Rotation of a Complex of the gamma  Subunit and c Ring of Escherichia coli ATP Synthase

THE ROTOR AND STATOR ARE INTERCHANGEABLE*

Mikio TanabeDagger , Kazuaki NishioDagger , Yuko IkoDagger , Yoshihiro SambongiDagger , Atsuko Iwamoto-Kihara§, Yoh WadaDagger , and Masamitsu FutaiDagger

From the Dagger  Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan and the § Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Tokyo 153-8902, Japan

Received for publication, January 12, 2001, and in revised form, February 12, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma  subunit and c ring (c10-14) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha 3beta 3 hexamer rotation; a biotin tag was introduced into the alpha  or beta  subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha  or beta  subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gamma epsilon c10-14 complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP is synthesized in chloroplasts, mitochondria, and bacteria by a ubiquitous ATP synthase (F0F1) coupled with an electrochemical proton gradient. The F0F1 of Escherichia coli consists of a catalytic sector, F1 (alpha 3beta 3gamma delta epsilon ), and a proton pathway, F0 (ab2c10-14) (for reviews, see Refs. 1-6). The amino- and carboxyl-terminal alpha  helices of the gamma  subunit occupy the central space of the alpha 3beta 3 hexamer, as shown by the high resolution structure (7). The catalytic sites in the three beta  subunits participate alternately in ATP synthesis and also in hydrolysis, as predicted from the binding change mechanism (6). The mechanism also proposes that the gamma  subunit rotation plays a major role in the conformational transmission among the beta  subunits.

The roles of the gamma  subunit in energy coupling and catalytic cooperativity have been shown by extensive genetic studies (2). Mutation and suppression studies have suggested that the gamma  subunit carboxyl-terminal domain and amino-terminal Met-23 interact through long range conformational transmissions involving the movement of the two helices (8, 9). gamma  subunit rotation has been suggested by beta -gamma cross-linking followed by dissociation and reconstitution (10) and the movement of a probe attached to the carboxyl terminus of the gamma  subunit (11). Continuous rotation has been observed directly as the movement of an actin filament connected to the gamma  subunit in the F1 sector (12-14). The rotation of the epsilon  subunit with the gamma  subunit was also shown subsequently (15). Thus, this enzyme can be defined as a biological nanomotor carrying out rotational catalysis (16).

In ATP hydrolysis, the mechanical work done by the gamma  subunit rotation should be transmitted to the F0 sector for ATP-dependent proton transport. During ATP synthesis, proton transport should drive the gamma  subunit rotation, which causes the beta  subunit conformational change to release the product ATP. Therefore, it is essential to determine how the gamma  subunit rotation is coupled to the proton transport through F0.

Studies on the F0 sector involving electron (17) and atomic force (18, 19) microscopy suggested that the c subunits form a ring structure and that subunits a and b are located outside the ring. A ring formed from 12 c subunits was proposed for the E. coli enzyme from the model of the solution structure (20, 21) and genetic fusion (22, 23), and the yeast structure formed from 10 monomers was observed by x-ray diffraction (24). A ring of 14 monomers was observed more recently for the chloroplast enzyme (25). The rotation of the c ring with the gamma  subunit has been proposed (26-29) and has been suggested by chemical cross-linking (30, 31). We recently provided the first direct evidence that the c ring rotates continuously together with the gamma  subunit in F0F1 (32); F0F1 was immobilized on a glass surface through His tags connected to the alpha  subunits, and an actin filament was attached to the c ring. Pänke et al. (33) have also demonstrated the c ring rotation more recently using a different experimental system. The x-ray structure of the yeast enzyme showed the tight association between the gamma  subunit and the c ring, also suggesting that they can rotate together as an assembly (24).

