Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA

Abstract

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.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene,CARS2,lead to a severe epileptic encephalopathy and complex movement disorder

“…The BN-PAGE gel analysis with in-gel activity staining showed the presence of additional bands reflecting incomplete assembly of complex V present in both skeletal muscle and cultured skin fibroblasts (figure 2A, B). Such bands of lower molecular mass in complex V have been shown to occur in patients with defects in complex V assembly, with mtDNA depletion, or in defects in mitochondrial transcription or translation 19. The combined deficiency in liver indicates that the deficiency is not limited to complex V assembly, and a western blot with an antibody against NDUFS2 indicated incomplete assembly of complex I (see online supplementary figure S1).…”

“…These patients each had a combined respiratory chain enzyme deficiency, fragmented complex V on BN-PAGE analysis and normal mtDNA analysis, thus indicating a defect in either mitochondrial transcription or translation 19. Primers were designed to amplify and sequence all exons and exon–intron boundaries of CARS2 and subjects were Sanger sequenced after PCR amplification.…”

Aberrant synthesis of ATP synthase resulting from a novel deletion in mitochondrial DNA in an African patient with progressive external ophthalmoplegia

“…Some activity remained in the holo-complex, which might be attributed to the 20% wild-type mtDNA present in the patient. This finding points to disturbed intra-mitochondrial protein synthesis, which can be attributed to the absence of the two mtDNA genes for complex V, ATPase6 and ATPase8 (Carrozzo et al 2006;Smet et al 2009). …”

Potentially diagnostic electron paramagnetic resonance spectra elucidate the underlying mechanism of mitochondrial dysfunction in the deoxyguanosine kinase deficient rat model of a genetic mitochondrial DNA depletion syndrome

“…Blue native PAGE analysis with in-gel activity staining was carried out as previously described (121–123). This allowed the identification of decreased synthesis of mitochondrial subunits (124). …”