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Torque Generation of Enterococcus hirae V-ATPase*

  • Hiroshi Ueno
    Footnotes
    Affiliations
    Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551, Japan,
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  • Yoshihiro Minagawa
    Footnotes
    Affiliations
    Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan,
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  • Mayu Hara
    Affiliations
    Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan,
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  • Suhaila Rahman
    Affiliations
    Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan,
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  • Ichiro Yamato
    Affiliations
    Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan,
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  • Eiro Muneyuki
    Affiliations
    Department of Physics, Faculty of Science and Engineering, Chuo University, Tokyo 112-8551, Japan,
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  • Hiroyuki Noji
    Affiliations
    Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan,
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  • Takeshi Murata
    Correspondence
    To whom correspondence may be addressed: Dept. of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan. Tel.: 81-43-290-2794; Fax: 81-43-290-2794;
    Affiliations
    Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan,

    JST, PRESTO, Chiba 263-8522, Japan,
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  • Ryota Iino
    Correspondence
    To whom correspondence may be addressed: Okazaki Inst. for Integrative Bioscience, Inst. for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan. Tel.: 81-564-59-5230;
    Affiliations
    Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan, and

    Department of Functional Molecular Science, School of Physical Sciences, The Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan
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  • Author Footnotes
    * This work was supported in part by Grants-in-aid for Scientific Research (70403003, 70403003, 24370062 to R.I., 26291009 to T.M.) and Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
    1 These authors equally contributed to this work.
    4 The abbreviations used are:VoV1V-ATPaseV1V1-ATPaseFoF1FoF1-ATP synthaseF1F1-ATPaseEhVoV1E. hirae V-ATPaseEhV1E. hirae V1-ATPaseSVoV1Saccharomyces cerevisiae V-ATPaseTtVoV1T. thermophilus V-ATPaseTtV1T. thermophilus V1-ATPaseTFoF1thermophilic Bacillus PS3 FoF1-ATP synthaseTF1thermophilic Bacillus PS3 F1-ATPaseEFoF1E. coli FoF1-ATP synthaseEF1E. coli F1-ATPaseNa+sodium ionDDMn-dodecyl β-d-maltosideNi2+-NTAnickel-nitrilotriacetic acidFTfluctuation theoremDCCDN, N′-dicyclohexylcarbodiimideLDAOlauryldimethylamine oxidefpsframes per secondrpsrevolutions per second.
Open AccessPublished:September 25, 2014DOI:https://doi.org/10.1074/jbc.M114.598177
      V-ATPase (VoV1) 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 VoV1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae VoV1 (EhVoV1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhVoV1 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, EhVoV1 only showed the “clear” state without apparent backward steps, whereas EhV1 showed two states, “clear” and “unclear.” Furthermore, EhVoV1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhVoV1 showed faster rotation than EhV1, and the torque of EhVoV1 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 EhVoV1. These results indicate that rotor-stator interactions of the Vo moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhVoV1 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 EhVoV1.

