Hybrid Rhodospirillum rubrumF0F1 ATP Synthases Containing Spinach Chloroplast F1 β or α and β Subunits Reveal the Essential Role of the α Subunit in ATP Synthesis and Tentoxin Sensitivity
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.906
ISSN1083-351X
AutoresWard C. Tucker, Ziyun Du, Ray Hein, Mark L. Richter, Zippora Gromet‐Elhanan,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoTrace amounts (∼5%) of the chloroplast α subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores ofRhodospirillum rubrum with the chloroplast β subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067–7072). To clarify the role of the α subunit in the rebinding of β, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast α and β subunits. The treated chromatophores were found to have lost, in addition to most of their β subunits, approximately a third of the α subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with theR. rubrum α and chloroplast β subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast α and β subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum α subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast α subunit is required together with the chloroplast β subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the β subunit on sensitivity to tentoxin. Trace amounts (∼5%) of the chloroplast α subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores ofRhodospirillum rubrum with the chloroplast β subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067–7072). To clarify the role of the α subunit in the rebinding of β, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast α and β subunits. The treated chromatophores were found to have lost, in addition to most of their β subunits, approximately a third of the α subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with theR. rubrum α and chloroplast β subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast α and β subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum α subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast α subunit is required together with the chloroplast β subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the β subunit on sensitivity to tentoxin. CF1, EcF1, and RrF1, F1-ATPases from mitochondria, chloroplasts, E. coli, and R. rubrum, respectively bacteriochlorophyll wild-type N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polymerase chain reaction high performance liquid chromatography The photosynthetic F0F1 ATP synthases found in the thylakoids of chloroplasts and in the cytoplasmic membranes of photosynthetic bacteria couple the movement of protons down an electrochemical proton gradient to the synthesis of ATP during photophosphorylation. The general structure of these ATP synthases is highly conserved, consisting of F0, the membrane-spanning proton channel, and F1, the peripheral membrane sector, which contains the catalytic sites for reversible ATP synthesis. The F0 is composed of four different subunits labeled a, b, b′, and c in photosynthetic bacteria (1.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-830Google Scholar) and I–IV in chloroplasts (2.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 807-839Google Scholar, 3.Richter M.L. Mills D.A. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 453-468Google Scholar, 4.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondrian Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar). The chloroplast F0 subunits IV, I, II, and III are analogous to the bacterial a, b, b′, and c, respectively, with a probable stoichiometry of a1b1b′1c9–12. F1 from all sources is composed of five different subunits designated α to ε in order of decreasing molecular weight with a stoichiometry of α3β3γ1δ1ε1.The x-ray crystal structure of bovine heart mitochondrial F1(MF1)1 at 2.8-Å resolution (5.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar) defined the three-dimensional structures of alternating α and β subunits as forming a closed hexamer having a portion of the γ subunit embedded in its central cavity. Among the nucleotide binding sites, which are located one at each of the six α/β interfaces, the three catalytic sites, located predominantly on β subunits, were found to exist in three different conformational states. This asymmetric feature is compatible with the binding change mechanism, which proposed that the catalytic sites interconvert between three different conformational states during ATP synthesis via energy-dependent affinity changes in substrate binding and product release (6.Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 7.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1566) Google Scholar). Several recent studies of isolated (8.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, 9.Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (461) Google Scholar, 10.Noji H. Yasuda R. Yoshida M. Kinosita K.J. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1940) Google Scholar) or membrane-bound (11.Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar) F1 have suggested that this is achieved via rotation of the γ subunit relative to the α3β3 subassembly. The MF1crystal structure identified a number of α-β, α-γ, and β-γ contacts, all or some of which may be responsible for dictating the asymmetric properties of each of the catalytic sites during catalysis. The importance of each of these contacts, and the steps involved in changing them, remain to be determined.Partially dissociated membrane ATP synthase complexes, which can be reassembled by adding isolated α and β subunits, can provide suitable tools for identifying and characterizing the interacting protein domains responsible for the binding change process. One such system, where both ATP synthesis and hydrolysis can be followed, was obtained by LiCl treatment of chromatophores isolated from the photosynthetic bacterium Rhodospirillum rubrum(12.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar). This treatment was found to release the bulk of their β subunits (12.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 13.Philosoph S. Gromet-Elhanan Z. Eur. J. Biochem. 1981; 119: 107-113Crossref PubMed Scopus (14) Google Scholar) resulting in the loss of over 90% of their ATP synthesis and hydrolysis activities. Both activities could be restored upon reconstituting the treated chromatophores with the released subunits. The treated chromatophores could also be reconstituted with native spinach chloroplast CF1 β, although the protein preparation was contaminated with trace amounts of CF1 α (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar). More recently it was shown that the presence of small amounts of the α subunit was a requirement for the reconstitution of a hybrid ATP synthase with the CF1 β subunits (15.Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7067-7072Abstract Full Text PDF PubMed Google Scholar, 16.Avni A. Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7317-7320Abstract Full Text PDF PubMed Google Scholar) or a native enzyme with RrF1 β (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, 1995: 51-54Google Scholar, 18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), suggesting the possibility that the LiCl treatment also removed some of the α subunit from the chromatophores. The identification of a small amount of an α1β1 dimer in addition to the large amount of β in the LiCl extract (19.Andralojc P.J. Harris D.A. FEBS Lett. 1992; 310: 187-192Crossref PubMed Scopus (13) Google Scholar) has confirmed this possibility. Further analysis (51.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 2000; 275: 901-905Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) has shown that the amount of α subunit, but not of β subunit, released by the LiCl treatment is dependent upon the concentration of the chromatophores during the treatment.The hybrid RrF0F1/CF1 containing the CF1 β subunit with trace amounts of the CF1 α subunit was shown to have little, if any, proton-coupled ATP synthesis but 30–40% of the normal MgATPase activity (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar). The hybrid MgATPase activity was fully sensitive to tentoxin, a specific CF1 inhibitor (20.Steele J.A. Uchytil T.F. Durbin R.D. Bhatnagar P. Rich D.H. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2245-2248Crossref PubMed Scopus (131) Google Scholar), whereas the control or restored native RrF0F1 ATP synthesis or hydrolysis activities were completely resistant to tentoxin (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar,21.Gromet-Elhanan Z. Avital S. Biochim. Biophys. Acta. 1992; 1102: 379-385Crossref Scopus (14) Google Scholar). This result suggested that the β subunit might be responsible for conferring tentoxin sensitivity to the F1 enzyme. An aspartic acid residue at position 83 of CF1 β was indeed implicated as being essential for tentoxin sensitivity of the chloroplast CF0F1 ATP synthase (22.Avni A. Anderson J.D. Holland N. Rochaix J.D. Gromet-Elhanan Z. Edelman M. Science. 1992; 257: 1245-1247Crossref PubMed Scopus (59) Google Scholar, 23.Hu D. Fiedler H.R. Golan T. Edelman M. Strotmann H. Shavit N. Leu S. J. Biol. Chem. 1997; 272: 5457-5463Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). However, the observation that the MgATPase activity of membranes isolated from an uncD-deleted Escherichia colistrain complemented with the chloroplast atpB gene was insensitive to tentoxin (24.Chen Z. Spies A. Hein R. Zhou X. Thomas B.C. Richter M.L. Gegenheimer P. J. Biol. Chem. 1995; 270: 17124-17132Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) suggests that additional CF1subunits might also be involved in conferral of this sensitivity.The fact that the LiCl treatment of R. rubrum chromatophores releases some of the α subunits as well as the β subunits has allowed us to examine the interplay between the α and β subunits, which results in coupled ATP synthesis/hydrolysis and in sensitivity to tentoxin. In this study we folded insoluble recombinant CF1β (25.Chen Z. Wu I. Richter M.L. Gegenheimer P. FEBS Lett. 1992; 298: 69-73Crossref PubMed Scopus (13) Google Scholar) into a fully functional monomer using the method developed for folding the recombinant RrF1 α subunit (26.Du Z. Gromet-Elhanan Z. Eur. J. Biochem. 