Evidence for rotation of V1-ATPase

Abstract

V(o)V(1)-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to F(o)F(1)-ATP synthase, V(o)V(1)-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V(1)-ATPase, a catalytic subcomplex of V(o)V(1)-ATPase. V(1)-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that V(o)V(1)-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Figure 1

(A) Experimental setup to observe rotation of V₁-ATPase. The V₁-ATPase was fixed on the glass surface with amino-terminal His-8 tags of the A subunits. A bead was attached to the D (Left) or F (Right) subunit through biotin-streptavidin linkage. Rotation of obliquely attached beads was observed. The arrows indicate the direction of rotation. (B) Biotinylation of the D and F subunits. The biotinylated V₁-ATPases were analyzed with 15% acrylamide gel electrophoresis in the presence of SDS. (Left) Protein staining with Coomassie brilliant blue. (Right) Western blotting stained by alkaline phosphatase-streptavidin conjugate. Lanes 1 and 5, V₁-ATPase that has the biotinylated D subunit; lanes 2 and 6, V₁-ATPase that has the biotinylated F subunit; lanes 3 and 7, nonbiotinylated V₁-ATPase; lanes 4 and 8, molecular mass standards (250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa).

Figure 2

Sequential images of a rotating bead at 4 mM ATP. A bead attached to the D subunit (A)and that attached to the F subunit (B) are shown. Centroid positions are shown above the images. The interval between images is 33 msec.

Figure 3

Time courses of rotation of beads attached to the D subunit. (A) Bead rotation at 4 mM ATP in the presence of sodium azide (0.5 mM). (B–D) Bead rotation in the absence of sodium azide at 4 (B), 0.5 (C), and 0.2 (D) mM ATP.

Figure 4

Time courses of rotation of beads attached to the F subunit. A bead was attached to the F subunit, and the rotation was observed at 4 mM ATP. The details of experimental conditions are as described in Materials and Methods.