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• W2095492608 abstract "We report evidence for catalysis-dependent rotation of the single ε subunit relative to the three catalytic β subunits of functionally coupled, membrane-bound FOF1-ATP synthase. Cysteines substituted at β380 and ε108 allowed rapid formation of a specific β-ε disulfide cross-link upon oxidation. Consistent with a need for ε to rotate during catalysis, tethering ε to one of the β subunits resulted in the inhibition of both ATP synthesis and hydrolysis. These activities were fully restored upon reduction of the β-ε cross-link. As a more critical test for rotation, a subunit dissociation/reassociation procedure was used to prepare a β-ε cross-linked hybrid F1 having epitope-tagged βD380C subunits (βflag) exclusively in the two noncross-linked positions. This allowed the β subunit originally aligned with ε to form the cross-link to be distinguished from the other two βs. The cross-linked hybrid was reconstituted with FO in F1-depleted membranes. After reduction of the β-ε cross-link and a brief period of catalytic turnover, reoxidation resulted in a significant amount of βflag in the β-ε cross-linked product. In contrast, exposure to ligands that bind to the catalytic site but do not allow catalysis resulted in the subsequent cross-linking of ε to the original untagged β. Furthermore, catalysis-dependent rotation of ε was prevented by prior treatment of membranes with N,N′-dicyclohexylcarbodiimide to block proton translocation through FO. From these results, we conclude that ε is part of the rotor that couples proton transport to ATP synthesis. We report evidence for catalysis-dependent rotation of the single ε subunit relative to the three catalytic β subunits of functionally coupled, membrane-bound FOF1-ATP synthase. Cysteines substituted at β380 and ε108 allowed rapid formation of a specific β-ε disulfide cross-link upon oxidation. Consistent with a need for ε to rotate during catalysis, tethering ε to one of the β subunits resulted in the inhibition of both ATP synthesis and hydrolysis. These activities were fully restored upon reduction of the β-ε cross-link. As a more critical test for rotation, a subunit dissociation/reassociation procedure was used to prepare a β-ε cross-linked hybrid F1 having epitope-tagged βD380C subunits (βflag) exclusively in the two noncross-linked positions. This allowed the β subunit originally aligned with ε to form the cross-link to be distinguished from the other two βs. The cross-linked hybrid was reconstituted with FO in F1-depleted membranes. After reduction of the β-ε cross-link and a brief period of catalytic turnover, reoxidation resulted in a significant amount of βflag in the β-ε cross-linked product. In contrast, exposure to ligands that bind to the catalytic site but do not allow catalysis resulted in the subsequent cross-linking of ε to the original untagged β. Furthermore, catalysis-dependent rotation of ε was prevented by prior treatment of membranes with N,N′-dicyclohexylcarbodiimide to block proton translocation through FO. From these results, we conclude that ε is part of the rotor that couples proton transport to ATP synthesis. carbonyl cyanide p-trifluoromethoxyphenylhydrazone N, N′-dicyclohexylcarbodiimide 5,5′-dithiobis(2-nitrobenzoate) dithiothreitol polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid. FOF1-ATP synthases are found embedded in the membranes of mitochondria, chloroplasts, and bacteria (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 2Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar). During oxidative phosphorylation and photophosphorylation, synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. The FO sector is composed of membrane-spanning subunits (ab2c12 in Escherichia coli) (3Fillingame R.H. Jones P.C. Jiang W. Valiyaveetil F.I. Dmitriev O.Y. Biochim. Biophys. Acta. 1998; 1365: 135-142Crossref PubMed Scopus (59) Google Scholar) that conduct protons across the membrane, whereas the F1 sector (α3β3γδε) is an extrinsic complex that contains catalytic sites for ATP synthesis. F1 can be removed from the membrane in a soluble form that functions as an ATPase, and rebinding F1 to FO in membranes restores the capacity to catalyze net ATP synthesis. A high-resolution structure for bovine F1 shows a hexamer of alternating α and β subunits surrounding a single γ subunit. The three catalytic sites of F1 are located on the three β subunits at α/β subunit interfaces (4Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2754) Google Scholar). A model for energy coupling by FOF1-ATP synthases that has gained widespread support is called the binding change mechanism (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar). According to this proposal, the major energy-requiring step is not the synthesis of ATP at catalytic sites but rather the simultaneous and highly cooperative binding of substrates to and release of products from these sites (5Boyer P.D. Cross R.L. Momsen W. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2837-2839Crossref PubMed Scopus (280) Google Scholar, 6Kayalar C. Rosing J. Boyer P.D. J. Biol. Chem. 1977; 252: 2486-2491Abstract Full Text PDF PubMed Google Scholar). Furthermore, it was proposed that these affinity changes are coupled to proton transport by the rotation of a complex of subunits that extends through FOF1. Rotation of the γ subunit in the center of F1 is thought to deform the surrounding catalytic subunits to give the required binding changes (7Boyer P.D. Kohlbrenner W.E. Selman B. Selman-Reiner S. Energy Coupling in Photosynthesis. Elsevier/North-Holland, New York1981: 231-240Google Scholar), whereas rotation of the c subunits relative to the single a subunit in FO is believed to be required for completion of the proton pathway (8Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 9Duncan 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 (459) Google Scholar, 10Hatch L.P. Cox G.B. Howitt S.M. J. Biol. Chem. 1995; 270: 29407-29412Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The rotary aspect of the binding change mechanism remained a popular but speculative idea for a number of years until a critical test became possible following the publication of a high resolution structure for F1 (4Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2754) Google Scholar). Focusing on a β/γ intersubunit point of contact identified in the structure, we introduced a Cys into the β subunit at a position (β380) that would place it in close proximity to a naturally occurring Cys on the γ subunit (γC87). When the resultant βD380C-F1 was exposed to an oxidant, a rapid and specific βD380C-γC87 disulfide cross-link was formed (9Duncan 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 (459) Google Scholar, 11Duncan T.M. Zhou Y. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochem. Soc. Trans. 1995; 23: 736-741Crossref PubMed Scopus (20) Google Scholar). Using a subunit dissociation/reassociation approach with the β-γ cross-linked enzyme, we incorporated radioisotope- or epitope-labeled β subunits into the two noncross-linked β subunit positions. Following reduction of the cross-link and a short burst of ATP hydrolysis (9Duncan 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 (459) Google Scholar, 12Zhou Y. Duncan T.M. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochim. Biophys. Acta. 1996; 1275: 96-100Crossref PubMed Scopus (69) Google Scholar) or synthesis (13Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar), labeled and unlabeled β subunits in the hybrid F1 showed a similar capacity to form a disulfide bond with the γ subunit indicating that γ had rotated relative to the three β subunits during catalysis. Subsequently, additional evidence for subunit rotation during ATP hydrolysis was obtained using immobilized chloroplast F1 with a spectroscopic probe attached near the C terminus of the γ subunit. Recovery of polarized absorption after photobleaching was used to monitor the rotational motion of γ during ATP hydrolysis by the tethered F1 on a time-resolved basis (14Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar). Finally, in a dramatic visual demonstration, a fluorescent actin filament attached to one end of the γ subunit of immobilized bacterial F1 was seen by fluorescence microscopy to undergo multiple unidirectional rotations during ATP hydrolysis (15Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar). Now that catalysis-dependent rotation of γ relative to the catalytic β subunits is well established (16Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 17Dimroth P. Kaim G. Matthey U. Biochim. Biophys. Acta. 1998; 1365: 87-92Crossref PubMed Scopus (29) Google Scholar, 18Elston T. Wang H. Oster G. Nature. 