      Introduction

      V-ATPase (VoV1)
      The abbreviations used are: VoV1
      V-ATPase
      V1
      V1-ATPase
      FoF1
      FoF1-ATP synthase
      F1
      F1-ATPase
      EhVoV1
      E. hirae V-ATPase
      EhV1
      E. hirae V1-ATPase
      SVoV1
      Saccharomyces cerevisiae V-ATPase
      TtVoV1
      T. thermophilus V-ATPase
      TtV1
      T. thermophilus V1-ATPase
      TFoF1
      thermophilic Bacillus PS3 FoF1-ATP synthase
      TF1
      thermophilic Bacillus PS3 F1-ATPase
      EFoF1
      E. coli FoF1-ATP synthase
      EF1
      E. coli F1-ATPase
      Na+
      sodium ion
      DDM
      n-dodecyl β-d-maltoside
      Ni2+-NTA
      nickel-nitrilotriacetic acid
      FT
      fluctuation theorem
      DCCD
      N, N′-dicyclohexylcarbodiimide
      LDAO
      lauryldimethylamine oxide
      fps
      frames per second
      rps
      revolutions per second.
      is an ATP-dependent ion pump. Acidification of vesicles by intracellular VoV1 is important for various cellular processes, including receptor-mediated endocytosis, membrane trafficking, and protein processing and degradation (
      • Nishi T.
      • Forgac M.
      The vacuolar H+-ATPases: nature's most versatile proton pumps.
      ). VoV1 also functions on the surface of certain cells, and because of its involvement in bone resorption and tumor metastasis (
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ), it is a drug target for osteoporosis and cancer treatments (
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Bowman E.J.
      • Bowman B.J.
      V-ATPases as drug targets.
      ). VoV1 is a large, multisubunit complex composed of a hydrophilic V1 moiety for hydrolyzing ATP and a membrane-embedded Vo moiety for transporting ions (
      • Nishi T.
      • Forgac M.
      The vacuolar H+-ATPases: nature's most versatile proton pumps.
      ,
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Marshansky V.
      • Futai M.
      The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function.
      ). The V1 and Vo moieties are connected by a central stalk and two or three peripheral stalks (
      • Nishi T.
      • Forgac M.
      The vacuolar H+-ATPases: nature's most versatile proton pumps.
      ,
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Marshansky V.
      • Futai M.
      The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function.
      ,
      • Stewart A.G.
      • Laming E.M.
      • Sobti M.
      • Stock D.
      Rotary ATPases: dynamic molecular machines.
      ,
      • Marshansky V.
      • Rubinstein J.L.
      • Grüber G.
      Eukaryotic V-ATPase: novel structural findings and functional insights.
      ). In the simplest bacterial VoV1, the catalytic core of V1 moiety consists of A, B, D, and F subunits, in which three alternately arranged A and B subunits form a hexameric A3B3 ring, and the central shaft, composed of D and F subunits, penetrates the central cavity of the A3B3 ring (
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ,
      • Maher M.J.
      • Akimoto S.
      • Iwata M.
      • Nagata K.
      • Hori Y.
      • Yoshida M.
      • Yokoyama S.
      • Iwata S.
      • Yokoyama K.
      Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus.
      ,
      • Nagamatsu Y.
      • Takeda K.
      • Kuranaga T.
      • Numoto N.
      • Miki K.
      Origin of asymmetry at the intersubunit interfaces of V-ATPase from Thermus thermophilus.
      ,
      • Numoto N.
      • Hasegawa Y.
      • Takeda K.
      • Miki K.
      Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase.
      ). ATP hydrolysis and synthesis occur on the catalytic sites that are located at the interfaces of the A and B subunits, with the majority of the catalytic residues residing in the A subunits (
      • Arai S.
      • Saijo S.
      • Suzuki K.
      • Mizutani K.
      • Kakinuma Y.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Iwata S.
      • Yamato I.
      • Murata T.
      Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.
      ). The membrane-embedded Vo consists of two different subunits, a and c subunits. The c subunits of the membrane-spanning ring are connected with the central DF axis via the d subunit, and the two peripheral stalks comprising the E and G subunits connect the channel-forming a subunit to the A3B3 ring (
      • Stewart A.G.
      • Laming E.M.
      • Sobti M.
      • Stock D.
      Rotary ATPases: dynamic molecular machines.
      ,
      • Stewart A.G.
      • Lee L.K.
      • Donohoe M.
      • Chaston J.J.
      • Stock D.
      The dynamic stator stalk of rotary ATPases.
      ,
      • Yokoyama K.
      • Imamura H.
      Rotation, structure, and classification of prokaryotic V-ATPase.
      ,
      • Murata T.
      • Yamato I.
      • Kakinuma Y.
      Structure and mechanism of vacuolar Na+-translocating ATPase from Enterococcus hirae.
      ). Enterococcus hirae VoV1 (EhVoV1), the target of this study, functions as a sodium ion (Na+) pump, similar in nature to eukaryotic VoV1 (
      • Murata T.
      • Yamato I.
      • Kakinuma Y.
      Structure and mechanism of vacuolar Na+-translocating ATPase from Enterococcus hirae.
      ,
      • Murata T.
      • Igarashi K.
      • Kakinuma Y.
      • Yamato I.
      Na+ binding of V-type Na+-ATPase in Enterococcus hirae.
      ,
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ,
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety.
      ). In EhVoV1, the membrane rotor ring consists of 10 c subunits. Each c subunit has four transmembrane α-helices, in which a Na+ can be bound to the specific binding pocket (
      • Mizutani K.
      • Yamamoto M.
      • Suzuki K.
      • Yamato I.
      • Kakinuma Y.
      • Shirouzu M.
      • Walker J.E.
      • Yokoyama S.
      • Iwata S.
      • Murata T.
      Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.
      ,
      • Murata T.
      • Yamato I.
      • Kakinuma Y.
      • Leslie A.G.
      • Walker J.E.
      Structure of the rotor of the V-type Na+-ATPase from Enterococcus hirae.
      ,
      • Murata T.
      • Yamato I.
      • Kakinuma Y.
      • Shirouzu M.
      • Walker J.E.
      • Yokoyama S.
      • Iwata S.
      Ion binding and selectivity of the rotor ring of the Na+-transporting V-ATPase.
      ).
      VoV1 is structurally similar to FoF1-ATP synthase (FoF1) (
      • Forgac M.
      Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.
      ,
      • Mulkidjanian A.Y.
      • Makarova K.S.
      • Galperin M.Y.
      • Koonin E.V.
      Inventing the dynamo machine: the evolution of the F-type and V-type ATPases.
      ,
      • Muench S.P.
      • Trinick J.
      • Harrison M.A.
      Structural divergence of the rotary ATPases.
      ). They also have a common rotary catalytic mechanism. The V1 (F1) moiety generates a torque at the interface between the rotor and stator by using the chemical free energy change resulting from ATP hydrolysis, which causes rotation of the rotor subunits relative to the stator subunits. This mechanical rotation accompanies active ion transport in the Vo (Fo) moiety through the aqueous access channels formed at the a-c interface. Inversely, when the magnitude of the electrochemical potential of an ion is large enough, the downhill flow of ions through Vo (Fo) causes rotation of the rotor subunits in the reverse direction, which forces V1 (F1) to synthesize ATP (
      • Boyer P.D.
      The binding change mechanism for ATP synthase: some probabilities and possibilities.
      ). Indeed, Thermus thermophilus VoV1 (TtVoV1) is known to function as an ATP synthase under physiological conditions (
      • Nakano M.
      • Imamura H.
      • Toei M.
      • Tamakoshi M.
      • Yoshida M.
      • Yokoyama K.
      ATP hydrolysis and synthesis of a rotary motor V-ATPase from Thermus thermophilus.
      ,
      • Toei M.
      • Gerle C.
      • Nakano M.
      • Tani K.
      • Gyobu N.
      • Tamakoshi M.
      • Sone N.
      • Yoshida M.
      • Fujiyoshi Y.
      • Mitsuoka K.
      • Yokoyama K.
      Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus.
      ,
      • Yokoyama K.
      • Muneyuki E.
      • Amano T.
      • Mizutani S.
      • Yoshida M.
      • Ishida M.
      • Ohkuma S.
      V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP.
      ); however, it remains unknown whether EhVoV1 can catalyze ATP synthesis.
      The rotation of V1 and F1 driven by ATP hydrolysis has been directly visualized under an optical microscope by attachment of a probe to the rotary shaft (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Imamura H.
      • Nakano M.
      • Noji H.
      • Muneyuki E.
      • Ohkuma S.
      • Yoshida M.
      • Yokoyama K.
      Evidence for rotation of V1-ATPase.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ,
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ,
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      Direct observation of the rotation of F1-ATPase.
      ). When viewed from the membrane side, T. thermophilus V1 (TtV1) is observed to rotate stepwise in a counterclockwise direction, consuming a single ATP molecule at each step (
      • Imamura H.
      • Nakano M.
      • Noji H.
      • Muneyuki E.
      • Ohkuma S.
      • Yoshida M.
      • Yokoyama K.
      Evidence for rotation of V1-ATPase.
      ). The step size is always 120° under conditions where ATP binding and/or hydrolysis (ATP cleavage and product release) are rate-limiting steps. This indicates that ATP binding and hydrolysis occur at the same angle in TtV1 (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), whereas these elementary reaction steps occur at different angles in thermophilic Bacillus PS3 F1 (TF1) and E. coli F1 (EF1) (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ,
      • Shimabukuro K.
      • Yasuda R.
      • Muneyuki E.
      • Hara K.Y.
      • Kinosita Jr., K.
      • Yoshida M.
      Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation.
      ,
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      ,
      • Adachi K.
      • Oiwa K.
      • Nishizaka T.
      • Furuike S.
      • Noji H.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      Coupling of rotation and catalysis in F1-ATPase revealed by single-molecule imaging and manipulation.
      ,
      • Ariga T.
      • Muneyuki E.
      • Yoshida M.
      F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits.
      ,
      • Watanabe R.
      • Iino R.
      • Noji H.
      Phosphate release in F1-ATPase catalytic cycle follows ADP release.
      ). Recently, we characterized the rotary dynamics of E. hirae V1-ATPase (EhV1) by single-molecule analysis (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). EhV1 rotated in basically the same manner as TtV1 and also showed no substeps (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), despite their difference in physiological function, suggesting that the 120° stepping rotation without substeps is a common property of V1. On the other hand, EhV1 also exhibited two characteristic rotational states, namely clear and unclear states, suggesting unstable interactions between the rotor and stator.
      Interactions between the rotor and stator will affect the torque generated by rotary ATPase. Because rotary ATPase interconverts the chemical free energy and the electrochemical potential via mechanical rotation, torque is an important factor affecting the energy conversion efficiency. Torque against viscous drag has been estimated for TtV1 (
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      ,
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ), TF1 (
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ,
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
      ), and EF1 (
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      ,
      • Noji H.
      • Häsler K.
      • Junge W.
      • Kinosita Jr., K.
      • Yoshida M.
      • Engelbrecht S.
      Rotation of Escherichia coli F1-ATPase.
      ,
      • Omote H.
      • Sambonmatsu N.
      • Saito K.
      • Sambongi Y.
      • Iwamoto-Kihara A.
      • Yanagida T.
      • Wada Y.
      • Futai M.
      The gamma-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli.
      ) (Table 1), all of which act as ATP synthases, and high energy conversion efficiency of TF1 under external torque was demonstrated (
      • Toyabe S.
      • Watanabe-Nakayama T.
      • Okamoto T.
      • Kudo S.
      • Muneyuki E.
      Thermodynamic efficiency and mechanochemical coupling of F1-ATPase.
      ). Therefore, it is intriguing to consider whether EhV1, which functions as an ion pump, might also generate a comparable torque.
      TABLE 1Torque of EhVoV1, EhV1, and other rotary ATPases
      ProteinTorqueReference
      Continuous rotationStepping rotation
      pNnm
      EhVoV123 ± 10
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      This study
      EhV113 ± 3
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      27 ± 5
      The values are the means ± S.D.
      ,
      The values were determined from the angular velocity and frictional drag coefficient of the probe, gold nanoparticle, or polystyrene beads.
      This study
      TtV135
      The values were determined from the angular velocity and frictional drag coefficient of the probe, gold nanoparticle, or polystyrene beads.
      Ref.
      • Imamura H.
      • Takeda M.
      • Funamoto S.
      • Shimabukuro K.
      • Yoshida M.
      • Yokoyama K.
      Rotation scheme of V1-motor is different from that of F1-motor.
      33 ± 2
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      Ref.
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      SVoV136
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      Ref.
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      TF139 ± 4
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      This study
      ∼40
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      ∼40
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      Ref.
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
      35 ± 3
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      38 ± 3
      The values were determined by the fluctuation theorem.
      ,
      The values are the means ± S.D.
      Ref.
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      EFoF150 ± 6
      The values were determined from the bending of the probe, actin filament.
      ,
      The values are the means ± S.E.
      Ref.
      • Pänke O.
      • Cherepanov D.A.
      • Gumbiowski K.
      • Engelbrecht S.
      • Junge W.
      Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.
      42
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      Refs.
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      and
      • Nishio K.
      • Iwamoto-Kihara A.
      • Yamamoto A.
      • Wada Y.
      • Futai M.
      Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring.
      EF1∼50
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      Ref.
      • Omote H.
      • Sambonmatsu N.
      • Saito K.
      • Sambongi Y.
      • Iwamoto-Kihara A.
      • Yanagida T.
      • Wada Y.
      • Futai M.
      The gamma-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli.
      ∼40
      The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      Ref.
      • Noji H.
      • Häsler K.
      • Junge W.
      • Kinosita Jr., K.
      • Yoshida M.
      • Engelbrecht S.
      Rotation of Escherichia coli F1-ATPase.
      ∼30
      The values were determined from the angular velocity and frictional drag coefficient of the probe, gold nanoparticle, or polystyrene beads.
      Ref.
      • Bilyard T.
      • Nakanishi-Matsui M.
      • Steel B.C.
      • Pilizota T.
      • Nord A.L.
      • Hosokawa H.
      • Futai M.
      • Berry R.M.
      High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase.
      a The values were determined by the fluctuation theorem.
      b The values are the means ± S.D.
      c The values were determined from the angular velocity and frictional drag coefficient of the probe, gold nanoparticle, or polystyrene beads.
      d The values were determined from the angular velocity and frictional drag coefficient of the probe, actin filament.
      e The values were determined from the bending of the probe, actin filament.
      f The values are the means ± S.E.
      The ATP-driven rotation of VoV1 and FoF1 has also been observed directly (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ,
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      ,
      • Yokoyama K.
      • Nakano M.
      • Imamura H.
      • Yoshida M.
      • Tamakoshi M.
      Rotation of the proteolipid ring in the V-ATPase.
      ,
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ,
      • Nishio K.
      • Iwamoto-Kihara A.
      • Yamamoto A.
      • Wada Y.
      • Futai M.
      Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring.
      ,
      • Ueno H.
      • Suzuki T.
      • Kinosita Jr., K.
      • Yoshida M.
      ATP-driven stepwise rotation of FoF1-ATP synthase.
      ). High spatiotemporal imaging employing a low load probe revealed the presence of short pauses and small steps that seem to reflect the interactions between the rotor (c subunit) and stator (a subunit) in Vo (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ) or Fo (
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ). However, no coupling between the pauses and proton transport was shown (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), and the observed short pauses were also found to be independent of proton transport (
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ). The torque has also been estimated for Saccharomyces cerevisiae VoV1 (SVoV1) (
      • Hirata T.
      • Iwamoto-Kihara A.
      • Sun-Wada G.H.
      • Okajima T.
      • Wada Y.
      • Futai M.
      Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits.
      ) and E. coli FoF1 (EFoF1) (
      • Nishio K.
      • Iwamoto-Kihara A.
      • Yamamoto A.
      • Wada Y.
      • Futai M.
      Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring.
      ,
      • Pänke O.
      • Cherepanov D.A.
      • Gumbiowski K.
      • Engelbrecht S.
      • Junge W.
      Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.
      ) (Table 1). However, the torque of V1 and VoV1 from the same species has not been compared quantitatively, and the effect of the peripheral stalks on torque generation has not been assessed. Although the torque of EFoF1 and EF1 has been reported to be comparable, no systematic comparison under the same experimental conditions has yet been carried out.
      In this study, we developed an expression system of recombinant EhVoV1 in E. coli and established a single-molecule rotation assay for EhVoV1. Recombinant EhVoV1 exhibited equivalent dependence of ATP hydrolysis activity on Na+ and ATP concentrations to that observed in native EhVoV1, indicating that recombinant EhVoV1 is as functional as native EhVoV1. We further used the single-molecule rotation assay to compare the rotational state and torque of EhVoV1 and EhV1 in the rotation driven by ATP hydrolysis. EhVoV1 showed only clear rotation and generated a larger torque than EhV1, indicating that the rotor-stator interactions in EhVoV1 are stabilized by two peripheral stalks to generate larger torque compared with that of isolated EhV1. However, the torque generated by EhVoV1 was substantially lower than those of other rotary ATPases, implying low energy conversion efficiency.