As shown previously by Fillingame et al. (34), the membrane ATPase activity of the c subunit cIle-28 (cI28) to the Thr (cT28) mutant was resistant to venturicidin, an effective inhibitor of the wild type. In this study, we observed that the c ring rotation became less sensitive to venturicidin when the cI28T mutation was introduced into the c subunit. Furthermore, the alpha 3beta 3 hexamer could rotate when F0F1 was immobilized through the c ring. These results indicate that a complex of the epsilon gamma subunit and the c ring is a mechanical unit of the nanomotor and can be a rotor or stator in an experimental system, depending on the subunit immobilized.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Plasmids, Bacterial Strain, and Growth Conditions-- A plasmid (pBWU13) carrying the entire gene for F0F1 (35) was engineered for the rotation of the alpha 3beta 3 hexamer. Codons for the (His)6-Leu-His (His tag) were introduced between Met-1 and Asn-3 of the c subunit, and codons for the 123 (Val-18-Tyr-140) and 105 (Lys-20-Leu-124) amino acid residues of the transcarboxylase biotin binding domain (biotin tag) (including multicloning sites from the PinPoint Xa-1 vector (Promega)) were inserted, respectively, into the amino-terminal regions of the alpha  and beta  subunits (both between the 1st and 2nd codons). Plasmid pBWU13 was also engineered for the c ring rotation, as described previously (32): a His tag, downstream of the initiation codon of the alpha  subunit; cGlu-2 right-arrow Cys in the c subunit; and gamma Cys-87 right-arrow Ala and gamma Cys-112 right-arrow Ala in the gamma  subunit (32). The cI28T (cIle-28 replaced by Thr) mutation was further introduced into the engineered F0F1 for the c ring rotation by a polymerase chain reaction-based method.

Recombinant plasmids were introduced into E. coli strain DK8 (Delta unc), which lacks the F0F1 gene (36). Cells were grown at 37 °C in a rich medium (L-broth) supplemented with ampicillin (50 µg/ml), a synthetic medium containing 0.5% glycerol for enzyme purification, or the same medium containing 0.5% succinate for testing growth as to oxidative phosphorylation (35). Biotin (2 µM) was included in the synthetic medium for growth of the strain expressing the enzyme with the biotin tag.

Preparations-- Membrane vesicles were prepared after the disruption of cells by passage through a French press (35). Nonengineered F0F1 was purified from membranes (DK8/pBWU13) as described previously (35). Modified procedures (32) were used for the engineered enzymes. Membrane vesicles were centrifuged and then suspended in buffer A (20 mM MES1-Tricine, pH 7.0, 5 mM MgCl2, and 100 mM KCl). The suspension (1 ml, 20 mg of protein) was added with 220 µl of 10% C12E8 (detergent, octaethylene glycol dodecyl ether). After a 10-min incubation at 4 °C, the suspension was centrifuged at 220,000 × g for 1 h. 6-{N'-[2-(N-maleimide)ethyl]-N-piperazinylamido}hexyl D-biotinamide (100 µM) was included in the suspension for the engineered F0F1 constructed for the c ring rotation. The supernatant (about 1 ml, 6-12 mg of protein) was applied to a TALON metal affinity column (1.4 × 1.5 cm; CLONTECH) and eluted with buffer A containing 50 mM imidazole, 10% glycerol, 0.3% C12E8, and 0.1% phosphatidylcholine (TypeII-S from soybean) (Sigma).

The eluate (500 µl) was applied to a glycerol gradient, 10-30% glycerol, in 10 mM MES-Tricine, pH 7.0, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.1% C12E8, and 0.02% phosphatidylcholine. After centrifugation at 340,000 × g for 5 h, the ATPase activity (40-150 µg of protein/ml, about 300 µl) was recovered in the 20-25% glycerol fractions. The F1 sector with the biotin tag in the beta  subunit was purified as described previously (13).