1998; 391: 510-513Crossref PubMed Scopus (446) Google Scholar, 19Kinosita Jr., K. Yasuda R. Noji H. Ishiwata S. Yoshida M. Cell. 1998; 93: 21-24Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), it is important to identify other components of the rotor. A likely candidate is the ε subunit. It forms a tight 1:1 complex with purified γ (20Dunn S.D. J. Biol. Chem. 1982; 257: 7354-7359Abstract Full Text PDF PubMed Google Scholar), and cross-linking ε to γ causes less than a proportional amount of inhibition (21Aggeler R. Chicas-Cruz K. Cai S.-X. Keana J.F.W. Capaldi R.A. Biochemistry. 1992; 31: 2956-2961Crossref PubMed Scopus (95) Google Scholar, 22Watts S.D. Tang C. Capaldi R.A. J. Biol. Chem. 1996; 271: 28341-28347Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 23Schulenberg B. Wellmer F. Lill H. Junge W. Engelbrecht S. Eur. J. Biochem. 1997; 249: 134-141Crossref PubMed Scopus (46) Google Scholar). In contrast, cross-linking ε to either β (24Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) or α (25Aggeler R. Capaldi R.A. J. Biol. Chem. 1996; 271: 13888-13891Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) in soluble F1 strongly inhibits ATPase activity. The fact that ε is randomly oriented in FOF1 relative to the α subunit that interacts with the single δ subunit is also consistent with ε being part of the rotor (26Aggeler R. Ogilvie I. Capaldi R.A. J. Biol. Chem. 1997; 272: 19621-19624Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Finally, it was recently reported that ATP hydrolysis promotes the rotation of ε in immobilized F1 from chloroplasts (27Häsler K. Engelbrecht S. Junge W. FEBS Lett. 1998; 426: 301-304Crossref PubMed Scopus (58) Google Scholar) and thermophilic bacteria (28Kato-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 (173) Google Scholar). In work presented here, we have extended our cross-linked hybrid approach to demonstrate rotation of ε relative to the three β subunits during catalysis by functionally coupled membrane-bound FOF1. NADH, ATP, ADP, phosphoenolpyruvate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),1 N,N′-dicyclohexylcarbodiimide (DCCD),N-ethylmaleimide, and hexokinase were supplied by Sigma; pyruvate kinase and lactate dehydrogenase were supplied by Boehringer Mannheim; 5,5′-dithiobis(2-nitrobenzoate) (DTNB) was supplied by Aldrich; and dithiothreitol (DTT) was supplied by American Bioanalytical (Natick, MA). Oligonucleotides for site-directed mutagenesis were synthesized by Life Technologies, Inc. PfuDNA polymerase I was from Stratagene, and all restriction endonucleases were from New England Biolabs. Anti-Flag M2 antibody was obtained from Eastman Kodak, 125I-labeled anti-mouse antibody was from Amersham Pharmacia Biotech, and [32P]Pi was from ICN. Other reagents and chemicals were the highest grade available. Mutant constructs p3UβD380C/γC87S and p3UβflagD380C/γC87S were described previously, and these combined mutations have minimal effects on the FOF1 function in vivo (normal phenotypic growth on succinate) or on ATPase activity of purified F1 (11Duncan T.M. Zhou Y. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochem. Soc. Trans. 1995; 23: 736-741Crossref PubMed Scopus (20) Google Scholar, 12Zhou Y. Duncan T.M. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochim. Biophys. Acta. 1996; 1275: 96-100Crossref PubMed Scopus (69) Google Scholar). The εS108C mutation, originally created by Aggeler et al. (21Aggeler R. Chicas-Cruz K. Cai S.-X. Keana J.F.W. Capaldi R.A. Biochemistry. 1992; 31: 2956-2961Crossref PubMed Scopus (95) Google Scholar), also has only minimal effects on the function of FOF1 in vivo or of purified F1. Here a polymerase chain reaction-based, site-directed method (29Landt O. Grunert H.-P. Hahn U. Gene (Amst.). 1990; 96: 125-128Crossref PubMed Scopus (639) Google Scholar) (Pfu DNA polymerase I) was used to introduce εS108C into p3U, which expresses all 8 FOF1 subunits. The antisense mutagenic primer, 5′-ATTAGCAGCTGTCACGGCGAC-3′, has a single base change that generates the εS108C mutation and creates a PvuII restriction site. The product was digested with KpnI and NdeI and cloned into the corresponding sites of the p3UβD380C/γC87S vector to produce p3UβD380C/γC87S/εS108C. The mutated region of uncC was sequenced to confirm the presence of εS108C and absence of any additional mutations. To express mutant FOF1, each construct was transformed into strain AN887, which has a Mu insertion that blocks expression of the chromosomal unc operon (30Gibson F. Downie J.A. Cox G.B. Radik J. J. Bacteriol. 