COMMUNICATION
Direct Observation of the Rotation of epsilon  Subunit in F1-ATPase*

Yasuyuki Kato-YamadaDagger §, Hiroyuki Noji§, Ryohei Yasuda§parallel , Kazuhiko Kinosita Jr.§parallel , and Masasuke YoshidaDagger §**

From the Dagger  Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan, the parallel  Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, Japan, and § CREST (Core Research for Evolutional Science and Technology) Genetic Programming Team 13, Teikyo University Biotechnology Research Center 3F, 907 Nogawa, Miyamae, Kawasaki 216-0001, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Rotation of the epsilon  subunit in F1-ATPase from thermophilic Bacillus strain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the gamma  subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302). The alpha 3beta 3gamma epsilon complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the epsilon  subunit through biotin-streptavidin. In the presence of ATP, the filament attached to epsilon  subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the gamma  subunit. The rotational velocity was slightly slower than the filament attached to the gamma  subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 19621-19624), the epsilon  subunit rotates with the gamma  subunit in F1-ATPase during catalysis.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

F0F1-ATP synthase catalyzes ATP synthesis coupled with the proton flow across the energy-transducing membranes such as the plasma membrane of bacteria, mitochondrial inner membrane, and thylakoid membrane of chloroplast (1-4). F1-ATPase is the water-soluble portion of F0F1-ATP synthase and contains a catalytic core for ATP synthesis and hydrolysis. The F1-ATPase consists of five kinds of subunits with a stoichiometry of alpha 3beta 3gamma 1delta 1epsilon 1. The catalytic sites of ATP synthesis and hydrolysis are located mainly on the beta  subunits, and noncatalytic nucleotide binding sites are located mainly on the alpha  subunits (5). The alpha 3beta 3gamma subcomplex of F1-ATPase is regarded as a minimum stable complex which has catalytic features similar to F1-ATPase (6-8). Three catalytic sites of F1-ATPase exhibit strong negative cooperativity in ATP binding and positive cooperativity in ATP hydrolysis. To explain these characteristics, a binding change mechanism was proposed (3, 4) and has been widely accepted. In the binding change mechanism, all three beta subunits in F0F1-ATP synthase are in different states at a given moment and alternately exchange their states during ATP synthesis and hydrolysis. The physical rotation of the gamma  subunit within the alpha 3beta 3 hexamer was hypothesized as a mechanism for the binding change to occur (3), and a crystal structure of bovine mitochondrial F1-ATPase in which a cylinder of the alpha 3beta 3 hexamer is penetrated by the coiled-coil structure of the gamma  subunit gave the hypothesis more reality (5). Biochemical (9, 10) and optical (11) analyses provided support for the rotation of the gamma  subunit, and finally, the rotation was directly observed as the rotation of a fluorescently labeled actin filament attached to the gamma  subunit (12). Driven by ATP hydrolysis, the gamma  subunit rotated for several minutes in the direction predicted from the crystal structure of bovine mitochondrial F1. To obtain further insight into the mechanism of this enzyme, it is necessary to identify each subunit of F0F1-ATP synthase as either a rotor or stator subunit.

The epsilon  subunit, the smallest subunit of bacterial and chloroplast F1-ATPases, is an endogenous ATPase inhibitor (14-16). According to recent structural analyses, the epsilon  subunit of Escherichia coli F1-ATPase consists of an N-terminal beta -sandwich and a C-terminal alpha -helical domain (17, 18). The epsilon  subunit interacts with the gamma  subunit (19) and the analysis of a chimeric complex from a thermophilic Bacillus PS3 (TF1)1 and chloroplast F1-ATPase indicated that the epsilon  subunit affects the ATPase activity of F1-ATPase through the gamma  subunit (20). The subunit interface between the gamma  and epsilon  subunits has been explored by the cross-linking and chemical modification (21, 22), and recent work by Aggeler et al. (23) suggested that the epsilon  subunit rotates together with the gamma  subunit. Previously we reported that the inhibitory effect of the epsilon  subunit on ATPase activity of TF1 was observed only at low concentrations of ATP. Unlike the case of E. coli F1-ATPase where the epsilon  subunit tends to dissociate from F1-ATPase during multiple turnovers of ATPase reaction, the epsilon subunit of TF1 remains associated with the alpha 3beta 3gamma portion during catalysis (24). Taking advantage of this stable association of the epsilon  subunit, we observed directly the rotation of the epsilon  subunit in TF1.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Preparation of the Mutant alpha 3beta 3gamma Complex and epsilon  Subunit of TF1-- Wild-type epsilon  subunit of TF1 does not contain cysteine. To ensure specific modification, a mutant epsilon  subunit (H38C) of TF1 was generated by the method of Kunkel et al. (25). A primer oligonucleotide (5'-AAGCGGAATGCATCCCGGCAAAATG-3') which contained substitution corresponding to H38C mutation and a new EcoT22I site, was used to introduce mutation to the expression plasmid of TF1epsilon , pTE2 (24). The mutant epsilon  subunit was expressed in E. coli BL21(DE3) and purified as described previously (20) except that all buffers contained 1 mM DTT. The expression plasmid for the cysteine-less, His-tagged mutant (alpha C193S, His10-tags in beta  N termini) alpha 3beta 3gamma complex was generated from the expression plasmid used for the observation of the rotation of the gamma  subunit (12). Its BglII-NheI fragment was exchanged by that of wild-type plasmid (pKABG1) (7), and the mutation gamma S107C was reverted to serine. The alpha 3beta 3gamma complex was purified as described previously (7).

