Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA

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Abstract
Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed ρ° cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in ρ° cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4–for ADP3–by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1‐ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin‐permeabilized mammalian ρ° cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in ρ° cells and an incomplete FoF1‐ATP synthase was also required. The maximum value of this potential was ≈110 mV in permeabilized cells and ≈ 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1‐ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear‐encoded proteins but ≈ 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of ρ° mitochondria showed that the azide‐sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1‐ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1‐ATP synthase in maintaining the mitochondrial membrane potential in ρ° cells.

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