1978; 134: 728-736Crossref PubMed Google Scholar). Expression of p3UβD380C/γC87S/εS108C yields membranes with normal levels of DCCD-sensitive ATPase and purified F1 with ATPase activity (35 μmol·min−1·mg−1) comparable with that of wild-type F1. Membranes were isolated and washed (31Senior A.E. Fayle D.R.H. Downie J.A. Gibson F. Cox G.B. Biochem. J. 1979; 180: 111-118Crossref PubMed Scopus (41) Google Scholar, 32Wise J.G. J. Biol. Chem. 1990; 265: 10403-10409Abstract Full Text PDF PubMed Google Scholar), and soluble F1 was purified as described (9Duncan 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 (459) Google Scholar). Membranes prepared from strain AN1460 (33Downie J.A. Langman L. Cox G.B. Yanofsky C. Gibson F. J. Bacteriol. 1980; 143: 8-17Crossref PubMed Google Scholar) were depleted of F1 with two additional washes with 10 mm Tris acetate, 1 mm EDTA, pH 8.0. Washed membranes were resuspended in TM buffer (50 mm Tris-Cl, 5 mmMgSO4, pH 7.5), quickly frozen, and stored at −70 °C. Aliquots of F1stock solutions were passed through centrifuge columns containing Sephadex G-50–80 (34Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar) equilibrated with MTKE buffer (20 mmMops-Tris, 50 mm KCl, 0.1 mm EDTA, pH 8.0). The enzymes were diluted to 1 mg/ml with the same buffer and treated with 50 μm DTNB for 40 min at 22 °C. Excess DTNB was removed by column centrifugation. β-ε cross-linked βD380C/γC87S/εS108C-F1 and epitope-labeled βflagD380C/γC87S-F1 were dissociated into subunits by repeated freezing and thawing in 50 mm MES, 1m LiCl, 5 mm ATP, 0.5 mm EDTA, pH 6.1. The dissociated enzymes were mixed in a 1:1 ratio and allowed to reassemble as hybrid complexes as described previously (9Duncan 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 (459) Google Scholar, 35Vogel G. Steinhart R. Biochemistry. 1976; 15: 208-216Crossref PubMed Scopus (196) Google Scholar). F1 hybrid containing the βD380C-εS108C disulfide cross-link can contain βflagD380C only in the two noncross-linked β positions. F1 containing the wild-type ε subunit is incapable of forming a β-ε cross-link and is therefore silent in subsequent experiments. Hybrid F1 (0.5 mg/ml) was rebound to FO in F1-depleted membranes (2 mg of protein/ml) by incubation in TM buffer for 30 min at 30 °C. Excess F1 was removed by centrifugation at 100,000 × g in a Beckman Airfuge for 1 min. The membrane pellet was resuspended and washed twice with TMG buffer (TM buffer containing 50 mm glucose) and finally resuspended in TMG buffer at about 4 mg of protein/ml. For the DCCD-inhibited control, membranes were incubated with 0.5 mm DCCD and 5 mm MgCl2 for 30 min at 22 °C, conditions that give specific modification of c subunits in FO (36Tommasino M. Capaldi R.A. Biochemistry. 1985; 24: 3972-3976Crossref PubMed Scopus (35) Google Scholar, 37Hermolin J. Fillingame R.H. J. Biol. Chem. 1989; 264: 3896-3903Abstract Full Text PDF PubMed Google Scholar). Enzyme for the “noncross-linked” control was prepared as described for hybrid F1 except that a β-ε cross-link was not formed prior to subunit dissociation by omitting DTNB treatment. Hence this form of F1 can have βflag at all three β subunit positions. SDS-PAGE was performed according to Laemmli (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) on 4–15% gradient gels (Ready Gels, Bio-Rad). For nonreducing conditions, samples were denatured in the presence of 0.5 mm N-ethylmaleimide instead of 2-mercaptoethanol to block sulfhydryls without cleaving disulfides. Proteins were transferred from the gel to a polyvinylidene difluoride membrane (Novex) in a Bio-Rad Mini Trans-Blot cell for 1 h at 250 mA using a buffer containing 25 mm Tris, 192 mmglycine, 10% methanol, and 0.005% SDS (39Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar). The blotted membrane was blocked for 1 h with 5% nonfat, dried milk in TBST (10 mm Tris-Cl, 150 mm NaCl, 0.05% Tween 20, pH 8.0), incubated for 2 h with anti-Flag M2 antibody (Eastman Kodak) at 0.4 mg/ml in TBST with 1% bovine serum albumin, and rinsed three times with TBST containing an additional 0.1 m NaCl. The immunoblot was then incubated for 1.5 h with 2 μCi of125I-labeled anti-mouse secondary antibody (19 μCi/μg, Amersham Pharmacia Biotech) in 10 ml of TBST with 1% bovine serum albumin, rinsed five times with TBST containing an additional 0.1m NaCl, air dried, and exposed to a Phosphor Screen for 12 h. 125I-Labeled bands were quantitated using a PhosphorImager (model 425E, Molecular