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 ∼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.
Department of Human Genetics and Montreal Neurological Institute, McGill University, Montreal, QC., Canada, Department of Children and Adolescents, Division of Pediatric Neurology, PEDEGO Research Group and Medical Research Center Oulu, University of Oulu, Oulu University Hospital, Oulu, Finland.
Reetta
R
Hinttala
Montreal Neurological Institute, McGill University, Montreal, QC., Canada, Division of Medical Genetics, Department of Pediatrics, CHU Sainte-Justine and Université de Montréal, Montreal, Que., Canada.
Florin
F
Sasarman
Montreal Neurological Institute, McGill University, Montreal, QC., Canada.
Tamiko
T
Nishimura
Montreal Neurological Institute, McGill University, Montreal, QC., Canada.
Hana
H
Antonicka
Division of Medical Genetics, Department of Pediatrics, CHU Sainte-Justine and Université de Montréal, Montreal, Que., Canada.
Catherine
C
Brunel-Guitton
Department of Human Genetics and.
Jeremy
J
Schwartzentruber
Department of Human Genetics and.
Somayyeh
S
Fahiminiya
Department of Human Genetics and.
Jacek
J
Majewski
Institut de Recherches Clinique de Montreal (IRCM), Montreal, Que., Canada.
Denis
D
Faubert
Department of Clinical Genetics, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark and.
Elsebet
E
Ostergaard
Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Mitochondrial Disorders, Nijmegen, The Netherlands.
Jan A
JA
Smeitink
Department of Human Genetics and Montreal Neurological Institute, McGill University, Montreal, QC., Canada, eric@ericpc.mni.mcgill.ca.
Eric A
EA
Shoubridge
D002478:Cells, Cultured; D002853:Chromatography, Liquid; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D059472:Exome; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006579:Heterozygote; D006801:Humans; D007888:Leigh Disease / Q000235:genetics; D008715:Methionine / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D010085:Oxidative Phosphorylation; D014176:Protein Biosynthesis; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D008715:Methionine; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D003576:Electron Transport Complex IV