Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ϵ subunit remains to be determined. To address this question, 11 mutants of the ϵ subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation. The ϵ subunit of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the ϵ subunit remains to be determined. To address this question, 11 mutants of the ϵ subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the α3β3γ complex of TF1. The ATPase activity of the resulting α3β3γϵ complexes was measured, and the extent of inhibition by the mutant ϵ subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the ϵ subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF1 by fixing the ϵ subunit into the folded conformation. F0F1-ATPase/synthase (F0F1) catalyzes ATP synthesis via coupling of the proton flow driven by the electrochemical gradient of protons or sodium. F0F1 consists of two rotary molecular motors: a water-soluble, ATP-driven F1 motor and a membrane-embedded, H+- or Na+-driven F0 motor. These molecular motors are connected together to couple ATP synthesis/hydrolysis and ion flow (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 2Kinosita Jr., K. Yasuda R. Noji H. Essays Biochem. 2000; 35: 3-18Crossref PubMed Scopus (40) Google Scholar, 3Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (713) Google Scholar, 4Senior A.E. Nadanaciva S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (340) Google Scholar). The F1-ATPase (α3β3δγϵ) hydrolyzes ATP into ADP and inorganic phosphate, and the hydrolysis of one ATP drives the discrete 120° rotation of the γϵ subunits relative to the other subunits (5Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 6Yasuda R. Noji H. Kinosita Jr., K. Yoshida M. Cell. 1998; 93: 1117-1124Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). As the smallest subunit of F1-ATPase, the ϵ subunit acts as an endogenous inhibitor of the ATPase activity in both the bacterial and chloroplast F1-ATPase, where it is believed to play a regulatory role in ATP synthase (7Smith J.B. Sternweis P.C. Heppel L.A. J. Supramol. Struct. 1975; 3: 248-255Crossref PubMed Scopus (48) Google Scholar, 8Laget P.P. Smith J.B. Arch. Biochem. Biophys. 1979; 197: 83-89Crossref PubMed Scopus (70) Google Scholar, 9Ort D.R. Oxborough K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 269-291Crossref Scopus (80) Google Scholar, 10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A recent single molecule study revealed its importance in efficient coupling in rotation and ATP synthesis (11Rondelez Y. Tresset G. Nakashima T. Kato-Yamada Y. Fujita H. Takeuchi S. Noji H. Nature. 2005; 433: 773-777Crossref PubMed Scopus (303) Google Scholar). The ϵ subunit consists of two distinct domains, an N-terminal β sandwich domain and a C-terminal α helical domain. Structural and biochemical studies have shown that the ϵ subunit adopts at least two different conformations in F1 and F0F1 (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (436) Google Scholar, 13Rodgers A.J.W. Wilce M.C.J. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar, 14Richter M.L. McCarty R.E. J. Biol. Chem. 1987; 262: 15037-15040Abstract Full Text PDF PubMed Google Scholar, 15Kato-Yamada Y. Bald D. Koike M. Motohashi K. Hisabori T. Yoshida M. J. Biol. Chem. 1999; 274: 33991-33994Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Tsunoda S.P. Rodgers A.J.W. Aggeler R. Wilce M.C.J. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (163) Google Scholar, 18Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The conformation that results in the inhibition of ATPase exists in an extended state, in which the C-terminal helical domain of the ϵ subunit unfolds to run parallel to the γ subunit. The conformation in which ATPase is active is known as the folded state and is characterized by C-terminal α helices folded into a hairpin configuration. The conformational change of the ϵ subunit is controlled by the concentration of both ATP and ADP as well as the membrane potential (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Tsunoda S.P. Rodgers A.J.W. Aggeler R. Wilce M.C.J. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (163) Google Scholar, 18Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 19Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The isolated ϵ subunit of F1 from the thermophilic Bacillus strain PS3 (TF1) 2The abbreviations used are: TF1F1-ATPase from thermophilic Bacillus PS3BF1F1-ATPase from B. subtilis;EF1, F1-ATPase from E. coliIC3-PE-maleimideN-ethyl-N′-{5-[N″-(2-maleimidoethyl)pyperazinocarbonyl]pentyl} indocarbocyanineWTwild typeTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidAMP-PNP5′-adenylyl-β,γ-imidodiphosphate was recently found to bind ATP (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Binding was so specific that GTP and ADP failed to