1999; 263: 430-437Crossref PubMed Scopus (13) Google Scholar) and prepared two different hybrid RrF0F1 enzymes containing RrF1 α and CF1 β or CF1 α and CF1 β subunits. The results reveal that (a) the CF1 β subunit can restore a significant amount of proton-coupled ATP synthesis to treated chromatophores but only when all three copies of the RrF1 α subunit are present, and (b) the CF1 α subunit is required, along with the CF1 β subunit, to confer sensitivity to inhibition by tentoxin as well as high (>8-fold) stimulation by sulfite of the restored MgATPase activity.DISCUSSIONPrevious work has shown that the MgATPase activity of LiCl-treated chromatophores could be restored by their reconstitution either with CF1 β containing trace amounts (∼5%) of CF1 α (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar), or with a preparation of CF1αβ containing an equimolar ratio of both subunits but not with a highly purified preparation of CF1 β (15.Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7067-7072Abstract Full Text PDF PubMed Google Scholar). High rates of MgATPase activity could, however, be restored to the treated chromatophores when the purified CF1 β was supplemented with small amounts of the CF1 αβ subunit preparation (16.Avni A. Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7317-7320Abstract Full Text PDF PubMed Google Scholar). We have shown here that LiCl treatment of the R. rubrum chromatophores releases a significant amount of the α subunit along with the β subunits, thus explaining the requirement for small amounts of α in addition to β subunits for restoration of ATP synthesis and hydrolysis in the treated chromatophores. In an accompanying paper (51.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 2000; 275: 901-905Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), it is further shown that the amount of α released increases with decreasing chromatophore concentration during the LiCl treatment, whereas the bulk of the β subunit is released at all tested concentrations. It is, therefore, also possible in this system to vary the ratio of the released α and β subunits.The preferential release of RrF1 β, which has been demonstrated by immunoblotting of the LiCl supernatant (51.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 2000; 275: 901-905Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) as well as of the treated chromatophores (Fig. 4, lanes 2 and3) is not readily explained by examining the crystal structure of MF1 in which the α3β3 hexamer appears to be stabilized by multiple contacts with the γ subunit (5.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). It can be better explained by the recently proposed structural models of the F0F1 ATP synthase, which suggest the presence of two connections between the F0 and F1 parts of the enzyme (7.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1566) Google Scholar, 41.Cross R.L. Duncan T.M. J. Bioenerg. Biomembr. 1996; 28: 403-408Crossref PubMed Scopus (67) Google Scholar, 42.Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (432) Google Scholar, 43.Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar). One connection involves the γ, ε, and c subunits and is proposed to function as the rotating portion of the enzyme. The second connection contains the δ subunit and the two b subunits and is suggested to act as a stator, which binds the F1 α3β3 hexamer to the F0 sector allowing the smaller subunits to rotate with respect to the hexamer. Earlier biochemical evidence has shown that a readily formed disulfide link between the EcF1 δ and one of the α subunits did not inhibit ATPase activity (44.Bragg P.D. Hou C. Biochim. Biophys. Acta. 1986; 851: 385-394Crossref PubMed Scopus (30) Google Scholar, 45.Tozer R.G. Dunn S.D. Eur. J. Biochem. 1986; 161: 513-518Crossref PubMed Scopus (31) Google Scholar). Furthermore, with bifunctional cross-linking reagents, α-δ dimers and α-α-δ trimers, but not β-δ dimers, were identified in EcF1 (46.Bragg P.D. Hou C. Arch. Biochem. Biophys. 1986; 244: 361-372Crossref PubMed Scopus (25) Google Scholar) and TF1 (47.Sone N. Hou C. Bragg P.D. Biochem. Cell Biol. 1986; 64: 229-237Crossref Scopus (2) Google Scholar). The formation of such dimers and trimers as well as b-δ and b-b dimers have recently been observed by cross-linking of introduced cysteine residues in EcF0F1 α, δ, and b subunits (48.Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272 (11656): 16652Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 49.Rodgers A.J. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The specific interactions between these subunits could stabilize a portion of the RrF1 α in the R. rubrum membranes during the LiCl treatment, thus resulting in the retention of at least one and possibly two α subunits per RrF0F1complex while nearly all of the RrF1 β subunits are removed.The treated R. rubrum chromatophores enabled us to form two types of RrF0F1/CF1 hybrids. One contained mostly CF1 β and exclusively RrF1α, while the other also contained CF1 α, which replaced the released portion of RrF1 α. A comparison of the activities of these two hybrids with control RrF0F1 demonstrated that F1 α plays an essential role in a number of catalytic properties. Two specific CF1 α functions were documented; it was absolutely required, together with CF1 β, for obtaining full inhibition of the restored MgATPase activity by tentoxin, as well as for the high stimulation of this restored activity by sulfite (Fig.6 and Table I).Earlier reports demonstrated that the binding of 1 mol of tentoxin/mol of heat-activated CF1 was sufficient for obtaining full inhibition of its ATPase activity (20.Steele J.A. Uchytil T.F. Durbin R.D. Bhatnagar P. Rich D.H. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2245-2248Crossref PubMed Scopus (131) Google Scholar). So, although the absence of any cross-reaction between RrF1 α antibodies and CF1 α (Fig. 4, lane 1) or vice versa (Fig. 6, inset) did not enable the determination of the ratio of the bound CF1 α to the remaining RrF1 α, the full inhibition of the hybrid containing both CF1 α and β by tentoxin sets a lower limit of 1 mol CF1 α bound/mol of reconstituted RrF0F1/CF1. CF1β-Asp83 was shown to be required for the inhibitory action of tentoxin and residues of different charge, such as lysine, or different spacer length such as glutamate, could not replace it (22.Avni A. Anderson J.D. Holland N. Rochaix J.D. Gromet-Elhanan Z. Edelman M. Science. 1992; 257: 1245-1247Crossref PubMed Scopus (59) Google Scholar,23.Hu D. Fiedler H.R. Golan T. Edelman M. Strotmann H. Shavit N. Leu S. J. Biol. Chem. 1997; 272: 5457-5463Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Table II illustrates that this aspartate is indeed essential for obtaining full inhibition by tentoxin in the presence of CF1 α. It could not be replaced even by the noncharged leucine or by the smaller alanine, thus suggesting the importance of the CF1 β-Asp83 charge and spacer length for tentoxin binding and/or inhibition.The mitochondrial equivalent of CF1 β-Asp83, MF1 β-Glu67, is located at an α/β interface in the bovine heart MF1 structure (5.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). If the CF1 β-Asp83 forms part of the tentoxin binding site, then CF1 α residues located near the CF1 β-Asp83 may also contribute to the binding of tentoxin. Comparison of known amino acid sequences of α subunits from both tentoxin-sensitive and -insensitive species revealed that, while all α subunits share considerable homology, a stretch of amino acid residues between 121 and 133 (chloroplast numbering) shows conservation only among the sensitive α subunits of spinach, pea, andC. reinhardtii. This stretch of amino acids is very divergent in the resistant α subunits of RrF1 and EcF1 with several amino acids differing in charge and size from the consensus sequence of sensitive CF1 α subunits. In addition, several of the residues in the equivalent stretch of amino acids in MF1 are located within 10 Å of Glu67on the β subunit. Two such residues in CF1, Ser131 and Pro132, are good candidates for having involvement in tentoxin binding. We are currently mutating these residues into RrF1 α to test this possibility.The MgATPase activity of treated chromatophores reconstituted with CF1 β containing ∼5% CF1 α were stimulated 5–7-fold by sulfite as compared with the 2–3-fold stimulation obtained with chromatophores reconstituted with RrF1 β (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar). These results could not specify whether the CF1 α or β or both were responsible for the extra high sulfite stimulation. The results presented in Table I indicate that, in the hybrid containing only CF1 β, the stimulation is very similar to the one obtained with the control R. rubrumchromatophores, but when CF1 α is also present, an 8-fold stimulation is obtained. This high stimulation was found to raise the level of the MgATPase activity restored in the hybrid enzyme formed with CF1 αβ to the activity restored with RrF1 α and β. The lower initial MgATPase activity of the hybrid containing CF1 α and β subunits may in part reflect the overall latency of the ATPase activity of CF1, which is considered necessary to limit wasteful ATP hydrolysis by CFoF1 in the dark (1.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-830Google Scholar, 2.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 807-839Google Scholar, 3.Richter M.L. Mills D.A. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 453-468Google Scholar).CF1 α exerts a negative effect on the restoration of proton-coupled ATP synthesis, which was obtained only when the treated chromatophores were reconstituted with RrF1 α in the presence of either RrF1 β or CF1 β (TableI). These results suggest that the chromatophores containing bound CF1 α are not properly coupled. In contrast, the hybrid chromatophores containing CF1 β in the presence of RrF1 α showed significant rates of ATP synthesis, indicating that CF1 β can replace at least some of the important energy coupling interactions of RrF1 β. The formation of hybrid RrF0F1/CF1 with chimeric CF1 α/RrF1 α and CF1β/RrF1 β, whose preparation is now under way, should help to identify the F1 α and/or β domains that are essential for the tight protein-protein interactions required for efficient proton-coupled ATP synthesis or hydrolysis.Interestingly, although CaATPase activity is not coupled to proton translocation (50.Gromet-Elhanan Z. Weiss S. Biochemistry. 