Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function.

Various human diseases are associated with mitochondrial DNA (mtDNA) mutations, but heteroplasmy-the coexistence of mutant and wild-type mtDNA-complicates their study. We previously isolated a temperature-lethal mtDNA mutation in Drosophila, mt:CoIT300I, which affects the cytochrome c oxidase subunit I (CoI) locus. In the present study, we found that the decrease in cytochrome c oxidase (COX) activity was ascribable to a temperature-dependent destabilization of cytochrome a heme. Consistently, the viability of homoplasmic flies at 29 degrees C was fully restored by expressing an alternative oxidase, which specifically bypasses the cytochrome chains. Heteroplasmic flies are fully viable and were used to explore the age-related and tissue-specific phenotypes of mt: CoIT300I. The proportion of mt: CoIT300I genome remained constant in somatic tissues along the aging process, suggesting a lack of quality control mechanism to remove defective mitochondria containing a deleterious mtDNA mutation. Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt: CoIT300I homoplasmy in the eye caused severe neurodegeneration at 29 degrees C. Degeneration was suppressed by improving mitochondrial Ca2+ uptake, suggesting that Ca2+ mishandling contributed to mt: CoIT300I pathogenesis. Our results demonstrate a novel approach for Drosophila mtDNA genetics and its application in modeling mtDNA diseases.