Preparation of Streptavidin-attached alpha 3beta 3gamma epsilon Complex of TF1-- Purified (H38C) epsilon  subunit was incubated at 23 °C with 2 mM DTT for 15 h and passed through a Sephadex G-25M column equilibrated with 50 mM Tris-HCl (pH 8.0) and 100 mM KCl. Then, 50 mM N'-[2-(N-maleimido)-ethyl]-N-piperazinyl D-biotinamide (Dojindo) dissolved in dimethyl sulfoxide was added to the epsilon  subunit solution (100 µM) to give a final concentration of 1 mM and incubated for 2 h at 23° C. The reaction was quenched by the addition of 7 mM DTT. The biotinylated epsilon  subunit was allowed to bind to the alpha 3beta 3gamma complex, and the alpha 3beta 3gamma epsilon complex formed was purified as described previously (24). The alpha 3beta 3gamma epsilon complex obtained was then mixed with 10 molar excess of streptavidin (SA) and incubated for 20 min at 23° C. Excess streptavidin was removed by G4000SWXL (Tosoh) gel filtration high performance liquid chromatography, and the fraction containing the alpha 3beta 3gamma epsilon SA (the superscript SA designates the subunit labeled with biotin-streptavidin) complex was concentrated by Microcon-100 (Amicon).

Observation of Rotation-- The rotation of the epsilon  and gamma  subunits was observed by the same experimental setup as that used for the rotation of the gamma  subunit in the previous report (12, 13, Fig. 1). The ATP concentration was fixed at 2 mM in an ATP regenerating system, containing 0.2 mg/ml creatine kinase and 2.5 mM creatine phosphate. Rotation was observed at 23° C on an inverted fluorescence microscope (IX70, Olympus), and images were recorded with an SIT camera (C2741-08, Hamamatsu Photonics) on an 8-mm video tape. The rotation angle of the filament was estimated from the circular movement of the centroid of the filament image calculated using a digital image processor (DIPS-C2000, Hamamatsu Photonics) (12, 13).

