Functional F1-ATPase Essential in Maintaining Growth and Membrane Potential of Human Mitochondrial DNA-depleted ρ° Cells

Abstract

F1-ATPase assembly has been studied in human °c ells devoid of mitochondrial DNA (mtDNA). Since, in these cells, oxidative phosphorylation cannot provide ATP, their growth relies on glycolysis. Despite the absence of the mtDNA-coded F0 subunits 6 and 8, °cells possessed normal levels of F1-ATPase ␣ and ␤ subunits. This F1-ATPase was functional and azide-or aurovertinsensitive but oligomycin-insensitive. In addition, aurovertin decreased cell growth in °cells and also reduced their mitochondrial membrane potential, as measured by rhodamine 123 fluorescence. Therefore, a functional F1-ATPase was important to maintain the mitochondrial membrane potential and the growth of these °c ells. Bongkrekic acid, a specific adenine nucleotide translocator (ANT) inhibitor, also reduced °cell growth and mitochondrial membrane potential. In conclusion, °cells need both a functional F1-ATPase and a functional ANT to maintain their mitochondrial membrane potential, which is necessary for their growth. ATP hydrolysis catalyzed by F1 must provide ADP 3؊ at a sufficient rate to maintain a rapid exchange with the glycolytic ATP 4؊ by ANT, this electrogenic exchange inducing a mitochondrial membrane potential efficient enough to sustain cell growth. However, since the effects of bongkrekic acid and of aurovertin were additive, other electrogenic pumps should cooperate with this pathway.The biogenesis of mitochondrial proteins is controlled by both nuclear and mitochondrial genomes. The proteins coded by the mtDNA are subunits of enzyme complexes involved in oxidative phosphorylation. Since all these complexes also contain proteins coded by the nuclear genome, mechanisms regulating the coordinated expression and assembly of the subunits of nuclear and mitochondrial origin must exist. In yeast cells, nuclear DNA-coded components continue to be synthesized and imported into mitochondria, even when the synthesis of mtDNA-coded subunits is blocked (cf. for review, Refs. 1 and 2). The groups of Schatz and co-workers (3) and Neupert (4) have shown that a mitochondrial membrane potential is a key requirement for protein import into mitochondria (5). In normal cells, the mitochondrial membrane potential is mainly maintained by transmembrane proton pumping occurring during electron transfer or during ATP hydrolysis catalyzed by the ATPase-ATP synthase. In °cells depleted of mtDNA, these complexes cannot be functional since all complexes involved in proton pumping contain mtDNA-coded subunits (6). However, proteins of nuclear origin are imported into the °cell mitochondria (7). Therefore, the mitochondrial membrane potential must be maintained by other electrogenic pumps. In yeast, it has been suggested that the adenine nucleotide translocator (ANT) 1 mediates an exchange of ATP 4Ϫ synthesized in the cytosol during glycolysis for ADP 3Ϫ to maintain this mitochondrial membrane potential (8). However, the exact mechanism that maintains this potential has not been thoroughly investigated. Since the mtDNA-coded subunits are essential components for ...

Resistance of mitochondrial DNA-deficient cells to TRAIL: role of Bax in TRAIL-induced apoptosis

“…This ®nding is consistent with the absence of cytochrome c translocation in r 0 cells, while dissociation between cytochrome c release and mitochondrial potential change has been reported (Bossy-Wentzel et al, 1998). Previous papers employing mammalian cells or yeast reported that mitochondrial potential is preserved in mitochondrial DNA-depleted cells, owing to compensatory adenine nucleotide translocator function allowing countertransport of adenine nucleotides across the mitochondrial membrane (Buchet and Gedinot, 1998;Drgon et al, 1991;Skowronek et al, 1992). No signi®cant basal and glucose-stimulated mitochondrial proton gradient previously observed in SK-Hep1 r 0 cells might be due to the di erence in the method of measurement between¯ow cytometry and quenching method (Park et al, 2001).…”

Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

“…This may explain why complementation by wild-type mtDNA is hampered at high doses of mutant mtDNA in vivo (Boulet et al, 1992;Nakada et al, 2001). In contrast, fusion capacity should not be affected in clones of 0-cells repopulated with mutant mtDNA (Bakker et al, 2000;Enriquez et al, 2000;Ono et al, 2001), because they are expected to maintain a ⌬⌿m similar to that of parental 0-cells (Buchet and Godinot, 1998). Therefore, mitochondrial fusion should not be the limiting factor for functional complementation between mitochondria carrying different mutants of mtDNA.…”

“…Because the available clones of 0-cells (HeLa-0 and 143B-0) maintain a ⌬⌿m that allows the import of nuclear-encoded proteins and the maintenance of several mitochondrial functions (Buchet and Godinot, 1998), we used specific inhibitors of energy metabolism to investigate the energetic requirements of mitochondrial fusion. We used 1) oligomycin to inhibit the mitochondrial ATP-synthetase; 2) the protonophore cccp to dissipate the ⌬⌿m and inhibit mitochondrial ATP-synthesis; and 3) deoxyglucose to sequester cytosolic ATP, block the glycolytic pathway, and inhibit cytosolic ATP synthesis.…”

Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology

“…According to this and previous studies (55), despite the failure to generate ATP oxidatively, rotenone only slightly depolarized mitochondria, maintaining more than 75% of control ⌬ m , even when CI was completely inhibited for long incubation periods (3 days). Glycolysis provides the cells with sufficient ATP, which can be used via the mitochondrial ADP/ATP translocator to create a ⌬ m across the mitochondrial inner membrane (56). Such a fall in ⌬ m may be insufficient to trigger apoptosis when cells grow in glucose medium, and additional biochemical insults (e.g.…”