Human Xenomitochondrial Cybrids

Abstract

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to intro- duce mtDNA from non-human apes into a human cell line lacking mtDNA (°), and succeeded in producing human-common chimpanzee, human-pigmy chimpan- zee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characteriza- tion of oxidative phosphorylation function in these cells. Mitochondrial complexes II, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as limiting substrates showed that the Vmax was decreased in HXC by approximately 40%, and the Km for the NADH was significantly in- creased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone pro- duced a mild effect in control cells (0–10% inhibition) but produced a marked inhibition of HXC respiration (50–75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their rela- tive amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of com- plex I deficiency in humans and underscore the impor- tance of nuclear and mitochondrial genomes co-evolu- tion in optimizing oxidative phosphorylation function.