In Vivo Regulation of Oxidative Phosphorylation in Cells Harboring a Stop-codon Mutation in Mitochondrial DNA-encoded Cytochrome c Oxidase Subunit I

• Published in: The Journal of biological chemistry 2001-12, Vol.276 (50), p.46925-46932
• Main Authors: D'Aurelio, Marilena, Pallotti, Francesco, Barrientos, Antoni, Gajewski, Carl D., Kwong, Jennifer Q., Bruno, Claudio, Beal, M. Flint, Manfredi, Giovanni
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - biosynthesis; Blood Platelets - metabolism; Blotting, Northern; Cell Line; Codon, Terminator; COX gene; DNA Mutational Analysis; DNA, Mitochondrial - genetics; Electron Transport; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - genetics; Galactose - pharmacology; Humans; Hybrid Cells; Immunoblotting; Mitochondria - metabolism; Mutation; Oxygen - metabolism; Oxygen Consumption; Phosphorylation; Polymorphism, Restriction Fragment Length; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; RNA - metabolism; RNA, Messenger - metabolism; Time Factors; Transcription, Genetic; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M106429200

The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochrome c oxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochrome coxidase subunit II was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at ∼40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.