New insights into S. cerevisiae F-ATP synthase and its role in the mitochondrial permeability transition

The mitochondrial Permeability Transition Pore (PTP) has been characterized in mammals as a Ca2+-activated channel whose opening allows solutes of molecular mass ≤ 1.5 kDa to equilibrate across the inner membrane, potentially causing matrix swelling, rupture of the outer membrane and release of pro-apoptotic factors. Together with Ca2+, the onset of PTP opening is favored by oxidative stress, membrane depolarization and Pi; conversely, acidic pH, adenine nucleotides and Mg2+ are the major physiological desensitizers. So far, cyclosporin A and derived compounds that prevent the binding of the positive effector cyclophilin D, as well as new compounds identified by our laboratory, have been discovered to be potent antagonists of the channel. Whether or not the PTP is a conserved feature in yeast has long been debated. Several studies suggested that yeast mitochondria might present different independent permeability pathways with peculiar features and modulations, including an ATP-activated pathway that might play a role as an energy-dissipating mechanism. A high-conductance channel considered to be the yeast equivalent of the PTP (yPTP) is inhibited rather than activated by Pi and is insensitive to CsA, and due to the lack of a mitochondrial Ca²⁺ uniporter in yeast, its Ca²⁺ dependence has been difficult to assess. In our recent paper (see Publication 1), we confirmed the existence of Ca²⁺-activated PTP in S. cerevisiae, as previously suggested by Shinohara and Coworkers, taking advantage of the Ca2+ ionophore ETH129, which allows the electrophoretic accumulation of added Ca2+. We found that yPTP opening is strongly sensitized by the thiol reagents phenylarsine oxide, copper-o-phenantroline and diamide, which are effective PTP inducers in mammals, and that this effect could be reverted by reducing reagents. Our laboratory recently proposed that F-ATP synthase dimers are the crucial component of mammalian PTP (mPTP). We carried out a Blue-Native PAGE separation of yeast mitochondrial proteins with digitonin, identified F-ATP synthase dimers through an in-gel activity assay for ATP hydrolysis, and reconstituted them in planar lipid bilayer for electrophysiology experiments. Our data show that dimers of yeast F-ATP synthase form a Ca²⁺- and oxidant-activated channel with “flickering” features resembling those of the mammalian PTP, although with a lower conductance of about 250-300 -pS. Like in mammals, activity of the channel was inhibited by ADP and Mg²⁺. These findings provide evidence that the PTP-forming properties of F-ATP synthase have been conserved in evolution. To verify the role of F-ATP synthase dimer in yPTP formation, we analyzed knock-out mutants for subunits reported to be involved in dimerization of the enzyme. These subunits, localized in the lateral stalk, are subunit e (TIM11) and- subunit g (ATP20). ΔTIM11, ΔATP20, as well as double null mutants, did not display the presence of dimers when analyzed with a BN-PAGE. Furthermore, the Ca2+ retention capacity (CRC, a measure of the propensity to pore opening) of mitochondria devoid of subunits e and/or g displayed an increased resistance to yPTP opening. BN-PAGE analysis after treatment with CuCl2, which promotes the formation of disulfide bridges between adjacent Cys residues, demonstrated that some dimers formed upon thiol oxidation even in absence of “dimerization” subunits, which may explain why the yPTP could open at high Ca2+ concentration even in their absence. Very interestingly, these dimers show a channel conductance of about 30 -pS, which is remarkably decreased compared to that measured in wild-type strain. Further analysis in single KO mutant for e- and g subunits is under way in order to define the contribution of single subunits to channel conductance. To identify the Cys residues affected by CuCl2 treatment (which should be involved in dimer formation following oxidation), we carried out site-directed mutagenesis of each of the Cys residues of F-ATP synthase in the e- and g- null genetic background. There are eight Cys residues in the yeast enzyme (six in subunits encoded by nuclear DNA and two in subunits encoded by mitochondrial DNA). We found that Cys23 of subunit a (ATP6) is the target of oxidation, and mediates formation of a disulfide bridge between the two monomers thereby stabilizing the dimeric form. Another important finding was that the unique Cys of OSCP, the subunit located on top of the lateral stalk of F-ATP synthase, appears to mediate the effect of diamide on PTP activity, at least at low concentrations. Indeed, CRC analysis revealed that OSCP C100S mutant are less sensitive specifically to diamide, whereas the sensitivity to other oxidants is unaffected. My results provide the first demonstration that yeast F-ATP synthase dimers form high conductance channels analogous to those formed by the mPTP and thus that channel formation is an evolutionarily conserved feature of F-ATP synthases. It is now possible to unravel the many open questions about structure and regulation of the PTP with the powerful methods of yeast genetics