Other Materials and Procedures-- For a control, alpha 3beta 3gamma SA in which streptavidin was attached to the biotinylated gamma -Cys-107 was prepared from the mutant (alpha C193S, gamma S107C, His10-tags in beta  N termini) alpha 3beta 3gamma complex as described (12, 13). A alpha 3beta 3gamma SAepsilon complex was reconstituted from alpha 3beta 3gamma SA and the wild-type epsilon  subunit (24). The purity of the complexes was checked by 6% polyacrylamide gel electrophoresis without a denaturing reagent (24). ATPase activity was measured at 23° C in the presence of an ATP regenerating system in 10 mM MOPS-KOH (pH 7.0) buffer containing 50 mM KCl, 4 mM MgCl2, 50 µg/ml pyruvate kinase, 50 µg/ml lactate dehydrogenase, 2.5 mM phosphoenolpyruvate, 0.2 mM NADH, and 2 mM ATP. Steady-state ATPase activities of the (alpha C193S, His-tag) alpha 3beta 3gamma complex and the (alpha C193S, His-tag) alpha 3beta 3gamma epsilon complex were almost the same; 57 s-1 and 58 s-1 (expressed as a turnover rate), respectively. Rabbit skeletal actin filaments were biotinylated and stained with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (as in Ref. 12 but without cross-linking). Streptavidin was purchased from Sigma as lyophilized powder. Experimental procedures of recombinant DNA were performed as described in a manual (26). E. coli strain JM109 (27) was used for the preparation of plasmids, and the strain CJ236 (25) was used for generating uracil-containing single-stranded plasmids for site-directed mutagenesis. Protein concentrations were determined by the method of Bradford (28) using bovine serum albumin as a standard or from the UV absorbance using an absorbance 0.45 at 280 nm for 1 mg/ml of the subunit complexes of TF1 (29). Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate was performed as described by Laemmli (30).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Experimental Setup-- Three kinds of subunit complexes were used for experiments; the (alpha C193S, epsilon H38C, His-tag) alpha 3beta 3gamma epsilon complex for observing the rotation of the epsilon  subunit, and the (alpha C193S, gamma S107C, His-tag) alpha 3beta 3gamma and the (alpha C193S, gamma S107C, His-tag) alpha 3beta 3gamma epsilon complexes for observing the rotation of the gamma  subunit. Ten histidine tags at the N termini of the beta  subunits were used for the immobilization of the complex on a solid surface, and alpha -C193S was for elimination of unwanted cysteine residues to ensure the specificity of the biotinylation (among five kinds of subunits of the wild-type TF1, only the alpha subunit contains a cysteine residue, alpha -Cys-193). In addition to these two mutations, we introduced a cysteine at the position of epsilon -His38 which is supposed to face the F0 side (opposite to the N termini of alpha  and beta  subunits) in the structure of F1-ATPase (31). The biotinylation at epsilon -Cys-38 did not impair the ability of the epsilon  subunit to associate with the alpha 3beta 3gamma complex to form the alpha 3beta 3gamma epsilon complex. Polyacrylamide gel electrophoresis without a denaturing reagent, in which two (alpha 3beta 3gamma and alpha 3beta 3gamma epsilon ) complexes were electrophoresed as separate bands (24), gave only a single band at a corresponding position, ensuring the homogeneity of each complex prepared. After streptavidin was bound to the introduced biotin, we fixed alpha 3beta 3gamma epsilon SA to the beads which adhered on the glass plate, and a fluorescently labeled, biotinylated actin filament was attached to the epsilon  subunit through biotin-streptavidin-biotin (Fig. 1). Fixation and actin filament attachment to alpha 3beta 3gamma SA and alpha 3beta 3gamma SAepsilon were carried out in the same way.


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Fig. 1.   Schematic illustration of experimental setup for observation of the rotation. a, rotation of the gamma  subunit (12, 13). The alpha 3beta 3gamma SA complex was fixed through His-tags to the surface of the Ni-NTA beads (13) adhered on the bottom cover glass. The biotinylated gamma -Cys-107 and a biotinylated actin filament, which was fluorescently labeled, were connected by streptavidin. The recorded images correspond to the view from the top in this figure (mirror image of the bottom view). The observed direction of the rotation was indicated by an arrow. The observation of the rotation of the gamma  subunit in alpha 3beta 3gamma SAepsilon was carried out in the same way. b, rotation of the epsilon  subunit. An actin filament was attached to the epsilon  subunit in alpha 3beta 3gamma epsilon SA. Other details are the same as in a.

Characteristics of the Rotation-- When 2 mM ATP was supplied, continuous rotation of the actin filament attached to alpha 3beta 3gamma epsilon SA was observed (Fig. 2). Rotation was absolutely dependent on ATP hydrolysis. In the absence of ATP, or in the presence of 2 mM ATP + 10 mM NaN3 which inhibits ATPase activity of TF1, no continuous rotation was observed (data not shown). The number of the rotating actin filaments per total actin filaments was less than 1%, 4- to 5-fold lower than the number observed for actin filaments attached to alpha 3beta 3gamma SA. If the connection between the gamma  and epsilon  subunits is weak, a rotating actin filament would be detached in a short time from the complex by the hydrodynamic frictional load. However, because the duration of the rotation which sometimes continued more than 5 min was apparently similar to the case when an actin filament was attached to the gamma  subunit, the gamma -epsilon intersubunit connection might be strong enough to bear the hydrodynamic friction on the actin filament. The rotation was anti-clockwise when viewed from the membrane side. This direction is the same as that observed for the rotation of an actin filament attached to the gamma  subunit (12). According to the crystal structure of the bovine mitochondrial F1 (5), when the epsilon  (and gamma ) subunit(s) rotates in this direction, one beta subunit experiences the transition in the order expected in the ATP hydrolysis reaction, AMPPNP-bound form, ADP-bound form, and empty form.


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Fig. 2.   Rotation of the actin filament attached to the epsilon  subunit in the presence of 2 mM ATP. a, an example of sequential images of a rotating actin filament attached to the epsilon  subunit in alpha 3beta 3gamma epsilon SA. Length of the filament from rotational axis to the tip was 1.6 µm. Rotational rate was 1.0 rps. Time interval between images was 100 ms. The scale bar denotes 5 µm. b, examples of time course of the rotation of the actin filament. Length of the filaments presented here is 0.5-1.4 µm. Only the filaments that rotated around one end are shown. Each line represents one filament. Solid lines indicate the rotation of the epsilon  subunit in alpha 3beta 3gamma epsilon SA while dotted lines and broken lines indicate the rotation of the gamma  subunit in alpha 3beta 3gamma SA and alpha 3beta 3gamma SAepsilon , respectively. c, same as b except that the length of the filaments is more than 1.5 µm. Details of the experiments are described under "Experimental Procedures."