Dynamics) and ImageQuant software. The ATPase activity of soluble F1was measured at 30 °C by a coupled enzyme assay (40Pullman M.E. Penefsky H.S. Datta A. Racker E. J. Biol. Chem. 1960; 235: 3322-3329Abstract Full Text PDF PubMed Google Scholar) using 5 mm ATP, 2 mm MgCl2, and 0.5 μg of F1/ml in MTK buffer (20 mm Mops-Tris, 50 mm KCl, pH 8.0). For measurement of ATP hydrolysis by membrane vesicles at 2–3 μg of protein/ml, 5 μm FCCP and 5 mm KCN were also added to prevent formation of a transmembrane proton gradient and to block NADH oxidation by the respiratory chain. The synthesis of ATP by E. coli membranes was determined by a coupled enzyme assay as described (13Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar). Protein concentrations were determined by a modified Lowry assay (41Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7134) Google Scholar). Our approach in testing for rotation of the ε subunit requires the reversible formation of a specific covalent linkage between a β subunit and the single copy of ε. Guided by previous cross-linking studies (24Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 42Dallmann H.G. Flynn T.G. Dunn S.D. J. Biol. Chem. 1992; 267: 18953-18960Abstract Full Text PDF PubMed Google Scholar), we combined the εS108C and βD380C mutations in a single construct. To prevent cross-linking between β and γ subunits, the γC87S mutation was also included (11Duncan T.M. Zhou Y. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochem. Soc. Trans. 1995; 23: 736-741Crossref PubMed Scopus (20) Google Scholar). As shown by nonreducing SDS-PAGE (Fig. 1), a rapid and near complete disappearance of ε is accompanied by the appearance of a new band at 67 kDa when F1 (lane 2 versus lane 1) or membrane-bound FOF1 (lane 7 versus lane 6) containing these mutations is oxidized by DTNB. The high yield of cross-linked product correlates to a 90–95% loss of ATPase activity, and the same results were obtained when samples were oxidized in the presence of Mg2+, MgATP, or MgADP/azide (data not shown). The apparent size of the cross-linked product (67 kDa) is consistent with the predicted molecular mass of 65 kDa for a 1:1 complex between β and ε. Furthermore, immunoblotting confirmed the presence of both ε and β in the 67-kDa band (data not shown). As expected for a disulfide linkage, cross-linking and inactivation are fully reversed by brief exposure to dithiothreitol (lane 3). Finally, oxidation of F1 lacking the εS108C mutation shows no 67-kDa band nor does the ε band disappear (lane 4). We conclude that the 67-kDa product results from formation of a specific disulfide cross-link between βD380C and εS108C. A second requirement for our F1-hybrid approach in testing for rotation of ε is that ε must not dissociate from F1 during the course of the experiment. If this occurred, ε could rebind in a manner that would allow it to cross-link to a different β than the one with which it was originally aligned, thus giving a false indication of subunit rotation. This requirement presented a potential problem in using soluble F1 because ε is known to undergo reversible dissociation from the E. coli enzyme (43Smith J.B. Sternweis P.C. Biochemistry. 1977; 16: 306-311Crossref PubMed Scopus (135) Google Scholar). Hence, the experiment presented in Fig. 2 was conducted to determine the rate of ε subunit exchange between members of an F1 population. The strategy behind this assay was to mix two different forms of the enzyme: one containing εS108C and β lacking the Flag epitope (βD380C/γC87S/εS108C-F1), and the other containing epitope-tagged β and wild-type ε (βflagD380C/γC87S-F1). Because wild-type ε cannot cross-link to βflag, the only way that a βflag-ε cross-link can form is if an εS108C subunit dissociates from the triple mutant enzyme and rebinds to a Flag-tagged double mutant F1 that has released its own wild-type ε. DTNB was added at the times indicated, and the Flag epitope in the cross-linked product was measured in the immunoblot shown in Fig. 2 A. As expected, the amount of Flag in the β-ε band increased with time. The t½ for the exchange of ε was found to be about 1 min (Fig. 2 B). Because this is on the same time scale as our assay for subunit rotation, we conclude that the soluble E. coli enzyme is unsuitable for this test. In contrast, ε does not dissociate from FOF1(as confirmed below in Fig. 3, lane 8) and in fact is required for binding F1 to FO (44Dunn S.D. Futai