Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel

Abstract

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) the 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, WT 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 WT 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.

ATP synthase c-subunit ring as the channel of mitochondrial permeability transition: Regulator of metabolism in development and degeneration

“…Further studies have shown that these proteins regulate channel behavior rather than constitute the pore of the mitochondrial permeability transition (mPTP) [44][45][46][47]. Mitochondrial F 1 F O ATP synthase was suggested to be a key player in mPTP formation according to several recent reports [28,30,[48][49][50][51][52][53][54][55][56][57]. The peptidyl-prolyl cis-trans isomerase cyclophilin D (CypD) is a well-known regulator of mPTP [58,59].…”

“…Multidisciplinary studies performed by Bernardi's group suggested that the channel is located between ATP synthase dimers with the direct involvement of F O subunits e and g [48][49][50][51][52]. Yeast mutant strains lacking subunits e and g, subunits that are necessary for dimer formation [61,62], displayed a striking resistance to mPTP opening suggesting at first glance that ATP synthase dimerization is required for pore formation in situ [49,50].…”

“…Yeast mutant strains lacking subunits e and g, subunits that are necessary for dimer formation [61,62], displayed a striking resistance to mPTP opening suggesting at first glance that ATP synthase dimerization is required for pore formation in situ [49,50]. The Arg-8 residue of subunit e was found to be crucial for stabilizing the ATP synthase dimers and for formation of full conductance mitochondrial megachannels in yeast [51]. Furthermore, channel formation was observed in detergent-solubilized tetramers and dimers but not in monomers of F 1 F O ATP synthase purified from bovine and porcine hearts [48,52].…”

F‐ATP synthase and the permeability transition pore: fewer doubts, more certainties

“…When extracted from BN gels and tested for Ca 2+ ‐dependent channel generation, wild‐type dimers displayed the high‐conductance channel activity expected for the PTP, whereas dimers (obtained at low digitonin concentrations) from the subunit e R8A/R8E variants displayed strikingly small conductance. Remarkably, the simultaneous subunit e R8E/subunit g E83K substitutions restored the high‐conductance channels, indicating that the interaction of subunit e with subunit g is important for generation of the full‐conductance PTP/MMC from F‐ATP synthase .…”

“…Individual substitutions R8A/R8E in subunit e, or E83A/E83K in subunit g decreased the stability of F-ATP synthase to digitonin, resulting in decreased dimer formation. Simultaneous subunit e R8E/subunit g E83K substitutions rescued digitonin-stable F-ATP synthase dimers, suggesting that electrostatic interaction between R8 of subunit e and E83 of subunit g contributes to dimer stability [106]. When extracted from BN gels and tested for Ca 2+ -dependent channel generation, wildtype dimers displayed the high-conductance channel activity expected for the PTP, whereas dimers (obtained at low digitonin concentrations) from the subunit e R8A/R8E variants displayed strikingly small conductance.…”