title: Torque generation of Enterococcus hirae V-ATPase.
abstract: V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1.
authors.affiliation: From the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueno
authors.affiliation: the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Minagawa
authors.affiliation: the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Hara
authors.affiliation: the Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan.
authors.forename: Suhaila
authors.initials: S
authors.lastname: Rahman
authors.affiliation: the Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
authors.affiliation: From the Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551, Japan.
authors.forename: Eiro
authors.initials: E
authors.lastname: Muneyuki
authors.affiliation: the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.affiliation: the Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan, JST, PRESTO, Chiba 263-8522, Japan, t.murata@faculty.chiba-u.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.affiliation: the Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan, and the Department of Functional Molecular Science, School of Physical Sciences, The Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan iino@ims.ac.jp.
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D002384:Catalysis; D016983:Enterococcus / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D016963:Thermus thermophilus / Q000201:enzymology; D019415:Torque; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012964:Sodium; D025262:Vacuolar Proton-Translocating ATPases
keywords: Bioenergetics; Membrane Protein; Molecular Motor; Single-molecule Biophysics; V-ATPase; Vacuolar ATPase