Channel formation by yeast F-ATP synthase and the role of dimerization in the mitochondrial permeability transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca(2+)-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca(2+) ionophore ETH129, which allows electrophoretic Ca(2+) uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains \u394TIM11 and \u394ATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ

Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability transition pore

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device

Do dimers of ATP synthase form the PTP in pea stem mitochondria?

In animal cells Ca2+ and ROS induce a sudden change in the inner mitochondrial membrane permeability, which has been named Permeability Transition (PT). Recently, it has been proposed that dimers of F-ATP synthase form the Permeability Transition Pore (PTP), the megachannel involved in this phenomenon [1]. This feature has not yet been characterized in plants, even if their mitochondria possess the candidate components for PTP formation. Therefore, wecharacterized the functional properties of PTP in plant mitochondria and verified if F-ATP synthase possesses channel function in electrophysiology experiments. Mitochondria isolated from pea stem underwent PT when Ca2+ was added in the presence of the ionophore ETH129. The membrane electrical potential was then collapsed and the phenomenon matched by Ca2+ release but not by mitochondrial swelling. As is observed with the PT of animal mitochondria, Cyclosporin A (CsA) significantly delayed occurrence of PT, which was inhibited by Mg2+-nucleotides and favored by benzodiazepine Bz-423 and oxidants, such as phenylarsine oxide and diamide. In electrophysiology experiments, F-ATP synthase dimers inserted into an artificial bilayer showed channel activity characterized by a rather small conductance, which could explain the inability of plant PTP to mediate mitochondrial swelling. These data suggest that F-ATP synthase is involved in PTP formation also in plant mitochondria

Collaboration between NDH and KEA3 allows maximally efficient photosynthesis after a long dark adaptation

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around photosystem I (CET). K+ efflux antiporter 3 (KEA3) is a putative thylakoid H+/K+ antiporter and allows an increase in membrane potential at the expense of the 06pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction 2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis thaliana. The NDH complex pumps protons during CET, further enhancing 06pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 \ub5mol photons m-2 s-1 after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the 06pH-dependent downregulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large 06pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 (PGR5)-overexpression line, in which the size of 06pH was enhanced. When photosynthesis was induced at 300 \ub5mol photons m-2 s-1, the contribution of KEA3 was negligible in the initial phase and the 06pH-dependent downregulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO2 fixation was delayed after overnight dark adaptation

Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and \u3c10 cells lacking subunits a and A6L. \u394g cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. \u394b and \u394OSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from \u3c10 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway