Human Xenomitochondrial Cybrids

Abstract

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (°), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characterization 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 V max was decreased in HXC by approximately 40%, and the K m for the NADH was significantly increased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced 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 relative amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.Mitochondria are organelles containing their own DNA (mtDNA) and are present in essentially all eukaryotic cells. It is believed that the presence of mitochondria resulted from an evolving symbiosis of infectious prokaryotes and their eukaryotic hosts, allowing eukaryotic cells to oxidize substrates to produce energy. The mitochondrial respiratory chain (MRC)

Mitonuclear interactions: evolutionary consequences over multiple biological scales

“…Empirical evidence supporting the notion that mitonuclear incompatibilities increase with genetic distance comes from research using human cell lines stripped of mtDNA and then repopulated with the mtDNA from increasingly distantly related taxa [81,82]. A first set of experiments paired a human nucleotype with the mtDNA haplotype of the common chimpanzee, pigmy chimpanzee and gorilla [82].…”

“…A first set of experiments paired a human nucleotype with the mtDNA haplotype of the common chimpanzee, pigmy chimpanzee and gorilla [82]. Interruption of the coevolved mitonuclear lineage resulted in decreases in the activity of complex I of the mETS of around 40% in cybrids where the human nuclear background was paired with foreign mitochondrial genotypes in comparison with the native mtDNA genotype, whereas complexes II, III, IV and V were not affected [82].…”

“…A first set of experiments paired a human nucleotype with the mtDNA haplotype of the common chimpanzee, pigmy chimpanzee and gorilla [82]. Interruption of the coevolved mitonuclear lineage resulted in decreases in the activity of complex I of the mETS of around 40% in cybrids where the human nuclear background was paired with foreign mitochondrial genotypes in comparison with the native mtDNA genotype, whereas complexes II, III, IV and V were not affected [82]. In a second study, mitonuclear combinations were extended by combining the human cell line with mitochondrial haplotypes from even more distant taxa-orangutan, species representative of Old-World monkeys, New-World monkeys and lemurs-all of which failed to restore any tangible OXPHOS activity [81].…”

Caspase-9 and effector caspases have sequential and distinct effects on mitochondria

“…FL5.12 Neo, Bcl-x L and C9DN cells incubated in the presence and absence of IL-3 for 24 h were freezethawed to disrupt the inner mitochondrial membrane. The function of complexes I þ III and II þ III was assayed by the ability of NADH and succinate, respectively, to reduce oxidized cytochrome c ( Figure 5a and b) (Barrientos et al, 1998). Both NADH and succinate are able to reduce cytochrome c in IL-3-withdrawn cell lysates, suggesting that complexes I-III are functional in the presence of caspase activity.…”

“…The function of complexes I þ III and II þ III was assayed as previously described on a Beckman DU-640 spectrophotometer (Barrientos et al, 1998). Briefly, after 48 h in the presence or absence of IL-3, cells were freeze-thawed in PBScontaining 10% glycerol.…”