ATP synthase dimers from pea stem mitochondria form the PTP

The Permeability Transition Pore (PTP) is a mitochondrial mega-channel well characterized in mammals, yeast and Drosophila. In these organisms, it has been recently demonstrated that the PTP is a channel formed by dimers of F-ATP synthase. Even if plant mitochondria possess all the components needed for PTP formation, the evidence for permeability transition (PT) in plants is still circumstantial. Therefore, this work was undertaken to characterize the plant mitochondria PTP and to verify if plant F-ATP synthase possesses features similar to those already seen in other species. This would help to understand the evolution of the F-ATP synthase channel functions. Pea stem mitochondria showed a substrate-dependent electrical potential difference formation. In the presence of the ionophore ETH129, trains of Ca2+ additions induced transient spikes, indicating a calcium uptake into the mitochondrial matrix. When Ca2+ concentration reached approx. 0.3 mM, an abrupt collapse of the membrane potential was observed, significantly delayed by Cyclosporin A (CsA) and Pi. Similarly to membrane potential, Ca2+ Retention Capacity (CRC) showed a trend suggesting that plant mitochondria, when permeabilized to Ca2+, can undergo a PT. Purified mitochondria were also subjected to Blue-native (BN)-PAGE and F-ATP synthase activity was detected in situ. The active bands, corresponding to the dimers, were eluted from the gel, and analyzed for the presence of F-ATP synthase components and of Cyclophylin D (CyPD). The active bands of the F-ATP synthase dimers were also inserted into an artificial bilayer, to evaluate the channel activity by electrophysiology experiments. This research is supported by MIUR grant PRIN 2010CSJX4F