control cells stained homogeneously and strongly. In fibroblasts from five out of seven patients with a severe deficiency of one of the OXPHOS complexes, a homogeneous reduction of cytoimmunoreactivity of the affected complex was observed. In five out of seven fibroblast strains harboring a mitochondrial tRNA mutation, a mosaic pattern of staining was observed for both complexes I and IV, reflecting the heteroplasmic nature of the defect. The proportion of deficient fibroblasts varied considerably between cell strains from different subjects. The method described offers a convenient and rapid approach to first-line screening of OXPHOS defects. In association with routine assays of enzyme activity, the technique is helpful in orienting molecular investigation further. (Pediatr Res 59: 2-6, 2006) R ecent remarkable progress in understanding the genetics and molecular physiology of OXPHOS has revealed both its intrinsic structural complexity and the causal heterogeneity of mutations affecting mitochondrial function adversely. Moreover, besides the so-called nosological classic clinical entities mainly caused by mutations in the mitochondrial tRNA genes, genotype-phenotype correlation remains difficult in many mitochondrial disorders. The OXPHOS is run by a set of five multiprotein complexes, embedded in the lipid bilayer of the inner mitochondrial membrane: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate:ubiquinone oxidoreductase), complex III (ubiquinol:cytochrome c oxidoreductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase). This structural system harbors at least 85 proteins, 13 of which are encoded by the small mitochondrial genome. Coordinated function of the complexes establishes a proton gradient between the mitochondrial matrix and the intermembrane space that, by the catalytic action of complex V, generates ATP.Besides many mutations reported in the structural complexes themselves (1,2), mutations were discovered in nuclear genes encoding nonstructural proteins essential for assembly, importation, and folding of functional OXPHOS complexes. Examples of the former have been reported for complex III (3), IV (4), and V (5). Also, mutations in heat shock protein 60, a mitochondrial matrix chaperone, have been identified as the cause of fatal systemic mitochondrial disease (6).As the phenotypic spectr...