Background/Aims: The permeability transition pore (PTP) is an unselective, Ca 2+ -dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca 2+ -activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu 2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu 2+ treatment and show PTP-dependent mitochondrial Ca 2+ release but not swelling. Cu 2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.

High-conductance channel formation in yeast mitochondria is mediated by F-ATP synthase e and g subunits / Carraro M.; Checchetto V.; Sartori G.; Kucharczyk R.; Di Rago J.-P.; Minervini G.; Franchin C.; Arrigoni G.; Giorgio V.; Petronilli V.; Tosatto S.C.E.; Lippe G.; Szabo I.; Bernardi P.. - In: CELLULAR PHYSIOLOGY AND BIOCHEMISTRY. - ISSN 1015-8987. - ELETTRONICO. - 50:5(2018), pp. 1840-1855. [10.1159/000494864]

High-conductance channel formation in yeast mitochondria is mediated by F-ATP synthase e and g subunits

Giorgio V.;
2018

Abstract

Background/Aims: The permeability transition pore (PTP) is an unselective, Ca 2+ -dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca 2+ -activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu 2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu 2+ treatment and show PTP-dependent mitochondrial Ca 2+ release but not swelling. Cu 2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.
2018
High-conductance channel formation in yeast mitochondria is mediated by F-ATP synthase e and g subunits / Carraro M.; Checchetto V.; Sartori G.; Kucharczyk R.; Di Rago J.-P.; Minervini G.; Franchin C.; Arrigoni G.; Giorgio V.; Petronilli V.; Tosatto S.C.E.; Lippe G.; Szabo I.; Bernardi P.. - In: CELLULAR PHYSIOLOGY AND BIOCHEMISTRY. - ISSN 1015-8987. - ELETTRONICO. - 50:5(2018), pp. 1840-1855. [10.1159/000494864]
Carraro M.; Checchetto V.; Sartori G.; Kucharczyk R.; Di Rago J.-P.; Minervini G.; Franchin C.; Arrigoni G.; Giorgio V.; Petronilli V.; Tosatto S.C.E.; Lippe G.; Szabo I.; Bernardi P.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/794412
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