Rotation scheme of V1-motor is different from that of F1-motor

Abstract

V(1), a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to F(1)-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the A(3)B(3) cylinder. In this study, we analyzed the rotation kinetics of V(1) in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120 degrees . The dwell time between steps revealed that V(1) consumes one ATP per 120 degrees step. V(1) generated torque of approximately 35 pN nm, slightly lower than the approximately 46 pN nm measured for F(1). Noticeably, the angles for both ATP cleavage and binding were apparently the same in V(1), in sharp contrast to F(1), which cleaves ATP at 80 degrees posterior to the binding of ATP. Thus, the mechanochemical cycle of V(1) has marked differences to that of F(1).

Fig. 1.

ATP-driven rotation of V₁. Rotation was visualized under a microscope by attaching a duplex of 340-nm beads to the D subunit. (a) ATP dependence of rotation speed and ATPase rate. Time-averaged rotation speed of the D subunit of single-molecule V₁ (blue circle) and one-third of bulk-phase ATPase rate (red circle) are plotted against MgATP concentration. By using the dependence of the rates observed on the concentration of ATP with the Michaelis–Menten equation, bead rotation (blue dotted line) occurred with a V_max of 8.1 Hz and a K_m of 107 μM, and bulk-phase ATP hydrolysis (red dotted line) occurred with a V_max of 24.5·s^–1 and a K_m of 333 μM. (b) Stepwise rotation of the D subunit at 1 μM MgATP recorded at 30 fps. (Insets b and c) The centroid of the rotating bead. (c) Stepwise rotation of the D subunit at 4 μM MgATP recorded at 30 fps. (d) Histogram of dwell time between successive steps at 1 μM MgATP (n = 306) is fitted with a single exponential: k_on = (4.17 ± 0.13) × 10⁵ M^–1·s^–1 (mean ± SE). (e) Histogram of dwell time between successive steps at 4 μM MgATP (n = 542) is fitted with a single exponential: k_on = (3.19 ± 0.39) × 10⁵ M^–1·s^–1 (mean ± SE).

Fig. 2.

Estimation of torque. (a) Magnification of step. To better resolve stepping rotations, rotation of a duplex of 340-nm beads was recorded with a high-speed camera at 1,000 fps. The concentration of MgATP used was 10 and 0.5 μM for V₁ and F₁, respectively. Steps distinguished from pauses by eye (red circle for step and black circle for pause) are fitted with linear segments (shown by thick blue line) to estimate the average angular velocity. (b) Torque generated in a step by V₁ and F₁. The torque in each step was calculated as ξω, where ξ is the frictional load of the bead, and ω is stepping angular velocity, estimated as in a. Frictional load ξ was calculated as described in ref. . Here, we assumed that the center of rotation is at the center of one of two beads, and that bead duplex is horizontal to the glass surface. A set of nine successive steps was chosen for each molecule. The box represents torque averaged over five molecules (45 steps) ±SEM.

Fig. 3.

ATPγS-driven rotation of V₁. Rotation was visualized under a microscope by attaching a duplex of 209-nm beads to the D subunit and recorded at 30 fps. (a) Dependence of rotation speed on concentration of ATPγS. (b) Rotation of the D subunit over time at 4 mM (green) and 200 μM (red) ATPγS. (Inset) Position of the bead centroid. (c) Observation of a single rotating V₁ in different conditions. After recording the rotation of the D subunit at 4 mM ATPγS(Left), buffer containing 10 μM ATP was infused into the flow cell (see Movie 1, which is published as supporting information on the PNAS web site), followed by further recording of the rotation (Right). See Movie 2, which is published as supporting information on the PNAS web site). (Inset) Position of the bead centroid. (d) Angular position of ATPγS cleavage relative to that of ATP binding. Histograms of angular distribution of the D subunit at 4 mM ATPγS(Upper) and at 10 μM ATP (Lower) of a single V₁ molecule shown in c. The center of each dwell position was estimated by fitting to a Gaussian equation (red line).

Fig. 4.

Possible rotation model. Rotation model for V₁ (a) and F₁ (b). Three catalytic sites are represented by blue (V₁) or green (F₁) filled circles, and the orientation of the D subunit or γ subunit is represented by arrows. Of the three catalytic sites, two are always occupied by nucleotides in this model. Other models such that three catalytic sites are occupied by one or two nucleotides (bi-site model) or by two or three nucleotides (tri-site model) might also be possible.