Structural characterization of an ATPase active F-1-/V-1-ATPase (alpha(3)beta(3)EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the α and β subunits of the F1-ATPase from the thermophilic Bacillus PS3 (TF1) resulted in an α3β3EG hybrid complex showing 53% of the ATPase activity of TF1. The α3β3EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2–2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the α3β3 subcomplex from TF1 demonstrates that a seventh mass is located inside the shaft of the α3β3 barrel and extends out from the hexamer. Furthermore, difference mapping of the α3β3EG oligomer with projections of the A3B3E and A3B3EC subcomplexes of the V1 from Caloramator fervidus shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active α3β3EG hybrid complex indicates that the coupling subunit γ inside the α3β3 oligomer of F1 can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and γ subunits are structurally similar, despite the fact that their genes do not show significant homology