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

Authors
  • Carraro, Michela
  • Checchetto, Vanessa
  • Sartori, Geppo
  • Kucharczyk, Roza
  • di Rago, Jean-Paul
  • Minervini, Giovanni
  • Franchin, Cinzia
  • Arrigoni, Giorgio
  • Giorgio, Valentina
  • Petronilli, Valeria
  • Tosatto, Silvio C.E.
  • Lippe, Giovanna
  • Szabó, Ildikó
  • Bernardi, Paolo
Type
Published Article
Journal
Cellular Physiology and Biochemistry
Publisher
S. Karger AG
Publication Date
Nov 13, 2018
Volume
50
Issue
5
Pages
1840–1855
Identifiers
DOI: 10.1159/000494864
PMID: 30423558
Source
Karger
Keywords
License
Green
External links

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) 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. 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.

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