Mutations in the β-Subunit Thr159 and Glu184 of the Rhodospirillum rubrumF0F1 ATP Synthase Reveal Differences in Ligands for the Coupled Mg2+- and Decoupled Ca2+-dependent F0F1 Activities
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.901
ISSN1083-351X
AutoresLubov Nathanson, Zippora Gromet‐Elhanan,
Tópico(s)Ion Transport and Channel Regulation
ResumoIn the crystal structure of the mitochondrial F1-ATPase, the β-Thr163 residue was identified as a ligand to Mg2+ and the β-Glu188 as directly involved in catalysis. We replaced the equivalent β-Thr159 of the chromatophore F0F1 ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu184 with Gln or Lys. The mutant β subunits were isolated and tested for their capacity to assemble into a β-less chromatophore F0F1 and restore its lost activities. All of them were found to bind into the β-less enzyme with the same efficiency as the wild type β subunit, but only the β-Thr159 → Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr159and Glu184 are not required for assembly and that Glu184 is indeed essential for all the membrane-bound chromatophore F0F1 activities. A detailed comparison between the wild type and the β-Thr159 → Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F0F1 proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg2+ and Mn2+, where threonine can be replaced by serine, and Ca2+, where only threonine is active, suggests that the β-subunit catalytic site has different conformational states when occupied by Ca2+ as compared with Mg2+. These different states might result in different interactions between the β and γ subunits, which are involved in linking F1 catalysis with F0proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F0F1catalytic activity. In the crystal structure of the mitochondrial F1-ATPase, the β-Thr163 residue was identified as a ligand to Mg2+ and the β-Glu188 as directly involved in catalysis. We replaced the equivalent β-Thr159 of the chromatophore F0F1 ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu184 with Gln or Lys. The mutant β subunits were isolated and tested for their capacity to assemble into a β-less chromatophore F0F1 and restore its lost activities. All of them were found to bind into the β-less enzyme with the same efficiency as the wild type β subunit, but only the β-Thr159 → Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr159and Glu184 are not required for assembly and that Glu184 is indeed essential for all the membrane-bound chromatophore F0F1 activities. A detailed comparison between the wild type and the β-Thr159 → Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F0F1 proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg2+ and Mn2+, where threonine can be replaced by serine, and Ca2+, where only threonine is active, suggests that the β-subunit catalytic site has different conformational states when occupied by Ca2+ as compared with Mg2+. These different states might result in different interactions between the β and γ subunits, which are involved in linking F1 catalysis with F0proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F0F1catalytic activity. RrF1α, CF1β, CF1α, EcF1β, MF1β, and TF1β, α and β subunits of the F1-ATPase of R. rubrum, chloroplasts, E. coli, mitochondria, and thermophilicBacillus PS3, respectively bacteriochlorophyll polymerase chain reaction phenylmethanesulfonyl fluoride wild type polyacrylamide gel electrophoresis The F0F1 ATP synthase-ATPase complexes, found in the inner membranes of mitochondria and chloroplasts and in bacterial plasma membranes, couple ATP synthesis and hydrolysis to electrochemical proton gradients. These complexes are composed of a membrane-intrinsic F0 sector, which mediates proton translocation, and an extrinsic F1 sector, which carries the catalytic sites. All isolated F1 complexes are composed of five subunits with a stoichiometry of α3β3γδε (1.Penefsky H.S. Cross R.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 173-214PubMed Google Scholar, 2.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-839Google Scholar, 3.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 4.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, 5.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldshmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar, 6.Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar). The crystal structure of bovine mitochondrial MF1 (7.