Immobilization of F0F1 and Observation of Rotation-- F0F1 was immobilized on a nitrocellulose-coated glass surface (cover glass) via the His tag and Ni2+-nitrilotriacetic acid horseradish peroxidase conjugate (13). The enzyme was then reacted with streptavidin for 5 min at 20 °C, followed by the attachment of a 10 nM fluorescent actin filament. The rotation was followed in 10 mM Hepes-NaOH (pH 7.8) containing 25 mM KCl, 6 mM MgCl2, 10 mg/ml bovine serum albumin (Sigma), 0.24 mM Triton X-100, pyruvate kinase (50 µg/ml), 1 mM phosphoenolpyruvate, 25 mM glucose, 1 µM biotin, 1% beta -mercaptoethanol, glucose oxidase (216 µg/ml), and catalase (36 µg/ml). Immediately after the addition of 5 mM ATP, the rotating filament in a 1-mm2 area was observed at 20 °C with a Zeiss Axiovert 135 equipped with an ICCD camera (Atto Instruments). The filament images were video recorded and analyzed (13).

ATPase Assay-- The ATPase activity of the purified F0F1 or F1 (0.8-1.0 µg of protein) was assayed in 200 µl of the buffer used for observing rotation (without phosphoenolpyruvate and pyruvate kinase) (32) or in standard buffer (10 mM Hepes/NaOH, pH 7.2, 6 mM MgCl2), and the reaction was initiated by adding 5 mM [gamma -32P]ATP. When indicated, various concentrations of venturicidin were included in the mixtures followed by incubation for 5 min before the addition of ATP. After 20 min of incubation at 20 °C, the reaction was terminated by adding 0.3 N trichloroacetic acid. Each mixture was centrifuged at 3000 × g for 5 min, and 100 µl of the supernatant was mixed with 250 µl of 1.25% ammonium molybdate containing 3.8% HCl. The [32P]phosphomolybdate was extracted in 500 µl of isobutyl alcohol-benzene-acetone (3:3:1, v/v), and then the radioactivity was determined with a liquid scintillation counter (37).

The dicyclohexylcarbodiimide (DCCD) sensitivity of the purified F0F1-ATPase activity was assayed in 10 mM Hepes/NaOH (pH 7.2), 6 mM MgCl2, and 5 mM Na2Tris ATP (pH 8.0). The F0F1 was incubated with DCCD (10-40 µM) for 10 min at 20 °C before starting the reaction.

Other Procedures and Materials-- Protein concentrations and the ATP-dependent formation of an electrochemical proton gradient were assayed as described previously (35). Fluorescently labeled biotinylated actin filaments were prepared as described (13). Venturicidin was kindly supplied by Dr. R. H. Fillingame. Triton X-100 was obtained from Nacalai Tesque (Kyoto, Japan). C12E8 was from Calbiochem. DNA-modifying enzymes were obtained from Takara Shuzo C., Ltd. or New England Biolabs (Beverly, MA). Other chemicals were of the highest grade commercially available.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Properties of the Engineered F0F1 for Rotation-- Plasmids for two types of engineered F0F1 were constructed in this study: (a) the replacement of Glu-2 with Cys in the c subunit and the introduction of a His tag into the alpha  subunit (alpha His tag) with or without the c subunit Ile-28 to Thr (cI28T) mutation2 and (b) the introduction of a His tag into the c subunit and a biotin tag into the alpha  (or beta ) subunit. The recombinant plasmids encoding these enzymes were introduced into E. coli strain DK8 lacking the entire F0F1 gene (36). The transformed cells could grow on succinate through oxidative phosphorylation similar to those harboring the control plasmid encoding the nonengineered F0F1 (Table I). These results indicate that the genetic modification for observing rotation did not affect the catalysis by or energy coupling of the enzyme.

                              
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Table I
Properties of F0F1 engineered for rotation
Cells were grown at 37 °C on a succinate plate for 2 days, and then the sizes of the colonies were determined. ATPase activity was assayed at 20 °C in the buffer used for rotation observation, and 1 unit of the enzyme released 1 µmol of phosphate in 1 min. F0F1 was incubated at 20 °C for 10 min in the presence of various concentrations of DCCD, and the inhibition with 40 µM DCCD is shown below.