      EXPERIMENTAL PROCEDURES

      Preparation of EhVoV1 Expressed in E. coli and EhV1 for Rotation Assays

      Recombinant EhVoV1 (ac10dE2G2-A3B3DF) was expressed in E. coli by using the expression plasmid pTR19-EhVoV1. We synthesized a DNA fragment containing nine genes of the ntp operon, ntp-FIKECGABD, and optimized its codon usage and ribosome-binding site for E. coli expression. This fragment was then cloned into the plasmid pTR19 (
      • Suzuki T.
      • Ueno H.
      • Mitome N.
      • Suzuki J.
      • Yoshida M.
      F0 of ATP synthase is a rotary proton channel. Obligatory coupling of proton translocation with rotation of c-subunit ring.
      ). For the rotation assay, a His3 tag and AviTag biotinylation sequence (GLNDIFEAQKIEWHE) (
      • Beckett D.
      • Kovaleva E.
      • Schatz P.J.
      A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.
      ) were introduced at the C terminus of the c subunit and at the N terminus of the A subunit by PCR-based mutagenesis, respectively. E. coli OverExpress C43(DE3) harboring pTR19-EhVoV1 was cultivated in Super broth (32 g/liter tryptone, 20 g/liter yeast extract, and 5 g/liter NaCl) containing 100 µg/ml ampicillin and 1 mm isopropyl-β-d-thiogalactopyranoside at 37 °C for 20 h. Cells were suspended in buffer A (10 mm HEPES-KOH, pH 7.5, 5 mm MgCl2, 10% glycerol) and disrupted by sonication. After removal of the cell debris by centrifugation (21,000 × g, 20 min, 4 °C), the membrane fraction was precipitated by centrifugation (>100,000 × g, 1 h, 4 °C). After washing the membranes with buffer A, the membranes were suspended in buffer B (50 mm potassium phosphate, pH 7.5, 100 mm KCl, 5 mm MgCl2, 20 mm imidazole, 10% glycerol), and then EhVoV1 was solubilized from the membranes by incubation with 2% n-dodecyl β-d-maltoside (DDM) for 30 min on ice. The insoluble fraction was removed by centrifugation (>100,000 × g, 30 min, 4 °C), and the supernatant was diluted 5-fold with buffer B. This suspension was applied to a nickel-nitrilotriacetic acid column (Ni2+-NTA Superflow; Qiagen) equilibrated with buffer B containing 0.05% DDM. After a wash with 10 column volumes of buffer B containing 0.05% DDM, recombinant EhVoV1 was eluted with buffer C (50 mm potassium phosphate, pH 7.5, 50 mm KCl, 5 mm MgCl2, 300 mm imidazole, 10% glycerol) containing 0.05% DDM. The eluted fractions were concentrated with an Amicon Ultra 100 K unit (Merck Millipore) and then passed through a gel filtration column (Superdex 200; GE Healthcare) equilibrated with buffer D (50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 10% glycerol, 0.05% DDM). The purified proteins were flash-frozen in liquid nitrogen and stored at −80 °C until use.
      EhV1 (A3B3DF) for the rotation assay was reconstituted from A3B3 and biotinylated DF subcomplexes. The A3B3 (A-His6 tags at the N terminus) and the biotinylated DF (D-T60C/R131C) subcomplexes were prepared separately as described previously (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). Purified A3B3 and biotinylated DF were mixed at a 1:2 molar ratio and incubated at room temperature for 2 h. Reconstituted EhV1 was purified using a gel filtration column (Superdex 200; GE Healthcare). Recombinant EhV1, which has no linker sequence for purification of the DF subcomplex, was purified as described previously (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ).
      The homogeneity of the purified proteins was confirmed by SDS-PAGE analysis (see Fig. 1). Specific biotinylation of the A or D subunit was confirmed by Western blotting using a streptavidin-alkaline phosphatase conjugate (see Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Gel electrophoresis. Lanes 1–4, SDS-PAGE of recombinant EhV1, reconstituted EhV1, native EhVoV1, and recombinant EhVoV1. A 16% gel was used, and 2 pmol of protein was loaded in each lane. The molecular masses of the a, A, B, d, D, E, c, G, and F subunits are 76, 66, 51, 38, 25, 23, 16, 14, and 11 kDa, respectively. The F subunit of reconstituted EhV1 has an additional linker sequence for purification of the DF subcomplex and caused a band shift to a higher molecular mass. Lanes 5–8, immunoblots stained by alkaline phosphatase-streptavidin conjugates, showing biotin labeling of the D subunit of reconstituted EhV1 and the A subunit of recombinant EhVoV1. Lane 5, nonbiotinylated recombinant EhV1. Lane 6, reconstituted EhV1 containing the biotinylated D subunit. Lane 7, nonbiotinylated native EhVoV1. Lane 8, recombinant EhVoV1 containing the biotinylated A subunit. Lanes 9 and 10, SDS-PAGE of native and recombinant EhVoV1, respectively. A 9% gel was used to resolve the band of the a subunit; 1 pmol protein was loaded in each lane. The a subunit migrated slightly faster than the A subunit as reported previously (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ).

