Abstract
Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) 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 V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ∼0.3 and ∼1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), 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 A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.
Highlights
The VoV1 is composed of the hydrophilic V1 and the membrane-embedded Vo
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 ϳ0.3 and ϳ1 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
Summary
The VoV1 is composed of the hydrophilic V1 and the membrane-embedded Vo. Results: Intact VoV1 and shaftless complexes can be reconstituted from individual subunits in vitro. Significance: The peripheral stalks are the major factor for the association of V1 with Vo. 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. The estimated dissociation constants Kd for VoV1 and A3B3D-Vo were ϳ0.3 and ϳ1 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. VoV1 and F-type ATPases (FoF1) are evolutionary related and share the rotary mechanism coupling ATP synthesis/hydrolysis and proton translocation across the membrane (1, 2) These two types of ATPase exhibit significant differences. A3B3 and A3B3D subcomplexes associated with Vo, suggesting that the peripheral stalks are mainly responsible for connecting V1 to Vo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
