• Orbit mini

    Simultaneous recording from four lipid bilayers

2019 - Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimersand to the generation of the full-conductance megachannel

 Icon Orbit Mini   Orbit mini publication in Journal of Biological Chemistry (2019)

Guo L., Carraro M., Carrer A., Minervini G., Urbani A., Masgras I., Tosatto S.C.E., Szabò I., Bernardi P., Lippe G.

Journal of Biological Chemistry (2019) doi: 10.1074/jbc.RA119.008775


The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis (i) substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation, as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, wild-type dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in wild-type enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

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