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

2001; Elsevier BV; Volume: 276; Issue: 18 Linguagem: Inglês

10.1074/jbc.m100289200

1083-351X

Mikio Tanabe, Kazuaki Nishio, Yuko Iko, Yoshihiro Sambongi, Atsuko Iwamoto-Kihara, Yoh Wada, Masamitsu Futai,

RNA modifications and cancer

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 γ 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 F0F1to see the α3β3 hexamer rotation; a biotin tag was introduced into the α or β 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 thec subunit, and an actin filament was connected to the α or β 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 γεc10–14 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. 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 γ 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 F0F1to see the α3β3 hexamer rotation; a biotin tag was introduced into the α or β 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 thec subunit, and an actin filament was connected to the α or β 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 γεc10–14 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. 4-morpholineethanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine dicyclohexylcarbodiimide ATP is synthesized in chloroplasts, mitochondria, and bacteria by a ubiquitous ATP synthase (F0F1) coupled with an electrochemical proton gradient. The F0F1 ofEscherichia coli consists of a catalytic sector, F1 (α3β3γδε), and a proton pathway, F0(ab2 c10–14) (for reviews, see Refs. 1Futai M. Noumi T. Maeda M. Annu. Rev. Biochem. 1989; 58: 111-136Crossref PubMed Scopus (458) Google Scholar, 2Futai M Omote H Handbook of Biological Physics.in: Konings W. N Kaback H. R Lolkema J. S Transport Processes in Eukaryotic and Prokaryotic Organisms. 2. Elsevier, Amsterdam1996: 47-74Google Scholar, 3Penefsky H.S. Cross R.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 173-214PubMed Google Scholar, 4Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (397) Google Scholar, 5Fillingame R.H. Curr. Opin. Struct. Biol. 1996; 6: 491-498Crossref PubMed Scopus (59) Google Scholar, 6Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1639) Google Scholar). The amino- and carboxyl-terminal α helices of the γ subunit occupy the central space of the α3β3 hexamer, as shown by the high resolution structure (7Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2797) Google Scholar). The catalytic sites in the three β subunits participate alternately in ATP synthesis and also in hydrolysis, as predicted from the binding change mechanism (6Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1639) Google Scholar). The mechanism also proposes that the γ subunit rotation plays a major role in the conformational transmission among the β subunits. The roles of the γ subunit in energy coupling and catalytic cooperativity have been shown by extensive genetic studies (2Futai M Omote H Handbook of Biological Physics.in: Konings W. N Kaback H. R Lolkema J. S Transport Processes in Eukaryotic and Prokaryotic Organisms. 2. Elsevier, Amsterdam1996: 47-74Google Scholar). Mutation and suppression studies have suggested that the γ subunit carboxyl-terminal domain and amino-terminal Met-23 interact through long range conformational transmissions involving the movement of the two helices (8Nakamoto R.K. Maeda M. Futai M. J. Biol. Chem. 1993; 268: 867-872Abstract Full Text PDF PubMed Google Scholar, 9Futai M. Omote H. J. Bioenerg. Biomembr. 1996; 28: 409-414Crossref PubMed Scopus (31) Google Scholar). γ subunit rotation has been suggested by β-γ cross-linking followed by dissociation and reconstitution (10Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (462) Google Scholar) and the movement of a probe attached to the carboxyl terminus of the γ subunit (11Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (466) Google Scholar). Continuous rotation has been observed directly as the movement of an actin filament connected to the γ subunit in the F1 sector (12Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (2005) Google Scholar, 13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar, 14Noji H. Hasler K. Junge W. Kinosita Jr., K. Yoshida M. Engelbrecht S. Biochem. Biophys. Res. Commun. 1999; 260: 597-599Crossref PubMed Scopus (86) Google Scholar). The rotation of the ε subunit with the γ subunit was also shown subsequently (15Kato-Yamada Y. Noji H. Yasuda R. Kinosita Jr., K. Yoshida M. J. Biol. Chem. 1998; 273: 19375-19377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Thus, this enzyme can be defined as a biological nanomotor carrying out rotational catalysis (16Wada Y. Sambongi Y. Futai M. Biochim. Biophys. Acta. 2000; 1459: 499-505Crossref PubMed Scopus (38) Google Scholar). In ATP hydrolysis, the mechanical work done by the γ subunit rotation should be transmitted to the F0 sector for ATP-dependent proton transport. During ATP synthesis, proton transport should drive the γ subunit rotation, which causes the β subunit conformational change to release the product ATP. Therefore, it is essential to determine how the γ subunit rotation is coupled to the proton transport through F0. Studies on the F0 sector involving electron (17Birkenhäger R. Hoppert M. Deckers-Hebestreit G. Mayer F. Altendorf K. Eur. J. Biochem. 1995; 230: 58-67Crossref PubMed Scopus (129) Google Scholar) and atomic force (18Takeyasu K. Omote H. Nettikadan S. Tokumasu F. Iwamoto-Kihara A. Futai M. FEBS Lett. 1996; 392: 110-113Crossref PubMed Scopus (117) Google Scholar, 19Singh S. Turina P. Bustamante C.J. Keller D.J. Capaldi R. FEBS Lett. 1996; 397: 30-34Crossref PubMed Scopus (105) Google Scholar) 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 (20Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Crossref PubMed Scopus (107) Google Scholar, 21Rastogi V.K. Girvin M.E. Nature. 