Effects of DCCD and Venturicidin on the Purified Engineered F0F1-- The engineered F0F1-ATP synthases could be purified by a method involving affinity chromatography and glycerol gradient centrifugation. They comprised eight subunits (Fig. 1) and showed substantial ATPase activities (30-50% of the nonengineered wild-type enzyme) (Table I), except that the engineered enzyme with an alpha  subunit biotin tag was difficult to purify for an unknown reason and was obviously less pure than the other enzymes (Fig. 1, lane 3). The positions of the alpha  and beta  subunits with a biotin tag and biotinylation of c subunit cE2C were confirmed by immunoblotting with streptavidin (data not shown). The c subunit with a His tag showed significantly stronger staining than that without the tag.


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Fig. 1.   Polyacrylamide gel electrophoresis of engineered F0F1. The engineered F0F1 (1.0 µg) was subjected to polyacrylamide gel electrophoresis (5-20% linear gradient) in the presence of 0.1% sodium dodecyl sulfate. The gel was stained with Coomassie Brilliant Blue: lane 1, nonengineered F0F1; lane 2, F0F1 with a His tag in the c ring and a biotin tag in the beta ; lane 3, F0F1 with a His tag in the c ring and a biotin tag in the alpha ; lane 4, F0F1 with cE2C and a His tag in the alpha ; and lane 5, F0F1 with cE2C, a His tag in alpha , and the cI28T mutation.

Similar to the nonengineered F0F1, the engineered F0F1 exhibited about 70-80% inhibition with 40 µM DCCD (assayed after a 10-min incubation without ATP). The enzyme with the cI28T mutation exhibited reduced sensitivity (Table I), confirming the previous results for membrane ATPase with the same mutation (34). However, all of the F0F1 was unaffected by DCCD when assayed in the buffer used for rotation, although it showed low but significant sensitivity after a 30-min incubation (about 30% inhibition with 40 µM DCCD). This apparent low DCCD sensitivity may be attributable to the high protein concentration in the buffer, including serum albumin and the enzymes to regenerate ATP, or to the presence of a sulfhydryl agent. Therefore, it was not possible to examine DCCD for the rotation of an actin filament connected to the c ring because the inhibitor should be effective right after the addition to the rotating filament.

The inhibitory effect of venturicidin on the ATPase activity was examined; about 50% of the activity of the engineered F0F1 (cE2C/alpha His tag) as to the c ring rotation was inhibited by 8 µM venturicidin, whereas ~30% of the activity of cI28T was inhibited (Fig. 2a), confirming the previous results for membrane enzymes (34). These enzymes became less sensitive when assayed in the buffer for rotation (Fig. 2b). However, this antibiotic can be considered as a specific inhibitor for the engineered F0F1 under the rotation conditions because it was less inhibitory for the enzyme with the cI28T mutation and not inhibitory for the F1 at all. It should be noted that specific inhibition was observed after a 5-min incubation, suggesting that venturicidin can be tested upon addition to the rotating enzyme.


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Fig. 2.   Effects of venturicidin on the ATPase activity of engineered F0F1. The ATPase activities of engineered F0F1 as to c ring and alpha 3beta 3 hexamer rotation were assayed in the presence of various concentrations of venturicidin: engineered F0F1 for c ring rotation, open circles; engineered F0F1 with the cI28T mutation for c ring rotation, closed circles; engineered F1 for gamma  subunit rotation, triangles; and F0F1 engineered for beta  subunit rotation, squares. Various concentrations of venturicidin were added 5 min before the addition of ATP. ATPase activity was examined under two sets of assay conditions: 5 mM ATP and 6 mM MgCl2 in Hepes buffer (a) and the same substrate concentration in the buffer used for rotation (b). The specific activities of all enzymes without venturicidin addition are shown in Table I.