      Na+ Concentration Dependence of the ATPase Activity of EhVoV1

      The following system was used to examine the effect of Na+ concentration on the ATPase activity of EhVoV1. The ATPase activity was determined by measuring the quantity of inorganic phosphate released during ATP hydrolysis. The concentration of inorganic phosphate was measured colorimetrically by using the molybdenum blue method in a 96-well plate (
      • Kakinuma Y.
      • Igarashi K.
      Purification and characterization of the catalytic moiety of vacuolar-type Na+-ATPase from Enterococcus hirae.
      ). The reactions were performed in 100 µl of reaction mixture containing 5 mm ATP, 100 mm Tris-HCl, pH 8.5, 5 mm MgCl2, 10% glycerol, 0.05% DDM, 20 nm EhVoV1, and various concentration of NaCl. ATP hydrolysis was initiated by the addition of ATP (final concentration, 5 mm). After incubation for 10 min at room temperature, the reaction was stopped by adding 50 µl of 20% SDS. To generate a standard curve for quantification, potassium phosphate monobasic (0.2, 0.5, 1.0, and 1.5 mm) was dispensed into the 96-well plate. Color development was initiated by adding 75 µl of color development reagent consisting of 5% (w/v) ferrous sulfate, 1.6% (w/v) ammonium molybdate, and 1 m sulfuric acid. The plate was read immediately for end point absorbance at 750 nm. The concentration of inorganic phosphate was calculated from the standard curve.

      Dependence of the ATPase Activity of EhVoV1 on ATP Concentration

      The effect of ATP concentration on the ATPase activities of EhVoV1 and EhV1 was determined using ATP-regenerating systems. For EhV1, this assay was performed as described previously (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). The reaction mixture for EhVoV1 contained 100 mm Tris-HCl, pH 8.5, 300 mm NaCl, 5 mm MgCl2, and 10% glycerol or 50 mm MES-KOH, pH 6.5, 300 mm NaCl, and 5 mm MgCl2, each containing 0.05% DDM, 2.5 mm phosphoenol pyruvate, 0.2 mg/ml NADH, 0.1 mg/ml pyruvate kinase, and 0.1 mg/ml lactate dehydrogenase in addition to various concentrations of ATP. ATP hydrolysis was initiated by the addition of EhVoV1 (final concentration, 5 nm). The rate of hydrolysis was monitored as the rate of NADH oxidation, which was measured by the absorbance decrease at 340 nm and determined as the highest rate for 10 s.

      Rotation Assay Using a Low Load Probe

      The rotation of EhV1 using a low load probe was observed as described previously (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). Briefly, the A3B3 stator ring was immobilized on a Ni2+-NTA-coated glass surface via a His6 tag introduced at the N terminus of the A subunit. A streptavidin-coated 40-nm gold colloid was then attached to the biotinylated cysteines in the rotor DF as a probe. The rotation of EhVoV1 using the low load probe was observed as follows. EhVoV1 was immobilized on a Ni2+-NTA glass surface through His3 tags in the c subunit, and a 40-nm gold colloid (British BioCell International) coated with streptavidin was attached to the AviTag in the A subunit as a low load probe. The flow cell was assembled from a Ni2+-NTA-coated glass and an uncoated coverglass. First, buffer E (buffer B containing 0.05% DDM, 5 mg/ml bovine serum albumin, and 250 mm NaCl) was infused into the flow cell to prevent nonspecific binding of the EhVoV1 and gold colloid. After incubation for 10 min, EhVoV1 (5–10 nm in buffer E) was infused into the flow cell. After incubation for 5 min, unbound EhVoV1 was washed out with buffer E, and then the gold colloid in buffer E was infused. After 10 min, any unbound gold colloid was washed out. Observations of the rotation of EhVoV1 and EhV1 were initiated after infusion of buffer F (50 mm MES-KOH, pH 6.5, 5 mm MgCl2, indicated concentrations of ATP, ATP-regenerating system) containing 300 mm NaCl and 0.05% DDM for EhVoV1 or 50 mm KCl for EhV1. The rotations of the gold colloid were visualized on an objective type total internal reflection dark field microscope constructed on an inverted microscope (IX-71; Olympus) (
      • Ueno H.
      • Nishikawa S.
      • Iino R.
      • Tabata K.V.
      • Sakakihara S.
      • Yanagida T.
      • Noji H.
      Simple dark-field microscopy with nanometer spatial precision and microsecond temporal resolution.
      ). The images were recorded with a high speed CMOS camera (FASTCAM-1024PCI; Photron) at 5,000–10,000 frames per second (fps).

      Torque Measurement

      The torque (N) was estimated from the rotation trajectories of the duplex beads (287 nm in diameter; Seradyn) attached to EhV1 or EhVoV1 by using the fluctuation theorem (FT) (
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ). Observation of rotation of EhV1 and EhVoV1 was performed basically the same procedure as described above. In the case of the rotary motor proteins that rotate in one direction, the following expression is derived from the FT,
      ln[P(Δθ)/P(Δθ)]=NΔθ/kBT
      (Eq. 1)


      where θ(t) is the rotary angle of the bead, Δθ = θ(t + Δt) − θ(t), and P(Δθ) is the probability distribution of Δθ. kB is the Boltzmann constant, and T is the room temperature (298 K). For molecules continuous rotating for at least 5 s, P(Δθ) was calculated for the case Δt = 10 ms, and then ln[P(Δθ)/P(−Δθ)] versus Δθ/kBT was plotted. The slope of the graph corresponds to the torque. The torque of each molecule was defined as the maximum value obtained from the FT analysis when employing a 5-s moving window, with windows starting at 1-ms intervals.

      Estimation of the Stepping Torque of EhV1

      The stepping torque was calculated from the frictional drag coefficient (Γ) and angular velocity (ω) of a 120° step between intervening pauses by using the equation n = Γω (
      • Sakaki N.
      • Shimo-Kon R.
      • Adachi K.
      • Itoh H.
      • Furuike S.
      • Muneyuki E.
      • Yoshida M.
      • Kinosita Jr., K.
      One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.
      ). The streptavidin-coated 50-nm gold colloid (British BioCell International) was used as a probe. The 50-nm gold colloid has two times larger Γ than that of 40-nm gold colloid. The Γ for a single bead was estimated using the equation Γ = 8πηa3 + 6πηax2, where a, x, and η are the radius of the gold colloid (a = 25 nm), the rotation radius, and the viscosity of the medium (η = 0.89 × 10−9 pNs/nm2 at 25 °C), respectively. The rotation radius, x, was determined from the long axis of the ellipse obtained by ellipse fitting to the x-y trajectories of the centroid of a rotating gold colloid (
      • Halır R.
      • Flusser J.
      Numerically stable direct least squares fitting of ellipses.
      ). The ellipse shape resulting from tilted EhV1 on the glass was transformed to a circle to obtain the angular velocity from the original 120° step. The angular velocity was estimated from the time course of stepping rotation obtained by the rotation assay as described previously (
      • Yasuda R.
      • Noji H.
      • Yoshida M.
      • Kinosita Jr., K.
      • Itoh H.
      Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase.
      ). The rotation assay was conducted as described above with slight modifications. The rotation of the gold colloid was recorded with a high speed CMOS camera (FASTCAM SA5; Photron) at 300,000–372,000 fps. To obtain the angular velocity, we first identified the main 120° steps observed within a continuous run by eye. Then the individual steps were aligned on the time and the angle axes by positioning a point closest to the midpoint of each 120° step. Then the average of the time courses of the individual steps was calculated. Finally, the angular velocity was determined by fitting the line to the linearly increasing region.

      Other Biochemical Assays

      The protein concentrations of EhV1 and EhVoV1 were determined based on absorbance at 280 nm using a molar extinction coefficient of 3.1 × 105 m−1 cm−1 calculated according to its amino acid sequence (ProtParam tool; ExPASy) and a BCA protein assay kit (Pierce) with bovine serum albumin as a standard, respectively. Inhibitory effect of N, N′-dicyclohexylcarbodiimide (DCCD) on ATPase activity of detergent-solubilized recombinant EhVoV1 was examined by incubating the enzyme (∼0.8 µm) in buffer D with 200 µm DCCD or solvent methanol only for indicated times at 25 °C and subsequent ATPase measurement using ATP-regenerating systems under the same buffer condition as rotation assay for EhVoV1 (50 mm MES-KOH, pH 6.5, 300 mm NaCl, 5 mm MgCl2, 4 mm ATP, 0.05% DDM) with or without 0.15% (w/v) lauryldimethylamine oxide (LDAO). 100% ATPase activity was obtained from the methanol only control. Residual activity was estimated as relative activity (%) against the 100% ATPase activity of the control. All measurements were carried out at 25 ± 1 °C.

