Functional and Molecular Mitochondrial Abnormalities Associated with a C 3 T Transition at Position 3256 of the Human Mitochondrial Genome THE EFFECTS OF A PATHOGENIC MITOCHONDRIAL tRNA POINT MUTATION IN ORGANELLE TRANSLATION AND RNA PROCESSING*

We have previously identified a mitochondrial DNA polymorphism (a C 3 T transition at position 3256, within the mitochondrial tRNA gene) in a patient with a multisystem disorder. Although there were several indicators suggesting a pathogenetic role for this mtDNA polymorphism, its heteroplasmic nature made functional and molecular studies difficult to interpret. We have now fused enucleated fibroblasts from the patient with a mtDNA-less cell line to generate transmitochondrial cybrids harboring different proportions of mutated and wild-type mtDNA. Individual clones harboring essentially 100% wild-type or >99% mutated mtDNAs were characterized and studied for respiratory capacity, respiratory chain enzymes activity, mitochondrial protein synthesis, and RNA steady-state levels and processing. Our results showed that cell lines containing exclusively mutated mtDNAs respire poorly, overproduce lactic acid, and have significantly impaired activity of respiratory complexes I and IV. Molecular studies showed that mutant clones have a decrease in steady-state levels of mitochondrial tRNA, and a partial impairment of mitochondrial protein synthesis and steady-state levels, suggesting that these molecular abnormalities are involved in the pathogenetic mechanism of the mtDNA 3256 mutation.