The engineered enzyme with the beta  biotin tag/cHis tag for alpha 3beta 3 hexamer rotation was significantly less sensitive to venturicidin; only about 15% inhibition was observed with excess venturicidin (Fig. 2a). This result may be attributable to the introduction of the His tag into the c subunit. Therefore, the antibiotic could not be tested for the rotation of this enzyme.

Rotation of an Actin Filament Connected to the c Ring and Effect of Venturicidin-- The rotation of actin filaments connected to the c ring was confirmed, and it was unlikely that the filaments were connected to the gamma  subunit of the contaminating F1, as discussed previously (32). As a control, F1 (with no cysteine in the gamma  subunit) was isolated from the engineered F0F1 to test whether the actin filament can bind to the Cys-less gamma  and rotate with the addition of ATP. We could find no rotating filament, indicating that the contaminating Cys-less gamma  F1, if any, is not responsible for the rotation observed.


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Fig. 3.   Rotation of an actin filament connected to the c ring with or without the cI28T mutation. a, time courses of the rotations of actin filaments (1.2 µm) connected to the c ring. The rotation of the actin filament connected to the cI28 (wild type) or cT28 (mutant) c ring of engineered F0F1 was followed before and after the addition of venturicidin (Vent). We slowly replaced the buffer for rotation with the same solution containing 70 µM venturicidin, waited about 30 s until the movement of the liquid had completely stopped, and then started recording the rotation. b, increase of pauses after venturicidin addition. The boxed areas (20 rounds) in panel a are expanded, and the beginnings of pauses are indicated by arrows. cI28, left; cT28, right; Vent (-), before venturicidin addition; and Vent (+), after the addition. c, examples of pauses in expanded scale. Boxed areas in panel b are further expanded, showing how pauses were defined. We defined pauses as described previously (32); the raw data plots (black dots) were median filtered (with rank 5) (gray line), and the resulting linear segments that had R2 values greater than 0.96 with a rotational rate between -0.2 and 0.2 (sec-1 and dwell durations longer than 67 ms were defined as pauses. Arrowheads pointing down and up indicate the beginning and end of pauses, respectively. (cI28, left; cT28, right; Vent (-), before venturicidin addition; and Vent (+), after the addition.) d, increase in pauses after venturicidin addition. The rotation of 10 actin filaments (<2 µm) connected to F0F1 (cI28, cT28) was recorded, and the numbers of pauses (per round) were recorded after and before venturicidin addition. The ratios of event numbers (pauses after venturicidin addition and pauses before venturicidin addition) are shown. e, rotations versus filament length. The rotational rates of actin filaments were obtained and plotted against filament lengths. Open circles, cI28; open triangles, cI28 after venturicidin addition; closed circles, cT28; and closed triangles, cT28 after venturicidin addition. The dotted lines represent the calculated rotational rate of filaments with a constant torque value of 40 piconewton·nm.

The rotation was also tested in the presence of venturicidin. On comparison of the filament rotation before and after the antibiotic addition, we found that venturicidin increased the pauses in the wild-type (cI28) engineered F0F1 (Fig. 3a, b, c), confirming the previous results (32). On the other hand, the filament connected to the c ring with the cI28T mutation showed apparently less frequent pauses after venturicidin addition; the ratios of events (pauses after venturicidin addition and pauses before venturicidin addition) were 2 for filaments with cI28T and 4 for those without (Fig. 3d).

We examined the effect of the cI28T mutation on the frictional torque generated by the c ring rotation. Torque can be determined from the filament rotation rate and viscous drag. Because the rotational rates of single filaments varied slightly and paused, as described above, we selected more than 20 linear segments with no pauses from the rotation time course of each filament and plotted the average rate (Fig. 3e). The cI28T mutation showed no significant effect on the filament rotation or torque generation (Fig. 3e). Furthermore, venturicidin showed no effect on the torque generation (Fig. 3e, open and closed triangles).