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar) presents the large α and β subunits arranged alternately in a closed hexamer around the N- and C-terminal helices of the γ subunit. The three catalytic β subunits show a clear difference in bound nucleotides resulting in different conformational states. The asymmetric structure imposed on this hexamer by the interaction of the γ-subunit with these different β-subunits, supports the binding change mechanism (8.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar), which proposed that ATP synthesis and hydrolysis involve transitions between different but interacting catalytic sites via rotation of the γ subunit relative to an α3β3 subassembly. Several models suggested that proton-translocation through F0 results in a coupled rotation of the F0-c and F1-γ subunits (8.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 9.Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 10.Cross R.L. Duncan T.M. J. Bioenerg. Biomembr. 1996; 28: 403-408Crossref PubMed Scopus (67) Google Scholar, 11.Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (439) Google Scholar). However full elucidation of the detailed mechanism of action of the F0F1 ATP synthase will depend on identification of the specific residues and/or whole domains that participate in proton-coupled ATP synthesis and hydrolysis as well as in the regulation of these reversible activities. Tight regulation of ATP hydrolysis is especially important in photosynthetic organisms where it prevents the depletion of essential cellular ATP pools in the dark (3.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 4.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, 5.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldshmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar). One stringent regulatory pathway operating in plant chloroplasts (12.Hochman Y. Lanir A. Carmeli C. FEBS Lett. 1976; 178: 10-14Google Scholar) as well as in bacterial chromatophores (13.Oren R. Gromet-Elhanan Z. Biochim. Biophys. Acta. 1979; 548: 106-118Crossref PubMed Scopus (13) Google Scholar, 14.Gromet-Elhanan Z. Weiss S. Biochemistry. 1989; 28: 3645-3650Crossref Scopus (17) Google Scholar) is their high sensitivity to inhibition by excess free Mg2+ ions, which results in optimal MgATPase activity at Mg2+/ATP ratios around 0.5 and its drastic decrease at higher ratios. F1-α, β, and γ subunits from photosynthetic sources are therefore very interesting targets for mutational analysis, based on the available MF1 crystal structure, of amino acid residues participating in coupled catalysis and its regulation. The F1-α and β subunits of the photosynthetic bacteriumRhodospirillum rubrum are most suitable for such studies. RrF1β1 was isolated in large amounts from the chromatophore membrane-bound RrF0F1 by a specific LiCl treatment (15.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar) and recently also cloned and expressed in soluble form (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) inEscherichia coli cells lacking the whole uncoperon. The recombinant WT RrF1β was found to be as active as the native β-subunit (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) in a large number of earlier developed in vitro assays, including the binding of nucleotides (19.Gromet-Elhanan Z. Khananshvili D. Biochemistry. 1984; 23: 1022-1028Crossref Scopus (39) Google Scholar) and rebinding to the β-less RrF0F1 resulting in restoration of both ATP synthesis and hydrolysis activities (15.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar, 16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). The rebinding of the highly purified native and WT RrF1β subunits was, however, found to require the presence of small amounts of RrF1α (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), which were released with the bulk of the β subunit from the LiCl-treated chromatophores (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar, 20.Andvalojc P.J. Harris D.A. Biochim. Biophys. Acta. 1993; 1143: 51-61Crossref Scopus (9) Google Scholar, 21.Gromet-Elhanan Z. Sokolov M. Photosynth. Res. 1995; 46: 79-86Crossref PubMed Scopus (6) Google Scholar). This finding provided a direct assay also for the isolated α-subunit. It has been used to follow the refolding of the recombinant RrF1α, expressed only in insoluble inclusion bodies (22.Du Z. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 27-30Google Scholar), into a functional monomer that assembles with the WT RrF1β monomer into active α1β1 dimers (23.Du Z. Gromet-Elhanan Z. Eur. J. Biochem. 