      RESULTS

      Recombinant EhVoV1 for Use in a Rotation Assay

      We developed an E. coli expression system for recombinant EhVoV1 to obtain mutant EhVoV1 for a rotation assay. The synthetic ntp genes coding for EhVoV1 were designed to optimize their codon usage for E. coli expression and were inserted into the plasmid pTR19 (
      • Suzuki T.
      • Ueno H.
      • Mitome N.
      • Suzuki J.
      • Yoshida M.
      F0 of ATP synthase is a rotary proton channel. Obligatory coupling of proton translocation with rotation of c-subunit ring.
      ). This recombinant EhVoV1 has His3 tags at the C terminus of the c subunits, one His3 tag on each of the 10 c subunits, to achieve immobilization onto the Ni2+-NTA glass surface, and an AviTag biotinylation sequence (
      • Beckett D.
      • Kovaleva E.
      • Schatz P.J.
      A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.
      ), a 15-amino acid sequence that is biotinylated by biotin ligase in E. coli, at the N terminus of the A subunit to attach the rotation probe through a biotin-streptavidin interaction. The enzyme was expressed in E. coli strain C43(DE3) and purified by Ni2+-NTA chromatography and subsequent gel filtration. The yield of the recombinant protein was ∼2–3 mg/liter of culture, which is sufficient for functional and structural analyses. Homogeneity of the enzyme and specific biotinylation of the A subunit was confirmed by SDS-PAGE and immunoblotting, respectively (Fig. 1). The recombinant EhVoV1 had the same subunit composition as the native EhVoV1 purified from E. hirae, although the His3 tag caused a band shift of the c subunit to a similar molecular weight of the G subunit (Fig. 1).

      Catalytic Properties of Recombinant EhVoV1

      To characterize the catalytic properties of recombinant EhVoV1, we measured the effect of Na+ on ATPase activity of the purified enzymes. To avoid contamination of Na+ in this experiment, ATPase activities were determined by measuring the concentrations of inorganic phosphate liberated from ATP rather than using an ATP-regenerating system. The recombinant EhVoV1 showed a ATP hydrolysis rate comparable with that of native EhVoV1 at all Na+ concentrations tested (Fig. 2A). As the Na+ concentration increased, the ATP hydrolysis rate of both enzymes increased, and partial and complete saturations occurred at about 1 and 200 mm Na+ concentrations, respectively. Double reciprocal plots of both data sets suggested the presence of two Km values for Na+ (Fig. 2B) as reported previously (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ); therefore, the data were fitted with the sum of two Michaelis-Menten equations (Fig. 2A, solid and dashed lines) assuming two independent binding sites of Na+. The Michaelis constants (Km1Na, Km2Na) for native and recombinant EhVoV1 were 20 ± 5 µm, 42 ± 8 mm and 13 ± 3 µm, 50 ± 9 mm (fitted value ± S.E. of the fit), respectively. The value of Km1Na (high affinity for Na+) was similar between recombinant and native EhVoV1 and was also close to the previously reported Km values (20 µm) for Na+-dependent ATPase activity of the purified native EhVoV1 in detergent (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ) and in liposomes (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety.
      ). At zero Na+ concentration, recombinant and native enzymes showed 20% of the maximum ATPase activity. However, it is difficult to completely prevent contamination of Na+ in the assay buffer (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ,
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety.
      ), which typically contains 5–10 µm Na+; therefore, the ATP hydrolysis of both enzymes is likely to be tightly coupled to Na+ transport. Although the values of Km2Na (low affinity for Na+) were larger than the reported value (4 mm) of the purified native EhVoV1 (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ), there was only a slight difference in the values of recombinant and native EhVoV1 in this study, which qualitatively indicates that recombinant and native enzymes have a similar low affinity binding site for Na+.
      Figure thumbnail gr2
      FIGURE 2Biochemical assay of the ATPase activity of EhVoV1. A, dependence of ATPase activity on Na+ concentration at 5 mm ATP. Average ATPase rates of native (open circles) and recombinant (open triangles) EhVoV1 are shown (n ≥ 3). The error bars represent standard deviations. The inset shows the expanded plot for lower Na+ concentrations. Solid and dashed curves show the fit of the model with a sum of two Michaelis-Menten equations assuming two independent binding sites of Na+: V = Vmax1[Na+]/(Km1Na + [Na+]) + Vmax2[Na+]/(Km2Na + [Na+]). The Km1Na, Km2Na, Vmax1, and Vmax2 values were 20 ± 5 µm, 42 ± 8 mm, 38 ± 1 s−1, and 55 ± 2 s−1 for native EhVoV1 and 13 ± 3 µm, 50 ± 9 mm, 37 ± 1 s−1, and 49 ± 2 s−1 for recombinant EhVoV1 (fitted value ± S.E. of the fit), respectively. B, double-reciprocal plots of A. C, dependence of ATPase activity on ATP concentration at high Na+ concentration (300 mm). Averages of one-third of the ATPase rate (corresponding to the rotation rate) for native (open circles) and recombinant (open triangles) EhVoV1 determined by a biochemical assay at pH 8.5 (n ≥ 3) are shown. The solid and dashed lines indicate the fit with the Michaelis-Menten equation: V = VmaxATP × [ATP]/(KmATP + [ATP]). The values of KmATP, VmaxATP, and the second order binding rate constant for ATP (3 × VmaxATP/KmATP) are shown in . D, inhibitory effect of DCCD on ATPase activity of detergent-solubilized recombinant EhVoV1. After incubation of recombinant EhVoV1 with 200 µm DCCD for indicated times, residual ATPase activity was examined (n ≥ 3) under the same buffer condition as the rotation assay at 4 mm ATP without (open triangles) or with 0.15% LDAO (filled triangles). Residual ATPase activities decreased to 24 ± 3 and 21 ± 3% after 60 and 90 min of incubation with DCCD, respectively (mean ± S.D.). Residual ATPase activities were recovered to 90 ± 1 and 88 ± 3% by the addition of LDAO, even after 60 and 90 min of incubation with DCCD, respectively. The error bars represent standard deviations.
      Next, we measured the dependence of the ATP hydrolysis rate on ATP concentration at saturated Na+ concentration (300 mm) by using an ATP-regenerating system. One-third of the ATP hydrolysis rates, which corresponded to the rotation rates, of native and recombinant EhVoV1 exhibited similar simple Michaelis-Menten dependence on ATP concentration (Fig. 2C). The Michaelis constants (KmATP) and the maximum rotation rates (VmaxATP) were 86 ± 7 µm and 40 ± 0.8 revolutions per second (rps) (fitted value ± S.E. of the fit), respectively, for native EhVoV1, and were 65 ± 2 µm and 37 ± 0.2 rps, respectively, for recombinant EhVoV1. The second order binding rate constants for ATP (konATP = 3 × VmaxATP/KmATP) were (1.4 ± 0.1) × 106 m−1 s−1 and (1.7 ± 0.1) × 106 m−1 s−1 for native and recombinant EhVoV1, respectively, and were comparable with that obtained for EhV1 (Table 2) (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). These results indicated that the Na+ coupling and catalytic properties of recombinant EhVoV1 for the rotation assay are almost identical to those of native EhVoV1.
      TABLE 2Kinetic parameters for ATP hydrolysis determined by a biochemical assay of EhVoV1 and EhV1
      ProteinpHNaClKmATPVmaxATPkonATP (3 × VmaxATP/KmATP)
      The second order binding rate constant for ATP (konATP) determined from 3 × VmaxATP/KmATP.
      Reference
      mmµmrpsm−1 s−1
      Native EhVoV18.530086 ± 740 ± 0.8(1.4 ± 0.1) × 106This study
      Recombinant EhVoV18.530065 ± 237 ± 0.2(1.7 ± 0.1) × 106This study
      Recombinant EhVoV16.5300134 ± 1259 ± 1.4(1.3 ± 0.1) × 106This study
      Recombinant EhV16.50 (50 mm KCl)221 ± 1773 ± 1.5(1.0 ± 0.1) × 106Ref.
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      a The second order binding rate constant for ATP (konATP) determined from 3 × VmaxATP/KmATP.
      Next, to confirm intactness and coupling of detergent-solubilized recombinant EhVoV1 under the same buffer condition as the rotation assay, we measured the inhibitory effect of DCCD on ATPase activity of recombinant EhVoV1. DCCD is known as an inhibitor of VoV1 (FoF1), which covalently reacts with an essential carboxyl group of the c-subunit in Vo (Fo) moiety (
      • Mizutani K.
      • Yamamoto M.
      • Suzuki K.
      • Yamato I.
      • Kakinuma Y.
      • Shirouzu M.
      • Walker J.E.
      • Yokoyama S.
      • Iwata S.
      • Murata T.
      Structure of the rotor ring modified with N,N′-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.
      ,
      • Pogoryelov D.
      • Krah A.
      • Langer J.D.
      • Yildiz Ö.
      • Faraldo-Gómez J.D.
      • Meier T.
      Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases.
      ), and if Vo (Fo) and V1 (F1) are coupled, it inhibits ATP hydrolysis by V1 (F1) moiety. So the inhibitory effect of DCCD on ATPase activity is generally used as an indicator of the intactness of VoV1 (FoF1) (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
      ,
      • Yokoyama K.
      • Nakano M.
      • Imamura H.
      • Yoshida M.
      • Tamakoshi M.
      Rotation of the proteolipid ring in the V-ATPase.
      ,
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ,
      • Nishio K.
      • Iwamoto-Kihara A.
      • Yamamoto A.
      • Wada Y.
      • Futai M.
      Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring.
      ,
      • Ueno H.
      • Suzuki T.
      • Kinosita Jr., K.
      • Yoshida M.
      ATP-driven stepwise rotation of FoF1-ATP synthase.
      ,
      • Tsunoda S.P.
      • Aggeler R.
      • Yoshida M.
      • Capaldi R.A.
      Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase.
      ,
      • Cook G.M.
      • Keis S.
      • Morgan H.W.
      • von Ballmoos C.
      • Matthey U.
      • Kaim G.
      • Dimroth P.
      Purification and biochemical characterization of the F1F0-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.
      ). In our preparation, ∼80% of the ATPase activity of the recombinant EhVoV1 was inhibited by DCCD after more than 60 min of incubation (Fig. 2D, open triangles). Furthermore, this inhibition was relieved by the addition of 0.15% (w/v) LDAO, and the residual activity was recovered to ∼90% (Fig. 2D, filled triangles), indicating that LDAO induced the uncoupling between Vo and V1 as reported in previous studies of FoF1 (
      • Cook G.M.
      • Keis S.
      • Morgan H.W.
      • von Ballmoos C.
      • Matthey U.
      • Kaim G.
      • Dimroth P.
      Purification and biochemical characterization of the F1F0-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.
      ,
      • Toei M.
      • Noji H.
      Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide.
      ), and most of the inhibitory effect is attributable to the labeling of the essential carboxyl group in the c subunit of Vo moiety, not in the catalytic sites of V1 moiety. Therefore, we concluded that most of the detergent-solubilized recombinant EhVoV1 used in our study is intact and fully coupled with Na+ transport.