M. J. Biol. Chem. 1980; 255: 113-118Abstract Full Text PDF PubMed Google Scholar). Hence, tests for ε subunit rotation were conducted with reconstituted hybrid FOF1.Figure 3Reorientation of ε relative to the β subunits in FOF1 during catalytic turnover. The presence of Flag epitope in the β-ε band following various treatments was detected by immunoblotting. Forlanes 1, 2, and 4–7, β-ε cross-linked hybrid F1 having Flag-tagged β exclusively in the two noncross-linked β positions was prepared and reconstituted with F1-depleted membranes as described under “Experimental Procedures.” Membranes were suspended at 4 mg of protein/ml in buffer containing 50 mm Tris acetate, 5 mm MgSO4, 50 mm glucose, 5 μm FCCP, pH 7.5 (TMGF buffer) and subjected to the following treatments. Lane 5, membranes were incubated with 10 mm DTT for 30 s, passed through a centrifuge column equilibrated with TMGF buffer, and collected in a tube containing DTNB (50 μm final), and incubated for 10 min at 22 °C.Lanes 4 and 7, same as for lane 5except that the incubation mixture and centrifuge column buffer also contained 1 mm ATP (lane 4) or 0.5 mm ADP and 0.5 mm NaN3 (lane 7). Lane 1, same as for lane 5 except that DTT was omitted. Lane 2, same as for lane 5except that the column effluent was collected in the absence of DTNB.Lane 6, same as for lane 4 except that membranes were pretreated with DCCD to modify FO as described under “Experimental Procedures.” Lane 3, same as forlane 4 except that FOF1 was reconstituted using F1 that could have βflagat all three β positions (see “Experimental Procedures”).Lane 8, as for lane 4 except that F1-depleted membranes were reconstituted separately with βD380C/γC87S/εS108C-F1 or βflagD380C/γC87S-F1 and then mixed in a 1:1 ratio. Hexokinase was included at 3 units/ml in samples 1, 2, 5, and 7 to prevent any potential ATP hydrolysis. The uncoupler FCCP was present in all samples to prevent formation of a transmembrane proton gradient. An aliquot of each sample containing 6 μg of membrane protein was subjected to SDS-PAGE under nonreducing conditions, and immunoblotting was performed as described under “Experimental Procedures.” The results shown are typical of three replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preliminary studies (not shown) confirmed that DTNB-treated βD380C/γC87S/εS108C-F1can be dissociated into subunits, reassembled, and reconstituted with FO in F1-depleted membranes without breaking the βD380C-εS108C disulfide bond. Membrane-bound, β-ε cross-linked F1 is catalytically inactive. However, treatment with DTT to reduce the disulfide cross-link restores ATP hydrolysis (9.5 μmol·min−1·mg−1) and synthesis (30 nmol·min−1·mg−1) activities. Furthermore, when reconstituted membranes were preincubated with DCCD, the hydrolysis and synthesis of ATP were inhibited by 80 and >98%, respectively. These results demonstrate that cross-linked βD380C/γC87S/εS108C-F1 can rebind to FOto form an FOF1 complex that is functionally coupled following reduction of the disulfide. Using a subunit dissociation/reassociation procedure developed previously (9Duncan 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 (459) Google Scholar), we formed a β-ε cross-linked hybrid F1containing βflagD380C subunits exclusively in the two noncross-linked β positions. The resulting hybrid provides a means of distinguishing the β subunit that is initially oriented to allow cross-linking to ε from the other two β subunits. Hybrid F1 was rebound to F1-depleted membranes, and excess soluble F1 was removed. To test for possible rotary movement of ε, the reconstituted membranes were briefly reduced with DTT, exposed to various ligands, and reoxidized with DTNB. In the absence of rotation, εS108C would be expected to cross-link to the original unlabeled β subunit. However, if ε has rotated, εS108C would be positioned to cross-link to Flag epitope-labeled β in a significant fraction of the FOF1 complexes resulting in the appearance of epitope in the β-ε band. The results are shown in the immunoblot in Fig. 3. When reconstituted membranes were reduced and exposed to conditions for ATP hydrolysis, a significant amount of Flag epitope was detected in the β-ε cross-linked band following reoxidation (Fig. 3, lane 4). In contrast, when reconstituted membranes were reduced and exposed to ligands that bind to catalytic sites but do not allow catalytic turnover, little Flag epitope was found in the β-ε bands (Fig. 3,lanes 5 