Rotation of an Actin Filament Connected to the alpha 3beta 3 Hexamer in F0F1-- The results obtained for the cT28 mutant strengthen the notion that a complex of the c ring and the gamma  subunit rotates during ATP hydrolysis, and this prompted us to examine a further possibility. If the gamma  subunit and c ring form a mechanical unit, the alpha 3beta 3 hexamer rotates when the c ring is immobilized on a glass surface (Fig. 4a). We introduced a His tag into the c subunit and a biotin tag into the alpha  or beta  subunit to examine this possibility. As expected, an actin filament connected to the alpha 3beta 3 hexamer rotated upon ATP addition (Fig. 4b). The rotation direction of the hexamer (counterclockwise viewed from the F1 side) was consistent with that of the c ring (clockwise from the F1) in F0F1 immobilized on a glass surface through the alpha  or beta  subunit. The rotation of the alpha 3beta 3 hexamer generated essentially the same torque (~40 piconewton·nm) as that of the c ring. An effect of venturicidin on the alpha 3beta 3 hexamer rotation was not apparent, which is consistent with its slight effect on the ATPase activity of the F0F1 engineered for alpha 3beta 3 rotation (Fig. 2). As a control, we purified F1 with a biotin tag in the alpha  subunit (but no His tag in the gamma  subunit) and connected an actin filament to it. We found no rotating filament among 10,000 attached nonspecifically to the glass surface, supporting the results described above.


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Fig. 4.   Rotation of an actin filament connected to the beta  or alpha  subunit. a, experimental system for observing the rotation of filaments connected to alpha  subunits. b, time courses of rotating filaments (1.3 and 1.9 µm) connected to c, alpha , and beta  subunits. c, effect of the actin filament length on the rotational rate. The rotational rates of actin filaments connected to beta  (open circles) or alpha  (closed circles) subunits were plotted against filament length. The dotted line represents the calculated rotational rate of the filaments with a constant torque value of 40 piconewton·nm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As predicted by the binding change mechanism for ATP synthesis and hydrolysis, the continuous rotation of the gamma  subunit in the F1 sector has been observed during ATP hydrolysis (12-14). The gamma  subunit rotation should be transmitted to the F0 sector for coupling with proton transport. We provided the first evidence that the c subunit ring rotates with the gamma  subunit in F0F1 (32). Pänke et al. (33) have also demonstrated the c ring rotation more recently. The present study further confirmed the co-rotation of the gamma  subunit with the c ring.

Fillingame et al. (34) have shown that membrane ATPase activity (attributable to F0F1) became less sensitive when the cI28T mutation was introduced. It should be noted, however, that this antibiotic was not a strong inhibitor even for the wild-type enzyme (maximum inhibition, about 60%). We observed lower but significant inhibition of the engineered F0F1 when ATPase activity was assayed under the rotation conditions. Consistent with the results for ATPase activity, this antibiotic did not have a strong effect on the rotation, such as immediate cessation upon its addition, but rather showed specific inhibition of F0F1. An actin filament connected to the c ring showed increased pauses upon the addition of venturicidin, and the cI28T mutation decreased the inhibitor sensitivity. As described above, this antibiotic had no effect on the ATPase activity or the gamma  rotation of the engineered F1. These results suggest that venturicidin inhibits F0F1 rotation by binding to the c ring and that a complex of the gamma  subunit and c ring was rotating.

We tested a different engineered enzyme for rotation by introducing a His tag into the c ring and a biotin tag into the alpha 3beta 3 hexamer. Thus, F0F1 was immobilized on a glass surface and connected with an actin filament without using chemistry such as cysteine residue modification with biotin-maleimide (12-15, 32). Upon the addition of ATP, the actin filament connected to the alpha 3beta 3 hexamer rotated counterclockwise when viewed from the F1 side and generated similar torque to the gamma  rotation. These results strongly suggest that the gamma epsilon c10-14 complex is a mechanical unit and rotates during ATP hydrolysis and that the rotor and stator of the entire complex are interchangeable experimentally, depending on the subunit immobilized.