1999; 402: 263-268Crossref PubMed Scopus (420) Google Scholar) and genetic fusion (22Jones P.C. Fillingame R.H. J. Biol. Chem. 1998; 273: 29701-29705Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Fillingame R.H Jiang W. Dmitriev O.Y. Jones P.C. Biochim. Biophys. Acta. 2000; 1458: 387-403Crossref PubMed Scopus (56) Google Scholar), and the yeast structure formed from 10 monomers was observed by x-ray diffraction (24Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1110) Google Scholar). A ring of 14 monomers was observed more recently for the chloroplast enzyme (25Seelert H. Poetsh A. Dencher N.A. Engel A. Stahlberg H. Müller D.J. Nature. 2000; 405: 418-419Crossref PubMed Scopus (423) Google Scholar). The rotation of the c ring with the γ subunit has been proposed (26Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 27Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 28Elston T. Wang H. Oster G. Nature. 1998; 391: 510-513Crossref PubMed Scopus (452) Google Scholar, 29Dimroth P. Wang H. Grabe M. Oster G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4924-4929Crossref PubMed Scopus (135) Google Scholar) and has been suggested by chemical cross-linking (30Watts S.D. Tang C. Capaldi R.A. J. Biol. Chem. 1996; 271: 28341-28347Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 31Schulenberg B. Aggeler R. Murray J. Capaldi R.A. J. Biol. Chem. 1999; 274: 34233-34237Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We recently provided the first direct evidence that the c ring rotates continuously together with the γ subunit in F0F1 (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar); F0F1 was immobilized on a glass surface through His tags connected to the α subunits, and an actin filament was attached to the c ring. Pänke et al. (33Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (180) Google Scholar) 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 γ subunit and thec ring, also suggesting that they can rotate together as an assembly (24Stock D. Leslie A.G.W. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1110) Google Scholar). As shown previously by Fillingame et al. (34Fillingame R.H. Oldenburg M. Fraga D. J. Biol. Chem. 1991; 266: 20934-20939Abstract Full Text PDF PubMed Google Scholar), 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 α3β3 hexamer could rotate when F0F1 was immobilized through the cring. These results indicate that a complex of the εγ 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. A plasmid (pBWU13) carrying the entire gene for F0F1 (35Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar) was engineered for the rotation of the α3β3hexamer. 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 α and β subunits (both between the 1st and 2nd codons). Plasmid pBWU13 was also engineered for the c ring rotation, as described previously (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar): a His tag, downstream of the initiation codon of the α subunit; cGlu-2 → Cys in the csubunit; and γCys-87 → Ala and γCys-112 → Ala in the γ subunit (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar). 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 (Δunc), which lacks the F0F1 gene (36Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar). 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 (35Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). Biotin (2 μm) was included in the synthetic medium for growth of the strain expressing the enzyme with the biotin tag. Membrane vesicles were prepared after the disruption of cells by passage through a French press (35Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). Nonengineered F0F1 was purified from membranes (DK8/pBWU13) as described previously (35Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). Modified procedures (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) were used for the engineered enzymes. Membrane vesicles were centrifuged and then suspended in buffer A (20 mmMES1-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}hexyld-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 mmMgCl2, 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 β subunit was purified as described previously (13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar). F0F1 was immobilized on a nitrocellulose-coated glass surface (cover glass) via the His tag and Ni2+-nitrilotriacetic acid horseradish peroxidase conjugate (13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar). 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 mmHepes-NaOH (pH 7.8) containing 25 mm KCl, 6 mmMgCl2, 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% β-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 (13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar). 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) (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) or in standard buffer (10 mm Hepes/NaOH, pH 7.2, 6 mmMgCl2), and the reaction was initiated by adding 5 mm [γ-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 0.3n 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 (37Futai M. J. Membr. Biol. 1974; 15: 15-28Crossref PubMed Scopus (126) Google Scholar). 