In the plot of rotational rate versus filament length (Fig. 3), we notice the tendency that the rotation of the filament attached to alpha 3beta 3gamma epsilon SA was somewhat slower than that of the filament attached to alpha 3beta 3gamma SA or to alpha 3beta 3gamma SAepsilon . As a consequence, the apparent torque needed for rotation (12, 32) of the filament attached to alpha 3beta 3gamma epsilon SA at the observed rate was at most ~25 pN·nm, smaller than the corresponding values for alpha 3beta 3gamma SA and alpha 3beta 3gamma SAepsilon , ~40 pN·nm (Fig. 3). Because the cross-linking between the gamma and epsilon  subunits has little effect on ATPase activity (33-35), the gamma  and epsilon  subunits are supposed to rotate together at the same angular velocity. The fact that rotational rates of the gamma  subunit in alpha 3beta 3gamma SAepsilon and in alpha 3beta 3gamma SA were the same with each other (Fig. 3) suggests that the presence of the epsilon  subunit in the alpha 3beta 3gamma epsilon complex does not impede the rotation of the gamma  subunit. Therefore, the apparent difference in the rotational rates between the epsilon -attached filament and the gamma -attached filament appears to be caused from an experimental artifact at present. If an actin filament can bind to the epsilon  subunit at Cys-38 only with nonhorizontal, downward angle (when a complex is viewed as in Fig. 1B), increased hydrodynamic friction near the surface (32) might slow the rotation of the filament. Another possible cause is the biotin-streptavidin connection through the single bond between the epsilon  subunit and the actin filament. In principle, a single bond allows free rotation around the bond axis, and the rotation of the epsilon  subunit in alpha 3beta 3gamma epsilon SA may not be transmitted at 100% efficiency to the rotation of the actin filament, resulting in the apparent slow rotation. This could happen to the rotation of the gamma  subunit-attached filament, but fortunately it seems not.


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Fig. 3.   Rotational rate versus length of the actin filament. Only the data for the filaments that rotated around one end are shown. Rotational rates were estimated by least square linear fitting on the time courses for more than 5 revolutions and expressed in rps. Closed circles, double circles, and open circles indicate results of alpha 3beta 3gamma epsilon SA, alpha 3beta 3gamma SA, and alpha 3beta 3gamma SAepsilon , respectively. Solid lines represent calculated rotational rate of the filaments with varying length which give a constant torque (hydrodynamic friction) value in pN·nm indicated on the lines. Hydrodynamic friction was calculated by (4pi /3)omega eta L3/[ln(L/2r- 0.447], where omega  is angular velocity, eta  (10-3 Nsm-2) the viscosity of the medium, L the length of actin filament, and r (5 nm) the radius of the actin filament (12, 13, 32).

In summary, we show here exclusive evidence that the epsilon  subunit rotates in F1-ATPase relative to the alpha 3beta 3 hexagon ring during catalysis. Further identification of the rotor and the stator subunits is required to know the coupling mechanism of F0F1-ATP synthase.

    FOOTNOTES

* This work was supported in part by Grant 07458158 from the Ministry of Education, Science, and Culture of Japan and grants from CREST (Core Research for Evolutional Science and Technology).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

** To whom correspondence should be addressed: Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, 226-8503, Japan. Tel.: 81-45-924-5233; Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.

1 The abbreviations used are: TF1, F1-ATPase from the thermophilic Bacillus PS3; MOPS, 3-(N-morpholino)propanesulfonic acid; alpha 3beta 3gamma SA, a mutant (alpha C193S, gamma S107C, His10-tag in N terminus of the beta  subunit) alpha 3beta 3gamma complex of TF1 with streptavidin bound to biotinylated gamma -Cys-107; alpha 3beta 3gamma SAepsilon , a complex of alpha 3beta 3gamma SA and the wild-type epsilon  subunit; alpha 3beta 3gamma epsilon SA, a mutant (alpha C193S, epsilon H38C, His10-tag in N terminus of the beta  subunit) alpha 3beta 3gamma epsilon complex of TF1 with streptavidin bound to biotinylated epsilon -Cys-38; SA, streptavidin; DTT, dithiothreitol; AMPPNP, adenosine 5'-(beta ,gamma -imino)triphosphate; Ni-NTA, nickel-nitrilotriacetic acid.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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