      Rotation Rate of EhVoV1 and EhV1 at Saturating ATP Concentration

      We observed the ATP-driven rotation of EhVoV1 in detergent to investigate the effect of the Vo moiety on the rotation driven by the V1 moiety. EhVoV1 was immobilized on a Ni2+-NTA glass surface through His3 tags in the c subunit, and a 40-nm streptavidin-coated gold colloid was attached to the A subunit as a low load probe (Fig. 3A). It is worth noting that in this “upside-down” configuration, the stator part (aE2G2-A3B3) rotates against the rotor part (c10d-DF) with the probe because the c ring is immobilized on a glass surface, and the direction of ATP-driven rotation is counterclockwise when viewed from the top as shown in Fig. 3A. To compare the results with those obtained in our earlier study of rotation of EhV1 (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ), whose ATPase activity decreased at pH >7 (
      • Kakinuma Y.
      • Igarashi K.
      Purification and characterization of the catalytic moiety of vacuolar-type Na+-ATPase from Enterococcus hirae.
      ), the rotation assay of EhVoV1 was performed using the same buffer condition (pH 6.5) as in our previous study but containing 0.05% DDM and 300 mm NaCl.
      Figure thumbnail gr3
      FIGURE 3Rotation rate of EhVoV1 and EhV1 at saturating ATP concentration and ATP concentration dependence of the ATPase activity. A, schematic image of the rotation assay of EhV1 (left panel) and EhVoV1 (right panel). The A3B3 ring of EhV1 or the c ring of EhVoV1 was fixed on the Ni2+-NTA glass surface with a His tag. A streptavidin-coated 40-nm gold colloid was attached to the biotinylated D subunit in EhV1 or the A subunit in EhVoV1. B, rotation rate at saturating ATP concentration and ATP concentration dependence of the ATPase activity. Average rotation rates for recombinant EhVoV1 (open red triangle, 45 ± 12 rps, mean ± S.D., n = 15) and reconstituted EhV1 (open blue triangle, 102 ± 13 rps, n = 6) determined by a rotation assay using a low load probe at 4 mm ATP and pH 6.5 are shown. Filled red and blue triangles indicate rotation rates for individual molecules. Averages of one-third of the ATPase activity (corresponding to the rotation rate) for recombinant EhVoV1 (open red circles) and reconstituted EhV1 (open blue circles) determined by a biochemical assay at pH 6.5 (n ≥ 3) are also shown. The solid lines indicate the fit with the Michaelis-Menten equation: V = VmaxATP× [ATP]/(KmATP + [ATP]). The values of KmATP, VmaxATP, and the second order binding rate constant for ATP (3 × VmaxATP/KmATP) are shown in .
      EhVoV1 rotated unidirectionally counterclockwise, similar to other rotary ATPases reported previously. At saturated ATP (4 mm), which is much higher than the KmATP values for EhVoV1 (134 µm) and EhV1 (221 µm) (Fig. 3B and Table 2) (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ), the rotation rate (45 ± 12 rps, mean ± S.D.) of EhVoV1 was about half that (102 ± 13 rps) of EhV1 (Fig. 3B, open red and blue triangles, respectively). Considering the averages and standard deviations of these rotation rates, almost all molecules of EhVoV1 analyzed here would be intact. If EhVoV1 was uncoupled and interactions between the rotor and stator in Vo were impaired, we should be able to find a fast rotating molecule like EhV1.

      Rotary Motion of EhV1 and EhVoV1

      We analyzed the in-depth rotary dynamics of EhVoV1 at 4 mm ATP by using a low load probe. Typical time courses of rotation of EhV1 (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ) and EhVoV1 are shown in Fig. 4 (A and E). As reported previously, EhV1 exhibited two reversible states of clear and unclear rotation (Fig. 4, A–D) (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). We assigned the rotation state based on the criteria if the centroid of the probe is distributed near the rotation center and that causes the apparent backward rotations. In the clear state (Fig. 4, A–D, panels 1a and 1c), the majority of the centroids of the probe were distant from the rotation center, and the time course showed clear unidirectional rotations. On the other hand, in the unclear state (Fig. 4, A–D, panels 1b), the centroids showed wide fluctuations toward the rotational center, and the time course resulted in some apparent backward rotations. In contrast to EhV1, EhVoV1 only showed the clear state exhibiting centroid distribution distant from the rotation center and clear unidirectional rotations (Fig. 4, E–H). In our previous report (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ), we proposed that the unclear state is caused by the unstable interactions between the rotor (DF) and stator (A3B3) in EhV1. We also expected that these unstable interactions would occur only in the isolated EhV1 and not in EhVoV1, because the two peripheral stalks composed of the E and G subunits should stabilize the rotor-stator interactions (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). The lack of an unclear state in EhVoV1 is consistent with these contentions.
      Figure thumbnail gr4
      FIGURE 4Low load rotation of EhVoV1 and EhV1. A and E, typical time course of rotation of EhV1 at 3 mm ATP recorded at 10,000 fps (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ) and of EhVoV1 at 4 mm ATP in the presence of 300 mm NaCl recorded at 5,000–10,000 fps by using a 40-nm gold colloid. The time course of EhV1 includes two reversible distinct states: clear (black) and unclear (gray) (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). B and F, the x-y trajectories of the centroid of the rotating gold colloid shown in A and E. C and G, the distribution of the rotary angle shown in B and F. D and H, the distribution of the distance of the centroid of the gold colloid from the rotation center. Average distance in unclear state (gray, ∼16 nm) is distinctly shorter than those in the clear state (black, 18–20 nm), and the histogram in the unclear state is distributed closer to the rotation center (∼13% within 10 nm) than that in clear state (<5% within 10 nm). The numbers in A–H indicate corresponding parts and molecules.
      In contrast to the relatively large difference between the rotation rate of EhV1 determined by a single-molecule assay (102 rps) and that determined with a biochemical assay (73 rps) at pH 6.5 in the presence of 4 mm ATP (Fig. 3B, open blue triangle and circles, respectively), the rotation rate of EhVoV1 was similar when determined by a single-molecule assay (45 rps) and by a biochemical assay (59 rps) (Fig. 3B, open red triangle and circles, respectively). This result also supports the hypothesis that EhVoV1 is more stable than isolated EhV1.
      Furthermore, interestingly, although EhVoV1 showed only clear rotation without apparent backward rotation (Fig. 4, E–H), three pauses and steps in the V1 moiety became obscure as compared with the clear rotation of EhV1 (Fig. 4, A–D). Considering the elastic coupling of rotary ATPase (
      • Wächter A.
      • Bi Y.
      • Dunn S.D.
      • Cain B.D.
      • Sielaff H.
      • Wintermann F.
      • Engelbrecht S.
      • Junge W.
      Two rotary motors in F-ATP synthase are elastically coupled by a flexible rotor and a stiff stator stalk.
      ), it is likely that the coupling with EhVo causes the multiple transient pausing positions corresponding to the pausing angle of the EhVo moiety in the catalytic dwell. However, under the present condition (300 mm Na+), no clear multiple pauses were resolved in the rotation of EhVoV1, whereas the ATP-driven rotation of TtVoV1 showed pauses separated by ∼30° (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), consistent with the 12-fold symmetry of the c ring. Further studies at lower Na+ concentrations with high spatiotemporal resolution will be necessary to resolve the clear small pauses and steps in EhVoV1.

