Characterization of F0F1 ATP synthase from pea stem mitochondria: does it form the mitochondrial permeability transition pore in plants?

Recent results suggest that the mitochondrial permeability transition pore (PTP) in mammals is actually formed by FOF1 ATP synthase dimers, showing electrophysiological properties identical to those of the mitochondrial megachannel (Giorgio et al., 2013; Bernardi et al., 2013). Nevertheless, in plant mitochondria the permeability transition (PT) is still elusive being evidenced only in few peculiar cases (Vianello et al., 2012). Therefore, the analysis of ATP synthase components and its activity in plant mitochondria is crucial to compare such an enzyme in relation to its mammalian counterpart. Purified mitochondria from pea stem were subjected to BN-PAGE and after the electrophoretic separation the gel was stained for ATPase activity. The active bands with an apparent molecular weight of ATP synthase dimers were then run under SDS-PAGE and blotted onto nitrocellulose. The immunodecoration with primary antibodies raised against mammalian cyclophilin D (CyPD) and oligomycin sensitivity-conferring protein (OSCP), respectively, strongly suggested the presence of such proteins associated to ATP synthase even if CyPD interaction with ATP synthase depended on the presence of inorganic phosphate. ATP synthase was also characterized in sub-mitochondrial particles (SMP), evaluating its activity both as ATP hydrolysis (scalar activity, detected as phosphate release) and proton pumping activity (vectorial activity, evaluated as acridine orange quenching). The scalar ATPase activity was dependent on divalent ions such as Mg2+, Mn2+ and even Ca2+. On the other hand, ATP induced the formation of a proton gradient in SMP in the presence of Mg2+ and Mn2+, while Ca2+ was ineffective. Oligomycin was always inhibitory on ATPase activity, regardless the divalent cation utilized, and on both scalar and vectorial activities. Our results show that ATP synthase in plant mitochondria possesses a similar structural and functional properties respect with the animal counterpart (Dabbeni-Sala et al., 2012). Nevertheless, even if CyPD seems to associate to the ATP synthase complex, there is still no evidence about the functional consequences on enzyme catalysis, in particular, on its capacity to modulate the ATP synthase switch to form the PTP in plant mitochondria.