Abstract

Abstract Vacuolar-type rotary H+-ATPase/synthase (VoV1) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V1 (A3B3D1F1) and Vo (C1E2G2I1L12) subcomplexes in vitro. A3B3D and A3B3 also reconstituted with Vo, resulting in a holoenzyme-like complexes. However, A3B3D-Vo and A3B3-Vo did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V1 or A3B3D and a donor dye attached to subunit C in Vo. The estimated dissociation constants Kd for VoV1 and A3B3D-Vo were ∼0.3 and ∼1 nm at 25 °C, respectively. These results suggest that the A3B3 domain tightly associated with the two EG peripheral stalks of Vo, even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A3B3 with Vo is much lower than that of the EG peripheral stalks.

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