      Torque Generation in EhV1 and EhVoV1

      The torque of EhV1 and EhVoV1 was measured and compared to examine the interactions between the rotor and stator in EhV1 and EhVoV1 (Fig. 5). We employed the FT to estimate the torque (
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ). This method estimates the torque only from the time courses of rotary angles without requiring an estimate of the frictional drag coefficient of the probe. In this experiment, large duplex beads (287 nm in diameter) were employed as the rotation probe instead of the low load probe shown in Fig. 3A, and we observed continuous rotation at 4 mm ATP.
      Figure thumbnail gr5
      FIGURE 5Rotation rate and torque of EhVoV1 and EhV1 determined by continuous rotation of a large probe. Rotation rate (A) and torque (B) estimated by FT analysis of EhVoV1 (columns 1 and 2), EhV1 (columns 3–5), and TF1 (columns 6). The rotation of EhVoV1 was observed at 50 mm KCl (column 1, n = 39) or 300 mm NaCl (columns 2, n = 25) in the presence of 0.05% DDM with 287-nm duplex beads. The rotation of EhV1 was observed at 50 mm KCl in the absence (columns 3, n = 9) or presence (columns 4, n = 4) of 0.05% DDM with 287-nm duplex beads or with 200-nm duplex beads (columns 5, n = 11). The rotation of TF1 was observed at 50 mm KCl without DDM with 287-nm duplex beads (columns 6, n = 8). ATP concentration was 4 mm in all conditions. Filled circles indicate rotation rates or torques for individual molecules. Columns show the averages. The error bars indicate standard deviations.
      In contrast to the results of rotation rate using the low load probe, EhVoV1 rotated faster than EhV1 when using the duplex beads (Fig. 5A, columns 1 and 3). Consistent with this difference in rotation rate, EhVoV1 generated a torque that was nearly twice that of EhV1 (23 ± 10 pNnm versus 13 ± 3 pNnm, mean ± S.D.) (Fig. 5B, columns 1 and 3, and Table 1). We also examined the rotation rate and torque of EhVoV1 with a high Na+ concentration (300 mm), and those of EhV1 with DDM, but there were no changes in the torque values (Fig. 5A and B, columns 2 and 4). The rotation assay of EhV1 using a slightly smaller probe (200 nm in diameter) resulted in an increase in the rotation rate (Fig. 5A, column 5), but no change in the torque was observed (Fig. 5B, column 5), indicating the reliability and reproducibility of torque estimation with FT.
      For comparison with other rotary ATPases under the same experimental conditions, we also measured the rotation rate and torque of TF1. The value of torque estimated for TF1 (39 ± 4 pNnm) was comparable with those previously reported (Table 1) (
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ,
      • Yasuda R.
      • Noji H.
      • Kinosita Jr., K.
      • Yoshida M.
      F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps.
      ), indicating the reliability of the estimated torque. TF1 rotated faster than EhVoV1 (Fig. 5A, column 6) and showed nearly twice the torque as that of EhVoV1 (Fig. 5B, column 6). It should be noted that the S.D. for the torque of EhVoV1 was relatively larger than those of EhV1 and TF1. We speculate that relatively large uncertainty for the torque of EhVoV1 results from the upside-down configuration of rotation assay for EhVoV1 (Fig. 3A, right panel). In this configuration, the a subunit rotates against the c ring very close to the glass surface and is more likely to nonspecifically interact with the glass surface during rotation as compared with that of the DF subunits in the rotation assay for EhV1 (Fig. 3A, left panel). The nonspecific interaction between the a subunit and the glass surface will occasionally cause transient pauses and/or restrict fluctuation of the probe, which results in the lower estimate of the torque using the fluctuation theorem (
      • Hayashi K.
      • Ueno H.
      • Iino R.
      • Noji H.
      Fluctuation theorem applied to F1-ATPase.
      ). This would result in the relatively large distribution (uncertainty) of the torque of EhVoV1. Therefore, the torque of EhVoV1 could be underestimated slightly.

      Stepping Torque of EhV1

      The lower torque of EhV1 than EhVoV1 as estimated from continuous rotation can be attributed to the transition between the clear and unclear states in rotation (Fig. 4, A–D), assuming that EhV1 can generate very low torque in the unclear state. To test this assumption, we determined the stepping torque of EhV1 in the clear state. Because observation of clear rotational steps using a large probe was difficult, we used a 50-nm gold colloid as a frictional probe. Rotation was observed at 3 mm ATP and was recorded at 300,000–372,000 fps. The torque was estimated from the angular velocity and the frictional drag coefficient of the probe. The 120° steps of individual EhV1 molecules are superimposed in Fig. 6. The angular velocity of the 120° step rotation was determined by a linear fit to the data (Fig. 6, dotted lines). The stepping torque of EhV1 was estimated to be 27 ± 5 pNnm (mean ± S.D.) and was higher than that of EhV1 (13 ± 3 pNnm) estimated from the continuous rotation (Table 1). On the other hand, the stepping torque of EhV1 was comparable with that of EhVoV1 (23 ± 10 pNnm), which showed only the clear state (Table 1). These results support our assumption that EhV1 in the unclear state generates lower torque than that in the clear state. Our results also indicate that even in the clear state, EhV1 and EhVoV1 generate lower torque than other rotary ATPases (Table 1).
      Figure thumbnail gr6
      FIGURE 6Stepping torque of EhV1 in clear rotation state. Time courses of the stepping rotation of four single EhV1 molecules at 3 mm ATP, captured at a rate of 300,000–372,000 fps. Each gray line shows 18 successive steps superimposed at the point closest to the midpoint of each 120° step, whereas black lines and dots show the averages of the 18 lines. The dotted lines show the fit with the linear function.