and 7). The amount of epitope in the β-ε band was also low when membranes were reduced and reoxidized in the presence of MgADP but absence of azide (not shown). An important control (the “nonhybrid” control) is shown in Fig. 3,lane 8. For this experiment, cross-linked βD380C/γC87S/εS108C-F1 and βflagD380C/γC87S-F1 remained separated during subunit dissociation/reassociation and were mixed in a 1:1 ratio only after reconstituting FOF1. The resulting sample was then reduced, exposed to MgATP, and reoxidized. The absence of βflag in the β-ε band (lane 8) excludes the possibility that the ε subunit can exchange between FOF1 complexes. An additional control of this type showed that even when the initial β-ε disulfide was reduced for 5 min prior to the addition of ATP, the exchange of ε between FOF1 complexes was not detectable (data not shown). Taken together, the results provide compelling evidence for rotation of ε relative to the β subunits during catalytic turnover. To determine the maximal level of Flag epitope expected in the β-ε band if εS108C has an equal chance of reacting with any of the three β subunits after catalytic turnover, a noncross-linked control was run (Fig. 3, lane 3). In this case, βD380C/γC87S/εS108C-F1 was dissociated in the presence of a source of βflag without forming an initial cross-link between ε and one of the β subunits. Consequently, during reassociation βflag could assemble in each of the three β subunit positions. When this enzyme was used to reconstitute FOF1, DTNB oxidation resulted in the highest level of βflag observed in the β-ε band (Fig. 3,lane 3). This value was used to calculate the maximal expected βflag in the β-ε band of hybrid F1 if the orientation of ε relative to the β subunits is randomized by catalysis-driven subunit rotation (Fig. 4, 100%). Conditions for ATP hydrolysis yielded 65% of this maximal value (Fig. 4, MgATP). A value less than the calculated maximum is not surprising in view of the expectation that not all FOF1 complexes in reconstituted membranes will be catalytically active during a brief exposure to MgATP. Of notable significance is the fact that very little βflag appeared in the β-ε band in the absence of catalytic turnover (Fig. 4, 5–7%). It is well known that covalent modification of one or more c subunits by DCCD blocks proton translocation through FO and inhibits both ATP synthesis and hydrolysis by FOF1 (37Hermolin J. Fillingame R.H. J. Biol. Chem. 1989; 264: 3896-3903Abstract Full Text PDF PubMed Google Scholar). In addition, we recently reported that DCCD modification of FO prevents the catalysis-dependent rotation of the γ subunit in membrane-bound FOF1 (12Zhou Y. Duncan T.M. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochim. Biophys. Acta. 1996; 1275: 96-100Crossref PubMed Scopus (69) Google Scholar,13Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar). A similar experiment was carried out here to test for functional coupling between ε rotation in F1 and proton conduction through FO. For this purpose, membranes were treated with DCCD under conditions that selectively modify the c subunits of FO. As shown in Fig. 3 (lane 6) and Fig. 4(+DCCD), exposure of DCCD-inhibited membranes to MgATP yielded only 3% of the calculated maximal amount of Flag epitope in the β-ε band. This emphasizes the tight functional coupling of subunit rotation in F1 to proton translocation through FO. The results presented demonstrate catalysis-dependent rotation of the ε subunit in functionally coupled membrane-bound FOF1. In view of earlier evidence for the rotation of γ in F1 (9Duncan 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 (459) Google Scholar, 14Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar, 15Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar) and in FOF1 (12Zhou Y. Duncan T.M. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochim. Biophys. Acta. 1996; 1275: 96-100Crossref PubMed Scopus (69) Google Scholar, 13Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar), we conclude that γ and ε constitute part of the rotor that couples proton transport through FO to the required binding changes in F1. The fact that DCCD modification of subunit c in FO prevents catalysis-dependent rotation of γ (12Zhou Y. Duncan T.M. Bulygin V.V. Hutcheon M.L. Cross R.L. Biochim. Biophys. Acta. 1996; 1275: 96-100Crossref PubMed Scopus (69) Google Scholar, 13Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar) and ε (Fig. 3, lane 6) supports the possibility that subunit rotation in F1 is coupled to subunit rotation in FO. However, it remains to be determined whether the c subunit complex of FO constitutes the remaining portion of the rotor (8Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 9Duncan 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 (459) Google Scholar,14Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar). The intact E. coli FOF1 complex was used in these studies to avoid two potential problems that might have been encountered with soluble F1. The first relates to the well established ability of ε to inhibit catalysis when F1 is separated from FO (43Smith J.B. Sternweis P.C. Biochemistry. 1977; 16: 306-311Crossref PubMed Scopus (135) Google Scholar). This could hinder attempts to detect catalysis-dependent subunit rotation, particularly if ε inhibits by preventing the rotation of γ. A second difficulty could arise from the fact that ε readily dissociates from F1. As noted earlier, when monitoring the orientation of ε relative to the three β subunits as a means of detecting subunit rotation, it is essential to rule out dissociation and rebinding of ε as an alternative cause for its reorientation. The use of FOF1 avoided both of these problems because in the native complex, ε does not inhibit activity (45Sternweis P.C. Smith J.B. Biochemistry. 1980; 19: 526-531Crossref PubMed Scopus (106) Google Scholar) nor does it dissociate from the complex (Fig. 3, lane 8). The t½ for exchange of ε between βD380C/γC87S/εS108C-F1 and βflagD380C/γC87S-F1 molecules was found to be about 1 min (Fig. 2), whereas at the same temperature, thet½ for dissociation of ε from our wild-type F1 is about 5 min. 2Y. M. Milgrom and R. L. Cross, unpublished data. This suggests that these mutations weaken the interaction of ε with F1resulting in an increased dissociation rate. This is not surprising in view of the fact that βD380 is in close contact with γC87 and εS108 as evidenced by the facility with which substituted cysteines can cross-link. The results imply that one or more of the native residues contributes to the stability of the interaction of ε with the rest of F1. We thank Dr. Yuantai Zhou for helpful suggestions and Marc Hutcheon for assisting with enzyme preparations. We also acknowledge the kind gift of anti-ε-subunit antibody by Professor Stanley Dunn." @default.
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• W2095492608 title "Rotation of the ε Subunit during Catalysis by Escherichia coli FOF1-ATP Synthase" @default.
• W2095492608 cites W145875931 @default.
• W2095492608 cites W1524836192 @default.
• W2095492608 cites W1525848520 @default.
• W2095492608 cites W1535089571 @default.
• W2095492608 cites W155146360 @default.
• W2095492608 cites W1556121347 @default.
• W2095492608 cites W1569645377 @default.
• W2095492608 cites W1570668744 @default.
• W2095492608 cites W1585590389 @default.
• W2095492608 cites W1592768266 @default.
• W2095492608 cites W1604233325 @default.
• W2095492608 cites W1661936255 @default.
• W2095492608 cites W1920400055 @default.
• W2095492608 cites W1964976203 @default.
• W2095492608 cites W1974430395 @default.
• W2095492608 cites W1980634926 @default.
• W2095492608 cites W1996653218 @default.
• W2095492608 cites W1998184406 @default.
• W2095492608 cites W2000809937 @default.
• W2095492608 cites W2006855005 @default.
• W2095492608 cites W2007055752 @default.
• W2095492608 cites W2007580517 @default.
• W2095492608 cites W2007858156 @default.
• W2095492608 cites W2019396695 @default.
• W2095492608 cites W2028416032 @default.
• W2095492608 cites W2033099907 @default.
• W2095492608 cites W2047459324 @default.
• W2095492608 cites W2048117183 @default.
• W2095492608 cites W2058415148 @default.
• W2095492608 cites W2067238665 @default.
• W2095492608 cites W2068634955 @default.
• W2095492608 cites W2071950147 @default.
• W2095492608 cites W2074033410 @default.
• W2095492608 cites W2082629421 @default.
• W2095492608 cites W2087553046 @default.
• W2095492608 cites W2100837269 @default.
• W2095492608 cites W2101108802 @default.
• W2095492608 cites W2102131116 @default.
• W2095492608 cites W2117109681 @default.
• W2095492608 cites W2119710448 @default.
• W2095492608 cites W2121884046 @default.
• W2095492608 cites W2161308640 @default.
• W2095492608 cites W2238897211 @default.
• W2095492608 cites W2403693452 @default.
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