title: Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus.
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.
authors.affiliation: Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Kishikawa
authors.forename: Ken
authors.initials: K
authors.lastname: Yokoyama
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C474451:F1F0-ATP synthase; D025262:Vacuolar Proton-Translocating ATPases; D025261:Mitochondrial Proton-Translocating ATPases