      DISCUSSION

      We developed an E. coli expression system for EhVoV1 using nine synthetic ntp genes in an ntp operon (Fig. 1). Recombinant EhVoV1 exhibited identical catalytic properties to native EhVoV1 (Fig. 2 and Table 2), which indicates that recombinant EhVoV1 is as functional as native EhVoV1. The dependence of the ATPase activity of both native and recombinant EhVoV1 on Na+ concentration showed biphasic Michaelis-Menten kinetics (Fig. 2, A and B). The values of Km1Na (20 µm for native and 13 µm for recombinant EhVoV1) for the high affinity Na+-binding site were close to the dissociation constant (15 µm) for Na+ (KdNa) of purified native EhVoV1 in detergent (
      • Murata T.
      • Igarashi K.
      • Kakinuma Y.
      • Yamato I.
      Na+ binding of V-type Na+-ATPase in Enterococcus hirae.
      ) and were comparable with the apparent Michaelis-Menten constant for Na+ transport, Kt (40 µm), measured as the half-maximal transport of Na+ by native EhVoV1 liposomes (
      • Murata T.
      • Takase K.
      • Yamato I.
      • Igarashi K.
      • Kakinuma Y.
      Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety.
      ). This suggests that ATP hydrolysis of detergent-solubilized EhVoV1 is tightly coupled to Na+ transport and that Na+ binding to the high affinity site in EhVo limits the ATP hydrolysis rate (rotation) at concentrations below the Km1Na value. There has been no report about the relationship between the low affinity site with higher Km2Na values (42 mm for native and 50 mm for recombinant VoV1) and Na+ transport. Therefore, it will be interesting to determine whether the Na+ bound to this low affinity site is transported or not. The mutagenesis approach of the recombinant EhVoV1 developed in this study will help to identify the residues that are important for Na+ binding and reveal the cause of the observed biphasic kinetics.
      We characterized the ATP-driven rotation of EhVoV1 using a low load probe, and its rotation behaviors were compared with those of EhV1 at the single-molecule level. Although EhV1 rotated with clear and unclear states (Fig. 4, A–D), EhVoV1 showed only the clear state (Fig. 4, E–H), as predicted based on the results of our previous study (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). Interestingly, the rotation rate of EhVoV1 was almost twice as slow as that of EhV1 at saturating ATP concentration (Fig. 3B) with no clear pauses separated by 120° observed for EhV1 (Fig. 4). We speculate that the interactions between the rotor (c ring) and stator (a subunit) in EhVo resist the rotation driven by EhV1, which results in the slow rotation of EhVoV1, as previously proposed for TtVoV1 (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ) and EFoF1 (
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ). It is also highly likely that the Na+ transport through EhVo limits the rotation; however, considering the Km1Na (13 µm), the reported KdNa values (15 µm) (
      • Murata T.
      • Igarashi K.
      • Kakinuma Y.
      • Yamato I.
      Na+ binding of V-type Na+-ATPase in Enterococcus hirae.
      ), and the Na+ concentration used in our rotation assay (300 mm), the slow rotation could be caused by the Na+ transfer process between the c ring and a subunit and/or Na+ release into the periplasm side rather than by Na+ binding onto the a or c subunit. Similar slow rotation caused by the effect of Vo has also been reported in the rotation of TtVoV1 (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ). The authors reported that the bumps caused by the Vo moiety obscured the ATP-waiting pauses (i.e. the catalytic pauses) separated by 120° observed in TtV1. Furthermore, they found that the bumps introduced by the Vo moiety led to ∼30° steps, which is consistent with the 12-fold symmetry of the c ring. In our rotation assay of EhVoV1, where catalytic events are expected to be rate-limiting, we also did not find the three clear pauses separated by 120° that were observed in EhV1 (Fig. 4). On the other hand, we did not observe small steps reflecting the c ring of the Vo moiety (Fig. 4, E–G). Because the c ring in EhVo consists of 10 c subunits that each have an essential glutamate for Na+ transport (
      • Murata T.
      • Yamato I.
      • Kakinuma Y.
      • Leslie A.G.
      • Walker J.E.
      Structure of the rotor of the V-type Na+-ATPase from Enterococcus hirae.
      ), it is possible that EhVoV1 exhibits ∼36° steps (360°/10). We are currently trying to observe the rotation of EhVoV1 at lower Na+ concentrations and resolve this small step.
      The values of torque for EhVoV1 and EhV1 determined in this study are summarized in Table 1 along with the reported values for various rotary ATPases. EhVoV1 only showed the clear rotation state without apparent backward rotations unlike EhV1, and when the torque was measured with the FT analysis of continuous rotation, EhVoV1 generated larger torque than EhV1. These results are consistent with our notion that two peripheral stalks stabilize the interactions between the rotor and stator (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ). This hypothesis is further supported by the result that the stepping torque of EhV1 measured in the clear rotation state was much higher than that measured by the FT analysis of continuous rotation. The difference between the torque of EhV1 estimated from continuous rotation and that from stepping rotation in the clear state strongly suggest that EhV1 in the unclear state generates much lower torque than that in the clear state. Given the ratio of the clear state to the total observation time (∼0.3) determined in a previous study (
      • Minagawa Y.
      • Ueno H.
      • Hara M.
      • Ishizuka-Katsura Y.
      • Ohsawa N.
      • Terada T.
      • Shirouzu M.
      • Yokoyama S.
      • Yamato I.
      • Muneyuki E.
      • Noji H.
      • Murata T.
      • Iino R.
      Basic properties of rotary dynamics of the molecular motor Enterococcus hirae V1-ATPase.
      ), the torque of EhV1 in the unclear state is estimated to be roughly 7 pNnm (i.e. [13 − 0.3 × 27]/0.7). A similar low torque property and rotation behavior as those observed for EhV1 showing the unclear state have been reported in TF1 mutants in which the N- and C-terminal residues of the rotor γ subunit were deleted (
      • Furuike S.
      • Hossain M.D.
      • Maki Y.
      • Adachi K.
      • Suzuki T.
      • Kohori A.
      • Itoh H.
      • Yoshida M.
      • Kinosita Jr., K.
      Axle-less F1-ATPase rotates in the correct direction.
      ).
      We also found that the torque of EhVoV1 was substantially lower than that of other rotary ATPases, although there is a possibility of underestimating the torque of EhVoV1 slightly as described above (Table 1). Thermophilic Bacillus PS3 FoF1 (TFoF1) mutants generating half the torque observed in the wild type was previously reported (
      • Usukura E.
      • Suzuki T.
      • Furuike S.
      • Soga N.
      • Saita E.
      • Hisabori T.
      • Kinosita Jr., K.
      • Yoshida M.
      Torque generation and utilization in motor enzyme F0F1-ATP synthase: half-torque F1 with short-sized pushrod helix and reduced ATP synthesis by half-torque F0F1.
      ). This half-torque TFoF1 mutant could carry out ATP-driven proton pumping and ATP synthesis, although these activities were low. Therefore, one important question arising from our results is the amount of Na+-motive force (electrochemical potential of Na+) that can be generated by EhVoV1. Resolving this issue will require the development of a quantitative method to accurately measure the Na+-motive force. Another important question is the capability of EhVoV1 to synthesize ATP. It has been reported that SVoV1 in the vacuolar membrane, which acts as a proton pump under physiological conditions, synthesized ATP when a proton-motive force was applied by the exogenously expressed pyrophosphatase (
      • Hirata T.
      • Nakamura N.
      • Omote H.
      • Wada Y.
      • Futai M.
      Regulation and reversibility of vacuolar H+-ATPase.
      ). However, a quantitative correlation between the amplitude of the proton-motive force and the ATP synthesis rate has not been determined. As previously described for EFoF1 (
      • Iino R.
      • Hasegawa R.
      • Tabata K.V.
      • Noji H.
      Mechanism of inhibition by C-terminal α-helices of the epsilon subunit of Escherichia coli FoF1-ATP synthase.
      ) and TFoF1 (
      • Soga N.
      • Kinosita Jr., K.
      • Yoshida M.
      • Suzuki T.
      Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis.
      ), our recombinant EhVoV1 will enable quantitative measurements of the ATP synthesis rate under defined conditions and should provide detailed insight to help resolve this issue.
      As described above, small steps have been observed in the rotation of TtVoV1 and EFoF1 driven by ATP hydrolysis, which is consistent with the structural symmetry of the rotor c ring (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ,
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ). However, in one study, no relationship between small steps and proton transport was shown (
      • Furuike S.
      • Nakano M.
      • Adachi K.
      • Noji H.
      • Kinosita Jr., K.
      • Yokoyama K.
      Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe.
      ), and the results of another study indicated that the observed short dwells were independent of proton transport (
      • Ishmukhametov R.
      • Hornung T.
      • Spetzler D.
      • Frasch W.D.
      Direct observation of stepped proteolipid ring rotation in E. coli F0F1-ATP synthase.
      ). The correlation between the small steps and proton transport could be established by using the single-molecule rotation assay with different proton concentrations at which proton binding/release becomes rate-limiting. However, large changes in proton concentration will also induce large changes in pH, which will greatly affect the stability of the sample. On the other hand, EhVoV1 has a significant advantage in that the concentration of the transport substrate, Na+, can be easily and widely changed without affecting the sample stability. This property will allow us to perform quantitative single-molecule analyses of EhVoV1 rotation coupled with ion binding/release events in the Vo moiety and help to further understand the mechano-electrochemical coupling mechanism in VoV1.

      Acknowledgments

      We thank Shou Furuike for valuable comments, as well as all the members of the laboratory for valuable discussions and comments.

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