form a complex with the ϵ subunit, as assayed by gel filtration. These results led to the suggestion that the ϵ subunit is both a regulator and sensor of cellular ATP concentration. ATP binding was also observed with the ϵ subunits of F1-ATPases from Bacillus subtilis (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google Scholar) and Escherichia coli (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). X-ray crystallographic analysis revealed that ATP is bound to the ϵ subunit in the folded state (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). As the ATP binding site consists of residues from the N-terminal domain and the first and second α helices of the C-terminal domain, ATP may bind to the ϵ subunit alone in the folded state. An NMR study revealed that in the absence of ATP, the ϵ subunit adopts a different conformation. ATP may stabilize the folded conformation of the ϵ subunit (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). ATP binding was also observed with the ϵ subunit in the γϵ subcomplex, indicating that ATP binding to the ϵ subunit can occur in ATP synthase, in which it may play a regulatory role (23Iizuka S. Kato S. Yoshida M. Kato-Yamada Y. Biochem. Biophys. Res. Commun. 2006; 349: 1368-1371Crossref PubMed Scopus (11) Google Scholar). F1-ATPase from thermophilic Bacillus PS3 F1-ATPase from B. subtilis;EF1, F1-ATPase from E. coli N-ethyl-N′-{5-[N″-(2-maleimidoethyl)pyperazinocarbonyl]pentyl} indocarbocyanine wild type 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid 5′-adenylyl-β,γ-imidodiphosphate To determine the role of ATP binding to the ϵ subunit in the regulation of the F0F1-ATP synthase, 11 mutants of the ϵ subunit were constructed, in which basic or acidic residues were substituted with alanine residues. Several mutants with impaired ATP binding were further reconstituted with α3β3γ, and the ATPase activities of these α3β3γϵ complexes were measured. The results clearly show that ATP binding to the ϵ subunit affects the regulation of ATPase activity. Materials—Wild-type and αK175A/T176A mutant (noncatalytic site-deficient mutant, ΔNC) 3As noted in Ref. 26Ono S. Hara K.Y. Hirao J. Matsui T. Noji H. Yoshida M. Muneyuki E. Biochim. Biophys. Acta. 2003; 1607: 35-44Crossref PubMed Scopus (27) Google Scholar, αK175A/T176A mutations are enough to suppress the nucleotide binding to the noncatalytic site. α3β3γ complexes of TF1 were prepared as described previously (24Matsui T. Yoshida M. Biochim. Biophys. Acta. 1995; 1231: 139-146Crossref PubMed Scopus (86) Google Scholar, 25Matsui T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). An expression plasmid for a mutant α3β3γ complex was prepared containing KT/AA substitutions in Walker A motifs (27Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar) in both the α and β subunits (αK175A/T176A and βK164A/T165A, noncatalytic and catalytic site-deficient mutants, ΔNC/ΔC); a DNA fragment containing the βK164A/T165A mutations was prepared using the overlap extension PCR method (28Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2102) Google Scholar, 29Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar) applied to the expression plasmid for the wild-type (WT) α3β3γ complex. A 900-bp fragment containing mutations was treated with restriction enzymes AgeI and MunI, and the resulting 624-bp fragment was purified and directly transferred to the respective sites of an expression plasmid for the ΔNC (αK175A/T176A) mutant α3β3γ complex. The mutant α3β3γ complex was purified in the same way as the wild-type complex. The wild-type and mutant a mutant ϵ subunit of TF1 in which and C-terminal can be ϵ subunits were prepared as previously described (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, T. Kato Y. Motohashi K. H. T. J. Biochem. 1997; PubMed Scopus Google Scholar). The expression for the mutant ϵ subunits were prepared by the overlap extension PCR method or PCR method U. Gene (Amst.). PubMed Scopus Google Scholar) applied to the plasmid (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The for was by the PCR method applied to the expression plasmid for the mutants in ATP were by DNA for the and mutants, the ϵ subunits were prepared as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, T. Kato Y. Motohashi K. H. T. J. Biochem. 1997; PubMed Scopus Google Scholar). was that the of a in a of bound ATP in the ϵ subunit (23Iizuka S. Kato S. Yoshida M. Kato-Yamada Y. Biochem. Biophys. Res. Commun. 2006; 349: 1368-1371Crossref PubMed Scopus (11) Google Scholar). The mutant ϵ subunit was prepared as were in and and by Cell was at for at The was applied to a with the same As the mutant ϵ subunit was the a gradient of was and the containing ϵ subunit were was to the to and the was applied to a with A containing a concentration of were with a to gradient of of the mutant, was in The α3β3γϵ complex was prepared by the purified α3β3γ complex and ϵ subunit to a of (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). ϵ subunit was for the ATPase the in ϵ subunit was from the α3β3γϵ complex by with an as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). of the α3β3γϵ complex was by gel a ATP Binding binding by gel-filtration a was as previously in a of and at (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). ATP binding by was as described R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) that was used of was in and with a of was with a The and ϵ subunit was in the and the in were of ATP. The were at with an and were and for and were change was for ATP and ATP concentration. ATPase activity was with an at Y. 1979; PubMed Scopus Google Scholar). The of and the concentration of The was by the of TF1 α3β3γϵ complex to the and in at were in a or were at the of the of ATPase activity was as that of of of the ϵ of a conformational change in the ϵ subunit to the of an was in the same way as described previously (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). and complexes were with or or was and the was for at The was by of and were to the and for at to residues that a The was by the of The were by The was by a with a and an The gel was with and the was with a As by the gel-filtration no in ATP binding was observed the mutant subunit and the wild-type of the α3β3γϵ complex by the ATP concentration was as The α3β3γϵ complex was with in a of and at for was in the which was with the α3β3γ complex. of the was to of ATPase ATP in a to a concentration of ATP at of the ϵ subunits were by the method of Biochem. PubMed Scopus Google Scholar) using as a and by by a of to the results of acid (20Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). of α3β3γ or α3β3γϵ complexes were by using at T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). were of the ATP Binding to the ϵ ϵ subunit mutants that showed ATP substitutions were in for the These are the basic or acidic residues in the C-terminal domain that are different in TF1 and B. subtilis F1-ATPase ATP binding to the ϵ subunit of was the for ATP was (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google Scholar). is that these residues may be for the and shown in in These are the residues that with ATP in the of the TF1 ϵ complex and with in and shown in in two of these and were to play an role in a study K.Y. Kato-Yamada Y. Y. Hisabori T. Yoshida M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). to with ATP bound to ϵ subunit in the (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar) to be in ATP ATP binding to these mutants was assayed by gel-filtration chromatography. The results are shown in mutants and showed the same as the wild and showed no ATP and the other and showed a of further four mutants and were that showed no ATP binding in the gel-filtration these residues are TF1 and which was in the previously ATP binding was found to be for ATP binding (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). As was also found to be for ATP binding (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The mutants selected by the TF1 and a ATP The in ATP binding TF1 and may be of the in other residues. As for E. coli F1-ATPase is ϵ a in of These may be the of the for ATP of the ϵ subunit (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The at in the the two C-terminal α was with was used to a of the ATP binding of the mutants that showed no ATP binding in the gel-filtration the extent of with mutants, of the in the and ATP concentration of the was observed The for ATP at of these mutants from to the that the and residues are for ATP the at was shown to have a analysis of ATP binding to the mutant ϵ subunits of TF1. ATP binding to the mutant ϵ subunits was a was at are described the of each and with and ATP, of the containing ϵ subunit are with The of the at are described of the mutant ϵ subunits for ATP. in the of mutant ϵ subunits of ATP were measured. ATP was to the ϵ subunit in are for ATP, to and ATP concentration. and were and and wild and with by a binding and of the mutants for ATP are as and ATPase with ϵ was no in ATPase activities of α3β3γϵ complexes containing mutant ϵ subunit at ATP concentration in ATP concentration in in mutant ATPase at ATP α3β3γϵ complexes containing showed ATPase activity the wild-type complex. ATP the were and the α3β3γϵ complex containing or also showed a in ATPase activity compared with the wild-type complex. The ATP concentration of the ATPase activity is in The ATP concentration that in activity may directly to the for ATP of the ϵ subunit activity was at a and at with the of inhibition was observed with ATP binding mutant ϵ subunit The of ATP binding were in the the activities were in