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The -Subunit Rotation and Torque Generation in F 1-Atpase from Wild-Type Or Uncoupled Mutant Escherichia Coli

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The -Subunit Rotation and Torque Generation in F 1-Atpase from Wild-Type Or Uncoupled Mutant Escherichia Coli Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7780–7784, July 1999 Biochemistry The ␥-subunit rotation and torque generation in F1-ATPase from wild-type or uncoupled mutant Escherichia coli (ATP synthase͞rotational catalysis͞frictional torque) HIROSHI OMOTE*, NORIKO SAMBONMATSU*, KIWAMU SAITO†,YOSHIHIRO SAMBONGI*, ATSUKO IWAMOTO-KIHARA‡, TOSHIO YANAGIDA§,YOH WADA*, AND MASAMITSU FUTAI*¶ *Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, CREST of Japan Science and Technology Corporation, Ibaraki, Osaka 567-0047, Japan; †Department of Physics, Kanazawa University, Kanazawa 920-1192, Japan; ‡Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Tokyo 153-8902, Japan; and §Department of Physiology, Osaka University Medical School, Suita, Osaka 565-0871, Japan Communicated by Paul D. Boyer, University of California, Los Angeles, CA, April 28, 1999 (received for review March 11, 1999) ABSTRACT The rotation of the ␥-subunit has been in- isolated by using a plasmid carrying the unc (or atp) operon for ͞ cluded in the binding-change mechanism of ATP synthesis FOF1, and their ATP synthesis, hydrolysis, and proton trans- hydrolysis by the proton ATP synthase (FOF1). The Escherichia location can be readily assayed (15, 16). Mutants mapped at or coli ATP synthase was engineered for rotation studies such near the catalytic site (15–20) or those defective in energy that its ATP hydrolysis and synthesis activity is similar to that coupling (21–26) may be useful for defining the mechanical- of wild type. A fluorescently labeled actin filament connected work step during ATP synthesis or hydrolysis. Thus, it is ␥ to the -subunit of the F1 sector rotated on addition of ATP. important to establish an assay system for observing the This progress enabled us to analyze the ␥M23K (the ␥-subunit mechanical work of the E. coli ␥-subunit. Met-23 replaced by Lys) mutant, which is defective in energy In this study, we observed that an actin filament connected coupling between catalysis and proton translocation. We to the E. coli ␥-subunit could rotate by using the energy of ATP found that the F1 sector produced essentially the same fric- hydrolysis. With this progress, we could analyze the energy- tional torque, regardless of the mutation. These results sug- coupling mutant, ␥M23K (␥-subunit Met-23 replaced by Lys), ␥ gest that the M23K mutant is defective in the transformation of which FOF1 shows low ATP-driven proton transport and of the mechanical work into proton translocation or vice versa. ATP synthesis (22–25). We found that the mutant ␥-subunit could generate essentially the same torque as that of the ␥ The ATP synthases (FOF1) of mitochondria, chloroplasts, and wild-type subunit. Thus, the major defect of the M23K Escherichia coli synthesize ATP coupled with an electrochem- mutant is in the transformation of mechanical work into ical gradient of protons (for reviews, see refs. 1–6). The proton transport. conserved basic structure of the enzyme consists of a catalytic ␣ ␤ ␥ ␦ ␧ sector, F1 or F1-ATPase ( 3 3 1 1 1), and a proton pathway, ␣ ␤ ␥ EXPERIMENTAL PROCEDURES FO (a1b2c12). The crystal structure of the bovine 3 3 complex indicates that the ␣- and ␤-subunits are arranged Bacterial Strain, Growth Conditions, and Plasmids. E. coli alternately around the amino- and carboxyl-terminal ␣-helices strain DK8 (27) lacking all genes for ATP synthase (⌬unc) was of the ␥-subunit (7). Catalytic sites are mostly comprised of used as a host for recombinant plasmids and grown at 37°C in residues from the ␤-subunit. These catalytic sites participate a rich medium (L broth) supplemented with ampicillin or a alternately in ATP synthesis as predicted by the binding- synthetic medium with 0.5% glycerol (15). The unc operon change mechanism (5). Consistent with this mechanism, the bearing plasmids pBWU17 (28) and pBMUG420-␥M23K (unc three ␤-subunits in the bovine structure have different con- operon plasmid with the ␥M23K mutation; A.I.