Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus.

The Journal of Biological Chemistry
Jun-ichi Kishikawa, Ken Yokoyama

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 th...Continue Reading

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Citations

Feb 11, 2014·Biochimica Et Biophysica Acta·Vladimir MarshanskyGerhard Grüber
Jan 10, 2018·Nature Communications·Atsuko NakanishiKen Yokoyama
Oct 30, 2019·Biophysics and Physicobiology·Atsuko NakanishiKen Yokoyama
Nov 20, 2013·The Journal of Biological Chemistry·Jun-ichi KishikawaKen Yokoyama

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Methods Mentioned

BETA
fluorescence resonance
FRET
electrophoresis

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