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. Protein concentrations and the ATP-dependent formation of an electrochemical proton gradient were assayed as described previously (35Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). Fluorescently labeled biotinylated actin filaments were prepared as described (13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar). 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. Plasmids for two types of engineered F0F1 were constructed in this study: (a) the replacement of Glu-2 with Cys in the csubunit and the introduction of a His tag into the α subunit (αHis tag) with or without the c subunit Ile-28 to Thr (cI28T) mutation 2The γ subunit cysteine residues were removed from this enzyme. and (b) the introduction of a His tag into the csubunit and a biotin tag into the α (or β) subunit. The recombinant plasmids encoding these enzymes were introduced into E. colistrain DK8 lacking the entire F0F1 gene (36Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar). The transformed cells could grow on succinate through oxidative phosphorylation similar to those harboring the control plasmid encoding the nonengineered F0F1 (TableI). These results indicate that the genetic modification for observing rotation did not affect the catalysis by or energy coupling of the enzyme.Table IProperties of F0F1 engineered for rotationF0F1Growth on succinatePurified F0F1 ATPaseDCCD inhibitionUnits/mg protein%Nonengineered+++14.484Engineered for c ring rotationαHis tag/cE2C+++7.885αHis tag/cE2C/cI28T+3.848Engineered for α3β3 hexamer rotationcHis tag/α biotin tag++5.775cHis tag/β biotin tag++7.175Cells 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. Open table in a new tab 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. 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 α 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 α and β subunits with a biotin tag and biotinylation ofc subunit cE2C were confirmed by immunoblotting with streptavidin (data not shown). The csubunit with a His tag showed significantly stronger staining than that without the tag. 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 (34Fillingame R.H. Oldenburg M. Fraga D. J. Biol. Chem. 1991; 266: 20934-20939Abstract Full Text PDF PubMed Google Scholar). 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/αHis tag) as to the c ring rotation was inhibited by 8 μmventuricidin, whereas ∼30% of the activity of cI28T was inhibited (Fig. 2 a), confirming the previous results for membrane enzymes (34Fillingame R.H. Oldenburg M. Fraga D. J. Biol. Chem. 1991; 266: 20934-20939Abstract Full Text PDF PubMed Google Scholar). These enzymes became less sensitive when assayed in the buffer for rotation (Fig. 2 b). However, this antibiotic can be considered as a specific inhibitor for the engineered F0F1under 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. The engineered enzyme with the β biotin tag/cHis tag for α3β3 hexamer rotation was significantly less sensitive to venturicidin; only about 15% inhibition was observed with excess venturicidin (Fig. 2 a). This result may be attributable to the introduction of the His tag into the csubunit. Therefore, the antibiotic could not be tested for the rotation of this enzyme. The rotation of actin filaments connected to thec ring was confirmed, and it was unlikely that the filaments were connected to the γ subunit of the contaminating F1, as discussed previously (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar). As a control, F1 (with no cysteine in the γ subunit) was isolated from the engineered F0F1 to test whether the actin filament can bind to the Cys-less γ and rotate with the addition of ATP. We could find no rotating filament, indicating that the contaminating Cys-less γ F1, if any, is not responsible for the rotation observed. 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. 3 a, b, c), confirming the previous results (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar). On the other hand, the filament connected to thec 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. 3 d). 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. 3 e). The cI28T mutation showed no significant effect on the filament rotation or torque generation (Fig. 3 e). Furthermore, venturicidin showed no effect on the torque generation (Fig. 3 e,open and closed triangles). The results obtained for thecT28 mutant strengthen the notion that a complex of the c ring and the γ subunit rotates during ATP hydrolysis, and this prompted us to examine a further possibility. If the γ subunit and c ring form a mechanical unit, the α3β3 hexamer rotates when the cring is immobilized on a glass surface (Fig.4 a). We introduced a His tag into the c subunit and a biotin tag into the α or β subunit to examine this possibility. As expected, an actin filament connected to the α3β3 hexamer rotated upon ATP addition (Fig. 4 b). 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 α or β subunit. The rotation of the α3β3 hexamer generated essentially the same torque (∼40 piconewton·nm) as that of the c ring. An effect of venturicidin on the α3β3hexamer rotation was not apparent, which is consistent with its slight effect on the ATPase activity of the F0F1engineered for α3β3 rotation (Fig. 2). As a control, we purified F1 with a biotin tag in the α subunit (but no His tag in the γ 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. As predicted by the binding change mechanism for ATP synthesis and hydrolysis, the continuous rotation of the γ subunit in the F1 sector has been observed during ATP hydrolysis (12Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (2005) Google Scholar, 13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar, 14Noji H. Hasler K. Junge W. Kinosita Jr., K. Yoshida M. Engelbrecht S. Biochem. Biophys. Res. Commun. 