-K., unpub- formations: empty, ATP-bound, and ADP-bound forms (7). lished work) was used. ‘‘Conformation transmission successively among three ␤ sub- Construction of unc Operon Plasmids Carrying a His-tag units through the mechanical ␥ subunit rotation’’ (5) has been and ␥-Subunit Ser-193 to Cys Replacement. Codons for the a fascinating model to include in the binding-change mecha- Met(His)6 sequence (His-tag) were introduced in front of the nism. The ␥-subunit rotation has been suggested by cryo- initiation codon of the ␣-or␤-subunit gene carried by electron microscopy studies of F1 (8), chemical cross-linking pBWU17; a double-stranded cassette [5Ј-CAT(CAC)5ATG- experiments on the subunits (9, 10), and analysis of polarized 3Ј͞3Ј-GTACGTA(GTG)4GT-5Ј] was introduced into a SphI absorption recovery after photobleaching of a probe linked to site located in the initiation codon of the ␣-subunit. For the the ␥-subunit (11, 12). Finally, the rotation of an actin filament ␤-subunit, the SphI–NarI fragment (60-bp upstream sequence attached to a thermophilic bacterial ␥-subunit was observed from the ␤Gly-10 codon) was replaced with a similar cassette directly and video-recorded (13). Thus, ATP synthesis or for Met(His)6 and then introduced into pBWU17. To construct hydrolysis is a combination of three different steps: catalysis, a ␥Ser-193 to Cys substitution of the ␥-subunit, the SalI–SpeI mechanical work (␥-subunit rotation and torque generation), segment was cloned; the ␥S193C (TCA 3 TGC) substitution and proton transport. During proton transport, rotational was introduced into the segment by PCR; and the RsrII–SpeI movement of the c subunit oligomer (a group of 12 c subunits) segment with the substitution was introduced into pBWU17 has also been proposed (5, 12, 14). engineered with a His-tag. The ␥M23K mutation was intro- The energy coupling of the three steps can be analyzed, duced by replacing Csp45I–RsrII (with unc operon segment taking advantage of the wealth of information on the E. coli between the ␣Phe-467 and ␥Ile-149 codons) of the engineered ATP synthase accumulated through the combined biochemical plasmid with the corresponding fragment from pBMUG420– and genetic approaches. Furthermore, mutants can be easily ␥M23K. Preparation of F1-ATPase. Membrane vesicles were pre- The publication costs of this article were defrayed in part by page charge pared from cells grown on 0.5% glycerol, suspended in 20 mM payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. e-mail: m-futai@ PNAS is available online at www.pnas.org. sanken.osaka-u.ac.jp. 7780 Downloaded by guest on September 28, 2021 Biochemistry: Omote et al. Proc. Natl. Acad. Sci. USA 96 (1999) 7781 Tris⅐HCl buffer (pH 8.0) containing 0.5 mM DTT, 140 mM finally the reaction mixture for rotation (50 ␮Mto5mM KCl, 1 mM EDTA, and 10% (wt͞vol) glycerol, and then Mg-ATP͞1 ␮M biotin͞50 ␮g/ml pyruvate kinase͞1 mM phos- centrifuged at 160,000 ϫ g for1htoremove the ␦-subunit. The phoenol pyruvate͞25 mM glucose͞1% ␤-mercaptoethanol͞ precipitate was incubated in 2 mM Tris⅐HCl (pH 8.0) contain- 216 ␮g/ml glucose oxidase͞36 ␮g/ml catalase in buffer A) was ing 1 mM EDTA for 10 min. After centrifugation, 1 M introduced into the flow cell. The cell was sealed with silicon Hepes-NaOH (pH 7.8) and 1 M MgSO4 were added to the grease, and the rotations were observed at 25°C by using a supernatant (final concentrations of 50 mM and 2 mM, Zeiss Axiovert 135 equipped with an ICCD camera (Atto respectively), and the mixture was incubated with 100 ␮M Instruments, Rockville MD) and video-recorded. The rotation biotin-PEAC5-maleimide (Dojindo, Kumamoto, Japan) for 30 angle of the filament was estimated from the centroid of the min. The mixture was applied to a Ϸ10–30% (wt͞vol) glycerol actin filament. Rotation (revolutions per second) was calcu- gradient [containing 10 mM Hepes-NaOH (pH 7.8) and 2 mM lated from the slope of the curves, as shown in Fig. 1. ϫ MgSO4] and then centrifuged at 350,000 g for 4 h. The Other Procedures. ATPase activity and the formation of an essentially pure biotinylated F1-ATPase was obtained in the electrochemical proton gradient were assayed under the con- fractions containing 20–25% (wt͞vol) glycerol. All solutions ditions used for the rotation assay. The measurement of contained 0.5 mM PMSF, 5 ␮g͞ml leupeptin, and 5 ␮g͞ml protein concentrations and other procedures were performed pepstatin, except that PMSF was omitted from the glycerol as described (15, 21). gradient. Materials. Actin filament and biotin-PEAC5-maleimide Assaying Actin Filament Rotation. Rotation was assayed by were incubated in a molar ratio of 1:5 at 25°C for 1 h. the slightly modified method previously reported (13). Buffer Biotinylated actin filaments were mixed with an equal amount ͞ ͞ ͞ A (10 mM Hepes-NaOH, pH 7.2 25 mM KCl 5 mM MgCl2 10 of native actin and then labeled with phalloidin tetramethyl mg/ml BSA) was included in all solutions used, unless other- rhodamine as described (29). DNA-modifying enzymes were wise specified. A flow cell Ϸ20–50 ␮m deep was constructed obtained from Takara Shuzo (Kyoto) or New England Biolabs. from nitrocellulose-coated cover glass by using Parafilm Other chemicals used were of the highest grade commercially (American National Can, Chicago, IL) and filled with 0.8 ␮M available. Ni-NTA HRP Conjugate (Qiagen, Germany) in buffer A without BSA. After a 5-min incubation at 25°C with Ni-NTA ͞ ␮ RESULTS HRP Conjugate, 10 mg ml BSA, 10 nM F1-ATPase, and 4 M streptavidin were successively introduced into the flow cell and Engineering of E. coli F1-ATPase.
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