Oxidative phosphorylation defect in the brains of carriers of the tRNAleu(UUR) A3243G mutation in a MELAS pedigree.

MELAS is a mitochondrial encephalomyopathy characterized clinically by recurrent stroke-like episodes, seizures, sensorineural deafness, dementia, hypertrophic cardiomyopathy, and short stature. The majority of patients are heteroplasmic for a mutation (A3243G) in the tRNAleu(UUR) gene in mitochondrial DNA (mtDNA). In cells cultured in vitro, the mutation produces a severe mitochondrial translation defect only when the proportion of mutant mtDNAs exceeds 95% of total mtDNAs. However, most patients are symptomatic well below this threshold, a paradox that remains unexplained. We studied the relationship between the level of heteroplasmy for the mutant mtDNA and the clinical and biochemical abnormalities in a large pedigree that included 8 individuals carrying the A3243G mutation, 4 of whom were asymptomatic. Unexpectedly, we found that brain lactate, a sensitive indicator of oxidative phosphorylation dysfunction, was linearly related to the proportion of mutant mtDNAs in all individuals carrying the mutation, whether they were symptomatic or not. There was no evidence for threshold expression of the metabolic defect. These results suggest that marked tissue-specific differences may exist in the pathogenic expression of the A3243G mutation and explain why a neurological phenotype can be observed at relatively low levels of heteroplasmy

Can the mudskipper, Periophthalmus chrysospilos, tolerate acute environmental hypoxic exposure?

10.1007/BF00004461Fish Physiology and Biochemistry83221-227FPBI

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was approximately 350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated