title: Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency.
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 (rho degrees), 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 Vmax was decreased in HXC by approximately 40%, and the Km 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.
authors.affiliation: Department of Neurology, University of Miami, School of Medicine, Miami, Florida 33136, USA.
authors.forename: A
authors.initials: A
authors.lastname: Barrientos
authors.forename: L
authors.initials: L
authors.lastname: Kenyon
authors.forename: C T
authors.initials: CT
authors.lastname: Moraes
mesh_terms: D000818:Animals; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002999:Clone Cells / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D019143:Evolution, Molecular; D015186:Hominidae; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000172:deficiency; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption / Q000502:physiology; D012402:Rotenone / Q000494:pharmacology
chemical_list: D004272:DNA, Mitochondrial; D012402:Rotenone; D009243:NAD; D016660:NAD(P)H Dehydrogenase (Quinone)