Genetic Fusions of Globular Proteins to the ε Subunit of theEscherichia coli ATP Synthase

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b 562, the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the ε subunit of the enzyme. According to the concept of rotational catalysis, because ε is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. colicells expressing the cytochrome b 562 fusion in place of wild-type ε grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochromeb 562 fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to ε generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of ε in F1F0 is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of γε in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of ε functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound. The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b 562, the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the ε subunit of the enzyme. According to the concept of rotational catalysis, because ε is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. colicells expressing the cytochrome b 562 fusion in place of wild-type ε grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochromeb 562 fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to ε generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of ε in F1F0 is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of γε in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of ε functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound. ATP synthase is the enzyme responsible for the production of ATP during oxidative phosphorylation. It is found throughout all forms of life, from the membranes of bacteria to the mitochondria and chloroplasts of eukaryotes. The enzyme can be easily dissociated into two components, a membrane integral F0 component that forms a proton-permeable pore through the membrane and a peripheral F1 component that houses the three catalytic sites responsible for the synthesis or hydrolysis of ATP. The F1portion of the Escherichia coli enzyme is composed of five subunits in the stoichiometry α3β3γδε, whereas the F0sector is made up of three subunits with stoichiometryab 2 c 10–14. The generally accepted mechanism of ATP synthase function is commonly referred to as “rotational catalysis.” In this mechanism proton translocation through F0 causes the rotation of thec 10–14γε complex with respect to the remainder of the enzyme. During the process of oxidative phosphorylation, movement of this “rotor” is believed to drive the sequential conformational changes in α3β3causing binding of the substrates, ADP and Pi, and the release of the product, ATP, as predicted by Paul Boyer's binding change mechanism. For the rotational mechanism to operate, a second, peripheral stalk composed of the two b subunits and δ must link the α3β3 hexamer of F1with the a subunit of F0, preventing rotation of either with c 10–14γε (for recent reviews see Refs. 1.Walker J.E. Biochim. Biophys. Acta. 2000; 1458: 2-3Google Scholar, 2.Peterson P.L. J. Bioenerg. Biomembr. 2000; 32: 4-5Google Scholar, 3.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1563) Google Scholar, 4.Nakamoto R.K. Ketchum C.J. Al-Shawi M.K. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 205-234Crossref PubMed Scopus (103) Google Scholar).The theory of rotational catalysis received substantial support when Walker and co-workers (5.Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2733) Google Scholar) solved the high resolution structure of the α3β3γ complex from beef heart mitochondria. The structure revealed a hexamer of alternating α and β subunits surrounding a central γ subunit. The three active sites for ATP synthesis/hydrolysis are located in clefts between α and β subunits and are occupied by different nucleotides. Since then, ATP-dependent rotational motion of the γε complex in the isolated F1 sector has been demonstrated by a number of methods (6.Duncan 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 (458) Google Scholar, 7.Hasler K. Engelbrecht S. Junge W. FEBS Lett. 1998; 426: 301-304Crossref PubMed Scopus (58) Google Scholar), culminating in the direct observation that filaments or beads attached to either γ or ε undergo continuing rotation when ATP is added (8.Yasuda R. Noji H. Yoshida M. Kinosita Jr., K. Itoh H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (703) Google Scholar, 9.Kato-Yamada Y. Noji H. Yasuda R. Kinosita K.J. Yoshida M. J. Biol. Chem. 1998; 273: 19375-19377Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10.Noji H. Yasuda R. Yoshida M. Kinosita K.J. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1938) Google Scholar). More recently, efforts have been directed toward demonstrating that this mechanism also functions in the holoenzyme,i.e. that ATP induces rotation of γεc 10–14 in ATP synthase (11.Panke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (178) Google Scholar, 12.Sambongi 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 (412) Google Scholar, 13.Tanabe M. Nishio K. Iko Y. Sambongi Y. Iwamoto-Kihara A. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 15269-15274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14.Tsunoda 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). Rotation of filaments linked to c was observed in these studies, but the interpretation is controversial because of concern that the F0 and F1 sectors were not fully coupled in these in vitro experimental systems. This concern was raised by the lack of sensitivity to F0-specific inhibitors (14.Tsunoda 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) and the tendency of the second stalk to dissociate from the purified yeast enzyme (15.Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1077) Google Scholar). In the absence of the proper interactions of thec oligomer with the a and b subunits, the enzyme would be uncoupled and rotation of the c ring would be expected, regardless of whether or not it occurred during coupled activity. In another approach to rotation in F1F0, specific sites on γ or ε could be linked through disulfide bonds to any of the three β subunits, provided ATP hydrolysis was allowed to proceed (16.Bulygin V.V. Duncan T.M. Cross R.L. J. Biol. Chem. 1998; 273: 31765-31769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17.Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar). Although consistent with rotation, this result could also be satisfied if γ and ε were to swivel in a reciprocating fashion through 240°. Such motion would allow γ and ε to sit in each of the three different positions they have been shown to occupy, while not actually rotating in a repeated circular fashion.Thus, questions remain regarding the rotational mechanism in intact ATP synthase (18.McCarty R.E. Evron Y. Johnson E.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 83-109Crossref PubMed Google Scholar). Furthermore, the mechanism has never been testedin vivo. In this work we describe an approach that allows tests of rotation and function both in vivo and in vitro. During rotational catalysis, the γε rotor must pass theb 2 stator; the size of the rotor is therefore limited by the space available. Any matter added to the rotor must also rotate within this confined space, sweeping out a larger volume of revolution. One would expect that if the added material is too large, it will sterically clash with b 2, and rotation will be blocked. As a result, both ATP synthesis/hydrolysis and proton pumping should be strongly or completely inhibited. We selected the C terminus of ε as the site to make additions, because deletion or mutation of residues in this region has only minimal effects on function in vivo (19.Kuki M. Noumi T. Maeda M. Amemura A. Futai M. J. Biol. Chem. 1988; 263: 17437-17442Abstract Full Text PDF PubMed Google Scholar, 20.Skakoon E.N. Dunn S.D. Arch. Biochem. Biophys. 1993; 302: 279-284Crossref PubMed Scopus (14) Google Scholar). In addition, effects on expression of ATP synthase subunits should be minor becauseuncC, encoding ε, is the last gene of the uncoperon. By fusing a series of proteins of increasing size to this site, we have altered the effective size of the rotor, allowing us to test rotation in vivo and to establish limits for the maximal volume of revolution. In addition, our studies indicate a novel role for the ε subunit in the holoenzyme, to increase the efficiency of ATP synthase by specifically preventing the uncoupled hydrolysis of ATP. ATP synthase is the enzyme responsible for the production of ATP during oxidative phosphorylation. It is found throughout all forms of life, from the membranes of bacteria to the mitochondria and chloroplasts of eukaryotes. The enzyme can be easily dissociated into two components, a membrane integral F0 component that forms a proton-permeable pore through the membrane and a peripheral F1 component that houses the three catalytic sites responsible for the synthesis or hydrolysis of ATP. The F1portion of the Escherichia coli enzyme is composed of five subunits in the stoichiometry α3β3γδε, whereas the F0sector is made up of three subunits with stoichiometryab 2 c 10–14. The generally accepted mechanism of ATP synthase function is commonly referred to as “rotational catalysis.” In this mechanism proton translocation through F0 causes the rotation of thec 10–14γε complex with respect to the remainder of the enzyme. During the process of oxidative phosphorylation, movement of this “rotor” is believed to drive the sequential conformational changes in α3β3causing binding of the substrates, ADP and Pi, and the release of the product, ATP, as predicted by Paul Boyer's binding change mechanism. For the rotational mechanism to operate, a second, peripheral stalk composed of the two b subunits and δ must link the α3β3 hexamer of F1with the a subunit of F0, preventing rotation of either with c 10–14γε (for recent reviews see Refs. 1.Walker J.E. Biochim. Biophys. Acta. 2000; 1458: 2-3Google Scholar, 2.Peterson P.L. J. Bioenerg. Biomembr. 2000; 32: 4-5Google Scholar, 3.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1563) Google Scholar, 4.Nakamoto R.K. Ketchum C.J. Al-Shawi M.K. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 205-234Crossref PubMed Scopus (103) Google Scholar). The theory of rotational catalysis received substantial support when Walker and co-workers (5.Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2733) Google Scholar) solved the high resolution structure of the α3β3γ complex from beef heart mitochondria. The structure revealed a hexamer of alternating α and β subunits surrounding a central γ subunit. The three active sites for ATP synthesis/hydrolysis are located in clefts between α and β subunits and are occupied by different nucleotides. Since then, ATP-dependent rotational motion of the γε complex in the isolated F1 sector has been demonstrated by a number of methods (6.Duncan 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 (458) Google Scholar, 7.Hasler K. Engelbrecht S. Junge W. FEBS Lett. 1998; 426: 301-304Crossref PubMed Scopus (58) Google Scholar), culminating in the direct observation that filaments or beads attached to either γ or ε undergo continuing rotation when ATP is added (8.Yasuda R. Noji H. Yoshida M. Kinosita Jr., K. Itoh H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (703) Google Scholar, 9.Kato-Yamada Y. Noji H. Yasuda R. Kinosita K.J. Yoshida M. J. Biol. Chem. 1998; 273: 19375-19377Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10.Noji H. Yasuda R. Yoshida M. Kinosita K.J. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1938) Google Scholar). More recently, efforts have been directed toward demonstrating that this mechanism also functions in the holoenzyme,i.e. that ATP induces rotation of γεc 10–14 in ATP synthase (11.Panke O. Gumbiowski K. Junge W. Engelbrecht S. FEBS Lett. 2000; 472: 34-38Crossref PubMed Scopus (178) Google Scholar, 12.Sambongi 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 (412) Google Scholar, 13.Tanabe M. Nishio K. Iko Y. Sambongi Y. Iwamoto-Kihara A. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 15269-15274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14.Tsunoda 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). Rotation of filaments linked to c was observed in these studies, but the interpretation is controversial because of concern that the F0 and F1 sectors were not fully coupled in these in vitro experimental systems. This concern was raised by the lack of sensitivity to F0-specific inhibitors (14.Tsunoda 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) and the tendency of the second stalk to dissociate from the purified yeast enzyme (15.Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1077) Google Scholar). In the absence of the proper interactions of thec oligomer with the a and b subunits, the enzyme would be uncoupled and rotation of the c ring would be expected, regardless of whether or not it occurred during coupled activity. In another approach to rotation in F1F0, specific sites on γ or ε could be linked through disulfide bonds to any of the three β subunits, provided ATP hydrolysis was allowed to proceed (16.Bulygin V.V. Duncan T.M. Cross R.L. J. Biol. Chem. 1998; 273: 31765-31769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17.Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar). Although consistent with rotation, this result could also be satisfied if γ and ε were to swivel in a reciprocating fashion through 240°. Such motion would allow γ and ε to sit in each of the three different positions they have been shown to occupy, while not actually rotating in a repeated circular fashion. Thus, questions remain regarding the rotational mechanism in intact ATP synthase (18.McCarty R.E. Evron Y. Johnson E.A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 83-109Crossref PubMed Google Scholar). Furthermore, the mechanism has never been testedin vivo. In this work we describe an approach that allows tests of rotation and function both in vivo and in vitro. During rotational catalysis, the γε rotor must pass theb 2 stator; the size of the rotor is therefore limited by the space available. Any matter added to the rotor must also rotate within this confined space, sweeping out a larger volume of revolution. One would expect that if the added material is too large, it will sterically clash with b 2, and rotation will be blocked. As a result, both ATP synthesis/hydrolysis and proton pumping should be strongly or completely inhibited. We selected the C terminus of ε as the site to make additions, because deletion or mutation of residues in this region has only minimal effects on function in vivo (19.Kuki M. Noumi T. Maeda M. Amemura A. Futai M. J. Biol. Chem. 1988; 263: 17437-17442Abstract Full Text PDF PubMed Google Scholar, 20.Skakoon E.N. Dunn S.D. Arch. Biochem. Biophys. 1993; 302: 279-284Crossref PubMed Scopus (14) Google Scholar). In addition, effects on expression of ATP synthase subunits should be minor becauseuncC, encoding ε, is the last gene of the uncoperon. By fusing a series of proteins of increasing size to this site, we have altered the effective size of the rotor, allowing us to test rotation in vivo and to establish limits for the maximal volume of revolution. In addition, our studies indicate a novel role for the ε subunit in the holoenzyme, to increase the efficiency of ATP synthase by specifically preventing the uncoupled hydrolysis of ATP. We thank Dr. Robert Nakamoto of the University of Virginia for the gift of plasmid pACWU1.2; Dr. Gilbert Privé of the Ontario Cancer Institute for the gift of the plasmid placYCH10; Drs. Gabriele Deckers-Hebestreit and Karlheinz Altendorf of Universität Osnabrück for providing the anti-bmonoclonal antibody; Drs. Robert Aggeler and Rod Capaldi of the University of Oregon for the gift of the anti-α monoclonal antibody; and Dr. Brian Shilton and Paul Del Rizzo of the University of Western Ontario for useful discussions. Some of the experiments described were carried out at the University of Western Ontario Biomolecular Interactions and Conformations Facility, which is supported by a MultiUser Maintenance and Equipment Grant from the Canadian Institutes of Heath Research.

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