One model for the interaction of the gamma  subunit with the c ring during ATP synthesis and hydrolysis predicts that the gamma  subunit interacts with the hydrophilic loop between the transmembrane helices of each c subunit one by one during rotation (discussed in Refs. 38 and 39). The rotation of the actin filament connected to the c ring indicates that this model will not work. However, it may be possible to argue that the c ring rotated with the gamma  subunit as a part of the fluorescent probe (actin filament). The rotation of the filament connected to the alpha 3beta 3 hexamer finally excluded this possibility because the model predicts that the filament should not rotate if the c ring is immobilized and the gamma  subunit moves on the ring.

Tsunoda et al. (40) have claimed that they could not connect an actin filament specifically to the c ring under their experimental conditions and criticized the experiments by Sambongi et al. (32) showing c ring rotation. The protocols used by the two groups are different, including the positions of Cys residues introduced into the c subunit (Sambongi et al. (32), Glu-2 right-arrow Cys; Tsunoda et al. (40), Cys inserted between Glu-2 and Asn-3), the conditions for maleimide modification (Sambongi et al. (32), 4 °C at pH 7.0; Tsunoda et al. (40), 25 °C at pH 7.5), and the detergent used for F0F1 preparation. Furthermore, in most experiments, Tsunoda et al. (40) reacted F0F1 with fluorescein-5-maleimide and then biotinylated anti-fluorescein IgG and the actin filament through streptavidin. On the other hand, Sambongi et al. (32) reacted F0F1 with biotin-maleimide and then connected an actin filament through streptavidin. Thus, the arguments raised by Tsunoda et al. (40), which are based on mostly negative observations, may reflect the differences in the experimental systems. As described in this study, the conclusion of Sambongi et al. (32) is supported by Pänke et al. (33) and the effect of venturicidin on the cI28T mutant, as described above. Furthermore, the actin filament connected to the alpha 3beta 3 hexamer in the immobilized F0F1 rotated, as discussed above.

In conclusion, we clearly showed that a complex of epsilon gamma and the c ring is a mechanical unit and that it rotates as an assembly. The preparations we used have all subunits of F0F1, and previous studies indicated that it is not easy to dissociate F0F1 even under conditions that release F1 from membranes (41). Genetic (42) and reconstitution (43) studies have suggested that the three F0 subunits (a, b, and c) are required for the formation of the F0F1 complex. However, we could not prove definitely that the rotating enzyme during video recording has the original integrity of F0F1. In this regard, Sambongi et al. (32) did not conclude that the rotating enzyme had all the subunits. Pänke et al. (33) stated more clearly that they might have observed the rotation of the c ring in incomplete F0F1. Although we accept this reservation as to the integrity of the rotating complex, we strongly suggest that the rotation of a complex of the gamma epsilon subunit and c ring is related to the fully functional F0F1. The obvious next step is to examine subunit rotation during ATP synthesis and to detect ATP synthesis when the c ring or gamma  subunit is artificially rotated.

    ACKNOWLEDGEMENTS

We thank Le Phi Nga for technical assistance during the early stage of this work.

    FOOTNOTES

* This work was supported in part by the Japanese Ministry of Education, Science, and Culture.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.

To whom correspondence should be addressed: Div. of Biological Sciences, ISIR, Osaka University, Ibaraki, Osaka 567-0047, Japan. Tel.: 81-6-6879-8480; Fax: 81-6-6875-5724; E-mail: m-futai@sanken.osaka-u.ac.jp.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100289200

2 The gamma  subunit cysteine residues were removed from this enzyme.

    ABBREVIATIONS

The abbreviations used are: MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DCCD, dicyclohexylcarbodiimide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Futai, M., Noumi, T., and Maeda, M. (1989) Annu. Rev. Biochem. 58, 111-136[CrossRef][Medline] [Order article via Infotrieve]
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