A Pathogenic 15-Base Pair Deletion in Mitochondrial DNA-encoded Cytochrome c Oxidase Subunit III Results in the Absence of Functional Cytochrome c Oxidase

Abstract

A 15-base pair, in-frame, deletion (9480del15) in the mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit III (COX III) gene was identified previously in a patient with recurrent episodes of myoglobinuria and an isolated COX deficiency. Transmitochondrial cell lines harboring 0, 97, and 100% of the 9480del15 deletion were created by fusing human cells lacking mtDNA ( 0 cells) with platelet and lymphocyte fractions isolated from the patient. The COX III gene mutation resulted in a severe respiratory chain defect in all mutant cell lines. Cells homoplasmic for the mutation had no detectable COX activity or respiratory ATP synthesis, and required uridine and pyruvate supplementation for growth, a phenotype similar to 0 cells. The cells with 97% mutated mtDNA exhibited severe reductions in both COX activity (6% of wild-type levels) and rates of ATP synthesis (9% of wild-type). The COX III polypeptide in the mutant cells, although translated at rates similar to wild-type, had reduced stability. There was no evidence for assembly of COX

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

“…A few examples in mammals suggest that translation of the COX1 mRNA might be reduced by defects associated with CcO assembly. It has been documented that a 15-base pair deletion in the human mitochondrial COX3 gene (46), as well as lack of cytochrome c in mouse fibroblasts (47) leads to a modest reduction of [ 35 S]methionine labeling of Cox1. However, these studies did not clearly distinguish whether decreased Cox1 labeling was due to reduced synthesis or increased turnover.…”

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNALeu(UUR) gene

“…Cells were incubated in DMEM lacking methionine and cysteine and containing 100 mg/mL emetine (Sigma) to inhibit cytoplasmic protein synthesis and labeled for 30 min or 60 min in the presence of 250 mCi of [ 35 S] Easy Tag EXPRE 35 S 35 S Protein Labeling Mix (>1000 Ci/mmole, Perkin-Elmer). Mitochondria were isolated from labeled cells by homogenization and differential centrifugation (Hoffbuhr et al 2000). Protein concentrations were determined using the DC protein assay (Bio-Rad).…”

“…Metabolic labeling of mtDNA-encoded proteins was performed as described (Hoffbuhr et al 2000). Cells were incubated in DMEM lacking methionine and cysteine and containing 100 mg/mL emetine (Sigma) to inhibit cytoplasmic protein synthesis and labeled for 30 min or 60 min in the presence of 250 mCi of [ 35 S] Easy Tag EXPRE 35 S 35 S Protein Labeling Mix (>1000 Ci/mmole, Perkin-Elmer).…”

Mitochondrial Biogenesis in Skeletal Muscle: Exercise and Aging

“…Some act as single protein subunits, but many are combined nuclear-encoded proteins to form multisubunit holoenzymes, like COX or NADH dehydrogenase (Figure-1). The function of these holoenzymes is clearly impaired if contributions from either genome is absent (5). Second, it is known that mtDNA transcription and replication require the import of nuclear gene products, which act as polymerases or transcription factors.…”