1989; 28: 3645-3650Crossref Scopus (17) Google Scholar) it was not restored by any hybrid RrF0F1/CF1 (Ref. 14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar and see TableI). Recent results obtained with an RrF1 β-T159S mutant (51.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 2000; 275: 901-905Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) have demonstrated that substitution of serine for threonine in the active site blocks the restoration of Ca-dependent ATPase activity while enabling full restoration of both Mg-dependent ATP synthesis and hydrolysis. These two sets of results likely demonstrate differences in the geometry of the active sites on CF1 β and RrF1 β, as well as on RrF1 β-T159S when occupied by CaATP as compared with MgATP. The photosynthetic F0F1 ATP synthases found in the thylakoids of chloroplasts and in the cytoplasmic membranes of photosynthetic bacteria couple the movement of protons down an electrochemical proton gradient to the synthesis of ATP during photophosphorylation. The general structure of these ATP synthases is highly conserved, consisting of F0, the membrane-spanning proton channel, and F1, the peripheral membrane sector, which contains the catalytic sites for reversible ATP synthesis. The F0 is composed of four different subunits labeled a, b, b′, and c in photosynthetic bacteria (1.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-830Google Scholar) and I–IV in chloroplasts (2.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 807-839Google Scholar, 3.Richter M.L. Mills D.A. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 453-468Google Scholar, 4.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldschmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondrian Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar). The chloroplast F0 subunits IV, I, II, and III are analogous to the bacterial a, b, b′, and c, respectively, with a probable stoichiometry of a1b1b′1c9–12. F1 from all sources is composed of five different subunits designated α to ε in order of decreasing molecular weight with a stoichiometry of α3β3γ1δ1ε1. The x-ray crystal structure of bovine heart mitochondrial F1(MF1)1 at 2.8-Å resolution (5.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar) defined the three-dimensional structures of alternating α and β subunits as forming a closed hexamer having a portion of the γ subunit embedded in its central cavity. Among the nucleotide binding sites, which are located one at each of the six α/β interfaces, the three catalytic sites, located predominantly on β subunits, were found to exist in three different conformational states. This asymmetric feature is compatible with the binding change mechanism, which proposed that the catalytic sites interconvert between three different conformational states during ATP synthesis via energy-dependent affinity changes in substrate binding and product release (6.Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 7.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1566) Google Scholar). Several recent studies of isolated (8.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, 9.Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (461) Google Scholar, 10.Noji H. Yasuda R. Yoshida M. Kinosita K.J. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1940) Google Scholar) or membrane-bound (11.Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar) F1 have suggested that this is achieved via rotation of the γ subunit relative to the α3β3 subassembly. The MF1crystal structure identified a number of α-β, α-γ, and β-γ contacts, all or some of which may be responsible for dictating the asymmetric properties of each of the catalytic sites during catalysis. The importance of each of these contacts, and the steps involved in changing them, remain to be determined. Partially dissociated membrane ATP synthase complexes, which can be reassembled by adding isolated α and β subunits, can provide suitable tools for identifying and characterizing the interacting protein domains responsible for the binding change process. One such system, where both ATP synthesis and hydrolysis can be followed, was obtained by LiCl treatment of chromatophores isolated from the photosynthetic bacterium Rhodospirillum rubrum(12.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar). This treatment was found to release the bulk of their β subunits (12.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 13.Philosoph S. Gromet-Elhanan Z. Eur. J. Biochem. 1981; 119: 107-113Crossref PubMed Scopus (14) Google Scholar) resulting in the loss of over 90% of their ATP synthesis and hydrolysis activities. Both activities could be restored upon reconstituting the treated chromatophores with the released subunits. The treated chromatophores could also be reconstituted with native spinach chloroplast CF1 β, although the protein preparation was contaminated with trace amounts of CF1 α (14.Richter M.L. Gromet-Elhanan Z. McCarty R.E. J. Biol. Chem. 1986; 261: 12109-12113Abstract Full Text PDF PubMed Google Scholar). More recently it was shown that the presence of small amounts of the α subunit was a requirement for the reconstitution of a hybrid ATP synthase with the CF1 β subunits (15.Av
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