Evidence for rotation of V<sub>1</sub>-ATPase

Hiromi Imamura

Masahiro Nakano

Hiroyuki Noji

Eiro Muneyuki

Shoji Ohkuma

Masasuke Yoshida

Ken Yokoyama

^{ATP System Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation (JST), 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan;}^{Precursory Research for Embryonic Science and Technology (PRESTO), JST, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan;}^{Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan; and}^{Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan}

^{To whom correspondence should be addressed. E-mail:}pj.ca.hcetit.ser@ar-amayokoyk.

Edited by Paul D. Boyer, University of California, Los Angeles, CA, and approved December 24, 2002

Received 2002 Nov 8

Abstract

V_oV₁-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to F_oF₁-ATP synthase, V_oV₁-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 V₁-ATPase, a catalytic subcomplex of V_oV₁-ATPase. V₁-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_oV₁-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Abstract

Two subclasses of the ATPase/ATP-synthase superfamily that catalyze the exchange of the energy of proton translocation across membranes and the energy of ATP hydrolysis/synthesis are V_oV₁-ATPase and F_oF₁-ATP synthase (1–3). V_o and F_o are the integral membrane proton channel portion. V₁ and F₁ are the soluble portion that contain catalytic sites for ATP hydrolysis/synthesis. V_oV₁-ATPases exist in membranes of various intracellular acidic compartments of eukaryotic cells (1, 4) and plasma membranes of Archaea and some eubacteria (5–7). F_oF₁-ATP synthases are responsible for ATP production in mitochondria, chloroplasts, and respiring bacteria (2). Several years ago, rotation of the centrally located γ subunit in the surrounding α₃β₃ hexamer cylinder in the isolated F₁ was video-imaged (8), and the rotary catalysis mechanism of F_oF₁-ATP synthase was established (3, 9). Given the functional and structural similarity between V_oV₁-ATPase and F_oF₁-ATP synthase, it has been assumed that V_oV₁-ATPase would use a similar rotary mechanism to the F_oF₁-ATP synthase. However, without a precise knowledge of the atomic structure or even accurate subunit arrangement in V_oV₁-ATPase, the attempts to prove this assumption have been unsuccessful.

We previously identified V_oV₁-ATPase in an aerobic thermophilic eubacterium, Thermus thermophilus (5, 10, 11). The V₁ portion of T. thermophilus, which is ATPase-active and hence called V₁-ATPase, is made up of four subunits: A (63.6 kDa), B (53.1 kDa), D (24.7 kDa), and F (11.7 kDa) with a stoichiometry of A₃B₃D₁F₁ (5). The A subunit contains a catalytic site, and the A and B subunits are arranged alternately, forming a hexameric cylinder similar to the α₃β₃ of F_oF₁-ATPase. The D subunit most likely fills the central cavity of the A₃B₃ cylinder (11). Cross-linking studies have suggested that the D subunit forms part of the central stalk, and the F subunit is assumed to be associated with the D subunit (12–14). Here we report the visual demonstration of ATP-dependent rotation of V₁-ATPase. The results also show that both D and F subunits constitute a rotor shaft with respect to the stator A₃B₃ cylinder.

Acknowledgments

We thank C. Ikeda for enzyme preparation and assays; T. Hisabori, K. Tabata, T. Ariga, K. Shimabukuro, and H. Ueno for critical discussions and technical advice; and B. Bernadette for critical assessment of the manuscript. This work was supported by Ministry of Education, Science, and Culture of Japan Grand-in-Aid for Scientific Research 12680629 (to K.Y.).

Acknowledgments

Abbreviation

rps	revolutions per second

Abbreviation

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

^{The viscous frictional load of the rotating bead at 2.6 rps is calculated to be ≈15 pN⋅nm (}18). The rotation is not impeded by the load of this magnitude, because the ATP hydrolysis rate of immobilized, bead-attached V₁-ATPase estimated from the rate of bead rotation is nearly the same as the ATP hydrolysis rate of free V₁-ATPase in the bulk medium, where the frictional load is negligible.

Footnotes

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