High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits.
Cell Physiol Biochem
; 50(5): 1840-1855, 2018.
Article
in En
| MEDLINE
| ID: mdl-30423558
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.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Saccharomyces cerevisiae
/
Mitochondrial Proton-Translocating ATPases
/
Saccharomyces cerevisiae Proteins
/
Mitochondria
Language:
En
Journal:
Cell Physiol Biochem
Journal subject:
BIOQUIMICA
/
FARMACOLOGIA
Year:
2018
Document type:
Article
Affiliation country:
Italy