1999; 260: 597-599Crossref PubMed Scopus (86) Google Scholar). The γ subunit rotation should be transmitted to the F0sector for coupling with proton transport. We provided the first evidence that the c subunit ring rotates with the γ subunit in F0F1 (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar). Pänke et al. (33Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (180) Google Scholar) have also demonstrated the c ring rotation more recently. The present study further confirmed the co-rotation of the γ subunit with the c ring. Fillingame et al. (34Fillingame R.H. Oldenburg M. Fraga D. J. Biol. Chem. 1991; 266: 20934-20939Abstract Full Text PDF PubMed Google Scholar) 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 γ rotation of the engineered F1. These results suggest that venturicidin inhibits F0F1 rotation by binding to the cring and that a complex of the γ 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 α3β3 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 (12Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (2005) Google Scholar, 13Omote H. Sambonmatsu N. Saito K. Sambongi Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (126) Google Scholar, 14Noji H. Hasler K. Junge W. Kinosita Jr., K. Yoshida M. Engelbrecht S. Biochem. Biophys. Res. Commun. 1999; 260: 597-599Crossref PubMed Scopus (86) Google Scholar, 15Kato-Yamada Y. Noji H. Yasuda R. Kinosita Jr., K. Yoshida M. J. Biol. Chem. 1998; 273: 19375-19377Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar). Upon the addition of ATP, the actin filament connected to the α3β3 hexamer rotated counterclockwise when viewed from the F1 side and generated similar torque to the γ rotation. These results strongly suggest that the γεc10–14 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 γ subunit with the cring during ATP synthesis and hydrolysis predicts that the γ subunit interacts with the hydrophilic loop between the transmembrane helices of each c subunit one by one during rotation (discussed in Refs. 38Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar and 39Altendorf K. Staltz W.D. Greie J.C. Deckers-Hebestreit G. J. Exp. Biol. 2000; 203: 19-28PubMed Google Scholar). The rotation of the actin filament connected to thec ring indicates that this model will not work. However, it may be possible to argue that the c ring rotated with the γ subunit as a part of the fluorescent probe (actin filament). The rotation of the filament connected to the α3β3 hexamer finally excluded this possibility because the model predicts that the filament should not rotate if the c ring is immobilized and the γ subunit moves on the ring. Tsunoda et al. (40Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar) have claimed that they could not connect an actin filament specifically to the c ring under their experimental conditions and criticized the experiments by Sambongiet al. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) showing c ring rotation. The protocols used by the two groups are different, including the positions of Cys residues introduced into the c subunit (Sambongiet al. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar), Glu-2 → Cys; Tsunoda et al. (40Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar), Cys inserted between Glu-2 and Asn-3), the conditions for maleimide modification (Sambongi et al. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar), 4 °C at pH 7.0; Tsunoda et al. (40Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar), 25 °C at pH 7.5), and the detergent used for F0F1 preparation. Furthermore, in most experiments, Tsunoda et al. (40Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar) 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. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) reacted F0F1 with biotin-maleimide and then connected an actin filament through streptavidin. Thus, the arguments raised by Tsunoda et al. (40Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar), which are based on mostly negative observations, may reflect the differences in the experimental systems. As described in this study, the conclusion of Sambongiet al. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) is supported by Pänke et al.(33Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (180) Google Scholar) and the effect of venturicidin on the cI28T mutant, as described above. Furthermore, the actin filament connected to the α3β3 hexamer in the immobilized F0F1 rotated, as discussed above. In conclusion, we clearly showed that a complex of εγ 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 (41Negrin R.S. Foster D.L. Fillingame R.H. J. Biol. Chem. 1980; 255: 5643-5648Abstract Full Text PDF PubMed Google Scholar). Genetic (42Friedl P. Hoppe J. Gunsalus R.P. Michelsen O. von Meyenburg K. Schairer H.U. EMBO J. 1983; 2: 99-103Crossref PubMed Scopus (54) Google Scholar) and reconstitution (43Schneider E. Altendorf K. EMBO J. 1985; 4: 515-518Crossref PubMed Scopus (123) Google Scholar) 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. (32Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (425) Google Scholar) did not conclude that the rotating enzyme had all the subunits. Pänke et al. (33Pänke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (180) Google Scholar) 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 γε subunit andc 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 γ subunit is artificially rotated. We thank Le Phi Nga for technical assistance during the early stage of this work.