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High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits
- Source :
- Cellular Physiology and Biochemistry, Vol 50, Iss 5, Pp 1840-1855 (2018)
- Publication Year :
- 2018
- Publisher :
- Cell Physiol Biochem Press GmbH & Co KG, 2018.
-
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.
- Subjects :
- 0301 basic medicine
Yeast mitochondria
Protein Structure
Secondary
Mitochondrial Proton-Translocating ATPase
Saccharomyces cerevisiae Proteins
Physiology
Protein subunit
Mutant
Saccharomyces cerevisiae
Mitochondrion
Membrane Potential
F-ATP synthase
Protein Structure, Secondary
lcsh:Physiology
Calcium
Mitochondrial megachannel
Permeability transition
Cryoelectron Microscopy
Dimerization
Membrane Potential, Mitochondrial
Mitochondria
Mitochondrial Proton-Translocating ATPases
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Protein Subunits
lcsh:Biochemistry
03 medical and health sciences
Site-Directed
lcsh:QD415-436
Inner mitochondrial membrane
Protein Subunit
Yeast mitochondria • Mitochondrial megachannel • Permeability transition • F-ATP synthase • Calcium
Membrane potential
ATP synthase
biology
lcsh:QP1-981
Chemistry
Mutagenesis
Transmembrane protein
Mitochondrial
030104 developmental biology
Biophysics
biology.protein
Tertiary
Subjects
Details
- Language :
- English
- ISSN :
- 14219778 and 10158987
- Volume :
- 50
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Cellular Physiology and Biochemistry
- Accession number :
- edsair.doi.dedup.....1a03929e38d72e2ca63fb9052f40b16d