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High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits

Carraro, Micaela and Checchetto, Vanessa and Sartori, Geppo and Kucharczyk, Roza and di Rago, Jean-Paul and Minervini, Giovanni and Franchin, Cinzia and Arrigoni, Giorgio and Giorgio, Valentina and Petronilli, Valeria and Tosatto, Silvio CE and Lippe, Giovanna and Szabó, Ildico and Bernardi, Polo (2018) High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits. Cellular Physiology and Biochemistry, 50 (5). pp. 1840-1855. ISSN 1015-8987

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Abstract

Abstract Background/Aims: The permeability transition pore (PTP) is an unselective, Ca2+-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 Ca2+-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) a-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. Cu2+ 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 Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ 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.

Item Type:Article
Subjects:Q Science > Q Science (General)
Q Science > QP Physiology
Divisions:Department of Genetics
ID Code:1638
Deposited By: Dr hab Roza Kucharczyk
Deposited On:29 Nov 2018 09:59
Last Modified:29 Nov 2018 09:59

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