the The of may be of the of in the of the ϵ subunit K.Y. Kato-Yamada Y. Y. Hisabori T. Yoshida M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google concentration of the ATPase activity. ATPase activities of the α3β3γϵ complexes are ATP concentration. were at the of the and α3β3γϵ complexes with mutant ϵ wild and ATPase activities of the α3β3γϵ complexes are to that of the α3β3γ complex at each ATP concentration. are the same as in ATP Binding to the ϵ a in a nucleotide binding to the β subunit was found to be for the conformational change in the ϵ subunit the conformational change was also observed in the noncatalytic site-deficient mutant in it was noted that also conformational change in the ϵ subunit (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). the that the ϵ subunit can bind ATP the that ATP binding may be a for the conformational change in the ϵ subunit. To this a mutant α3β3γ complex was prepared that KT/AA substitutions in Walker A motifs (27Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar) in both the α and β subunits The KT/AA mutations are known to in the of ATP binding in T. Muneyuki E. Honda M. Allison W.S. Dou C. Yoshida M. J. Biol. Chem. 1997; 272: 8215-8221Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Motohashi K. Yoshida M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The complex showed no ATPase activity of of the wild the of that no ATP was bound to the catalytic sites a in the that the ϵ subunit was a folded state, a and With the ΔNC mutant, of the in the subunit was observed (16Kato-Yamada Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; 275: 35746-35750Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). no of was observed with the mutant The results that ATP bind to the ϵ subunit, to the second or catalytic may be the for from inhibition by the ϵ subunit. the ϵ subunit is in the extended state, the ATP binding site is and ATP the residues as are the ATP binding to the β subunit may conformational in the β subunit, resulting in of the ϵ subunit from the the C-terminal helices are from the ATP can the binding site the ϵ subunit. by ATP of ATP the ATPase activities of the α3β3γϵ complexes containing the wild-type or mutant ϵ subunit were found to be the ATPase activities at of ATP from the ATP concentration was from to to determine the of ATP from the ϵ subunit in the α3β3γϵ complex. The α3β3γϵ complex was with for at As this the α3β3γϵ complex (10Kato Y. Matsui T. Tanaka N. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; 272: 24906-24912Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). of the was into of ATPase to a ATP concentration of to the ATPase with ATP the α3β3γϵ complex showed ATPase activity to the α3β3γ complex was observed with and for which the for ATP was The ATPase activity in the α3β3γϵ complex may have been the wild-type ϵ subunit for ATP, and ATP bound at a concentration of ATP Binding to the ϵ the and ATP binding form of the ϵ subunit was revealed as the folded state (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google Scholar). The binding site consists of both the and C-terminal may be no for ATP to bind to the ϵ subunit in the extended as the C-terminal helices of the ϵ subunit are by the and γ The results also that of the ϵ subunit folded and extended in the absence of ATP binding to the β The role of ATP binding to the ϵ subunit is to stabilize the ϵ subunit of complex in the folded state. A is shown in The TF1 complex is by binding of ATP to the β the ϵ subunit its conformation from the extended to the folded state. in the absence of bound ATP, the folded state is (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google and the C-terminal domain of the ϵ subunit has the to to its extended ATP binding to the ϵ subunit the folded state, the the and active of and results in a of active complex. may be for ATP, the membrane potential is of an F0F1 may as a proton to membrane potential at the of ATP. the absence of ATP binding to the ϵ subunit, F0F1 may the conformation the ATP. be noted that for ATP of the ϵ subunit is at and the cellular ATP concentration may in this R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). the of the for ATP is in the of (21Kato-Yamada Y. FEBS Lett. 2005; 579: 6875-6878Crossref PubMed Scopus (40) Google the regulatory may be the same as that of TF1. As for the of ϵ was to be at (22Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11233-11238Crossref PubMed Scopus (95) Google and this for ATP may be the of the of ϵ compared with that of TF1 the study that the ϵ subunit as a regulator also as an ATP sensor to activity to cellular the role of ATP binding to the ϵ subunit in the ATP synthesis are and Akutsu and Kajiwara for the ATP binding site of the TF1 ϵ subunit.

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