1999; 263: 430-437Crossref PubMed Scopus (13) Google Scholar). If the released α/β ratio can be controlled, the α-depleted β-less chromatophores together with the recombinant RrF1α and β monomers could provide suitable systems for studying the effect of mutagenized α as well as β on the in vitro assembly and activity of both the membrane-bound F0F1 and soluble F1 complexes. In this investigation we have defined the conditions for LiCl treatment of R .rubrum chromatophores that release the bulk of their RrF1β together with specific amounts of RrF1α. β-less chromatophores, which lost at least a third of this α subunit were used for preparing hybrid RrF0F1/CF1 complexes containing either only CF1β or CF1β and at least one copy of CF1α (50.Tucker W.C. Du Z. Hein R. Richter M.L. Gromet-Elhanan Z. J. Biol. Chem. 1999; 274: 906-912Google Scholar). β-less chromatophores containing 90% of their α subunit were used here for testing the effect of mutations in RrF1β-Thr159 and Glu184, which are equivalent to MF1β-Thr163 and Glu188 in the catalytic nucleotide binding site (7.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). These fully conserved F1β residues have been mutated only in respiratory F1-ATPases (6.Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar), except for the equivalent CF1β-Thr168 of Chlamydomonas reinhardtii, which has recently been mutated to serine and shown to increase dramatically the MgATPase activities of the soluble CF1 and α3β3γ complexes (24.Hu D. Strotmann H. Shavit N. Leu S. FEBS Lett. 1998; 421: 65-68Crossref PubMed Scopus (12) Google Scholar). Our studies revealed that in the membrane-bound F0F1 the RrF1β-T159S, but not the T159A or T159V, could restore the proton-coupled Mg- and Mn-dependent ATP synthesis and hydrolysis activities to the extent restored by WT RrF1β. Moreover, even the active β-T159S did not restore the proton-decoupled CaATPase activity. These results indicate that the conserved WT β-Thr159 is an absolutely essential ligand for Ca2+, which could not be replaced even by serine. The RrF1β-E184Q and E184K mutants did bind to the β-less chromatophores, but the assembled mutant RrF0F1s were completely inactive. R. rubrum cells were grown as described previously (15.Philosoph S. Binder A. Gromet-Elhanan Z. J. Biol. Chem. 1977; 252: 8747-8752Abstract Full Text PDF PubMed Google Scholar). The E. coli LM3115 strain (25.Jensen P.R. Michelsen O. J. Bacteriol. 1992; 174: 7635-7641Crossref PubMed Google Scholar) lacking the unc operon was used as host for the recombinant plasmids (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) carrying the WT and mutated RrF1β genes. This strain was found to express large amounts of RrF1β as a soluble protein when grown in LB medium, supplemented as described by Nathanson and Gromet-Elhanan (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), at 22 °C to about A 0.65. The RrF1β-Thr159 mutants were obtained by a modification of the PCR-based mutagenesis method of Ho et al. (26.Ho 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). For each mutation the start and end fragments of the cloned WT RrF1β gene (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) were amplified separately in two independent PCR reactions, using the start-forward primer with the earlier introduced EcoRI site (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar), and the reverse primer with a newly introduced BamHI site. Each PCR reaction contained a complementary mutagenic primer (26.Ho 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). The following mutagenic primers for T159V, T159A, and T159S-forward were, respectively: 5′-C-GTC-GGC-AAG-GT A - GTA -CTG-ATC-CAG-G-3′; 5′-C-GTC-GGC-AAG-GC A -GTA -CTG-ATC-CAG-G-3′ and 5′-C-GTC-GGC-AAG-TC A -GTA -CTG-ATC-CAG-G-3′; and for T159V, T159A and T159S-reverse were, respectively: 5′-C-CTG-GAT-C AG - TAC - T AC-CTT-GCC-GAC-G-3; 5′-C-CTG-GAT-CAG - TAC - T GC-CTT-GCC-GAC-G-3′ and 5′-C-CTG-GAT-CAG - TAC - T GA-CTT-GCC-GAC-G-3′. Each primer contained a new, underlined site for ScaI, introduced by two base changes, indicated by bold letters as the bases changed to give the Val, Ala, or Ser codons. Amplification was carried out as described earlier (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar), except that 50 pmol of each set of primers, 4 ng of pBSKS+-WTβ, and 1.25 units ofPwo DNA polymerase were used, in the buffer supplied with the enzyme. The two amplified fragments of each mutated gene were cut by EcoRI and ScaI or ScaI andBamHI, ligated with each other and with the pBSKS+ plasmid between its EcoRI andBamHI sites. The resulting recombinant plasmids were transformed into E. coli HB101 cells (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar), and plasmids fromamp-resistant colonies were screened for the mutations byScaI restriction analysis and by full DNA sequencing. All three mutants had only the above stated nucleotide changes. For mutagenesis of RrF1β-Glu184, the pBSKS+-WTβ plasmid (17.Nathanson L. Gromet-Elhanan Z. Mathis P. Photosynthesis: From Light to Biosphere. III. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 51-54Google Scholar) was transformed into E. coli CJ236 and single-stranded uracil-containing DNA was prepared after infection with helper phage VCS M13 (27.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY1989Google Scholar). Site-directed mutagenesis was performed as described by Kunkel et al. (28.Kunkel T.A. Benebek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (633) Google Scholar), using the following oligonucleotides to introduce the β-E184Q and E184K mutations, respectively: 5′-GCC-CTC-ACG-CGT -GCG-CTG-GCC-GAC-GCC-G and 5′-GCC-CTC-ACG-CGT -GCG-CTT-GCC-GAC-GCC-G. They also contained a new, underlined site for MluI, introduced by a single base change, indicated by a bold letter as the bases changed to give the Glu or Lys codons. The mutagenized DNA was transformed into E. coli HB101 cells, and the mutations were confirmed by MluI restriction analysis and full DNA sequencing. The β-E184K gene had only the stated mutation. However, β-E184Q had one additional change in nucleotide 249, altering the codon from GGC to GGT, both coding for glycine. All the expressed WT and mutant RrF1β subunits were isolated from the cytoplasmic fraction of the E. coli LM3115 cells and purified as described previously (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). LiCl treatment of the chromatophores was carried out as outlined by Gromet-Elhanan and Khananshvili (16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar) with the following modifications. 1 mm protease inhibitor PMSF was present in the final 1.9 m LiCl buffer, and the concentration of the treated chromatophores was varied as stated in the text. The LiCl supernatant was separated from the treated chromatophores, and the dissolved 60% ammonium sulfate precipitate (16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar) was subjected to SDS-PAGE (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar, 30.Flings S.P. Gregerson D.S. Anal. Biochem. 1986; 155: 83-88Crossref PubMed Scopus (784) Google Scholar), transferred to nitrocellulose (31.Gershoni J.M. Methods Biochem. Anal. 1988; 33: 1-58PubMed Google Scholar), and probed with antibodies raised against RrF1α and β subunits. Chromatophores treated with 1.9m LiCl at 1.2 mg of BChl/ml were washed to remove all traces of LiCl (16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar), reconstituted with WT or mutated RrF1β in presence of RrF1α at a ratio of α/β of 0.2, and assayed for restored ATP synthesis and hydrolysis as described previously (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Published methods were used for measurements of protein concentration (32.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar, 33.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18643) Google Scholar) and the BChl content of chromatophores (34.Clayton R.K. Gest H. San Pietro A. Vernon L.P. Bacterial Photosynthesis. Antioch Press, Yellow Springs, OH1963: 495-500Google Scholar). E. coli LM3115 was a gift of Dr. P. R. Jensen (The Netherlands Cancer Institute, Amsterdam). Oligonucleotides were synthesized by Dr. Ora Goldberg (Biological Services, Weizmann Institute of Science, Rehovot, Israel). Restriction enzymes, T4 DNA polymerase, and ligase were from New England Biolabs.Pwo DNA polymerase, dNTP, and pBTacI were purchased from Roche Molecular Biochemicals. Plasmid pBSKS+ and helper phage VCS M13 were from Stratagene. [32P]Piwas obtained from the Nuclear Research Center, Negev, Israel. All other reagents were of the highest purity available. Earlier preparations of β-less chromatophores were obtained by treatment with 2m LiCl and no added protease inhibitors (16.Gromet-Elhanan Z. Khananshvili D. Methods Enzymol. 1986; 126: 528-538Crossref Scopus (24) Google Scholar). Similar treatments of spinach (35.Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7067-7072Abstract Full Text PDF PubMed Google Scholar) as well as lettuce and tobacco (36.Avni A. Avital S. Gromet-Elhanan Z. J. Biol. Chem. 1991; 266: 7317-7320Abstract Full Text PDF PubMed Google Scholar) chloroplasts were also found to release some CF1α together with all the CF1β. Furthermore Western immunoblots probed with antibodies raised against spinach CF1α and β subunits revealed that some of the released CF1α was nicked by proteases and ran in SDS-PAGE together with the β subunit. This proteolysis was fully blocked only in the presence of a mixture of three protease inhibitors (37.Sokolov M. Gromet-Elhanan Z. Biochemistry. 1996; 35: 1242-1248Crossref PubMed Scopus (23) Google Scholar). Since antibodies against CF1α show no cross-reaction with RrF1α (50.Tucker W.C. Du Z. Hein R. Richter M.L. Gromet-Elhanan Z. J. Biol. Chem. 1999; 274: 906-912Google Scholar), we have raised antibodies against RrF1α and found proteolyzed RrF1α in LiCl extracts of earlier treated chromatophores. Addition of 1 mm PMSF blocked completely this proteolysis, thus enabling a clear determination of the relative amounts of the RrF1α and β subunits released from chromatophores treated by 1.9 m LiCl at 0.4, 0.8, or 1.2 mg of BChl/ml (Fig. 1, lanes 2–4). About 0.65, 0.32, and 0.13 of their RrF1α were, respectively, released as compared with practically all their β subunit (Fig. 1 and Fig.2, lane 5). Chromatophores treated with LiCl at 1.2 mg of BChl/ml were used for evaluating the effect of the β-T159 and β-E184 mutants when reconstituted with the various β subunits in presence of RrF1α at a ratio of α/β of 0.2.Figure 2Binding of WT and Thr159 mutant RrF1β to the β-less chromatophores. Reconstitution of the β-less chromatophores obtained by LiCl treatment at 1.2 mg of BChl/ml was carried out as described under "Experimental Procedures." Washed reconstituted chromatophores corresponding to 3 μg of BChl were incubated with 1% SDS at 100 °C for 5 min, applied on SDS-PAGE, and probed with antibodies raised against RrF1α and β subunits as described in Fig. 1. Lanes 1–4, β-less chromatophores reconstituted with WT, T159A, T159V, and T159S RrF1β, respectively; lane 5, unreconstituted β-less chromatophores.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RrF1β-Thr159 is the last residue in the glycine rich p-loop sequence found in α and β subunits of all F1-ATPases and many other nucleotide-binding proteins (38.Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Abstract Full Text PDF PubMed Scopus (1748) Google Scholar). The parallel MF1β Oγ-T163 was identified in the crystal structure as a ligand to Mg2+ (7.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). In the GTP-binding Ras protein, a serine, which replaces the threonine in this sequence, was also found to be a ligand to Mg2+ (39.Tong L. deVos A.M. Milburn M.V. Kim S.H. J. Mol. Biol. 1991; 217: 503-516Crossref PubMed Scopus (216) Google Scholar). We have mutated the RrF1β-Thr159 into serine (β-T159S) as well as alanine (β-T159A) or valine (β-T159V). All of them were found to assemble into the β-less R. rubrumchromatophores as efficiently as WT RrF1β (Fig. 2,lanes 1–4). However, the RrF1β-T159V and T159A mutants were unable to restore any ATP synthesis or hydrolysis activities, whereas the RrF1β-T159S mutant restored ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis (Figs.3 and4).Figure 4Divalent cation requirement of ATP hydrolysis restored in β-less chromatophores reconstituted with WT and mutant RrF1β at position 159. Washed chromatophores reconstituted with WT (■), T159S (▴), T159A (○), and T159V (⋄) were assayed for restored ATP hydrolysis using 4 mm ATP and the indicated concentrations of MgCl2 (A), MnCl2 (B), and CaCl2 (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ATP synthesis restored by the β-T159S was similar or slightly higher than the WT rate and showed a similar MgCl2requirement (Fig. 3). Values of K m of 173 and 238 μm MgCl2 and V max of 352 and 429 μmol of ATP formed/h per mg of BChl were calculated for chromatophores reconstituted with the WT and the T159S mutant, respectively. The maximal rate of this restored ATP synthesis, as that observed in control R. rubrum chromatophores (14.Gromet-Elhanan Z. Weiss S. Biochemistry. 1989; 28: 3645-3650Crossref Scopus (17) Google Scholar), was obtained at MgCl2 concentrations below 10 mmand Mg2+/ADP ratios below 5. At 40 mmMgCl2, the rates restored by the WT and T159S mutant were inhibited by 50% and 30%, respectively (Fig. 3). The lower sensitivity of the mutant to inhibition by increasing MgCl2concentrations could explain its capacity to restore somewhat higher maximal rates of ATP synthesis. A very similar pattern was observed also for restoration of MgATPase activities by the WT and T159S mutant (Fig. 4 A). Mg- and Mn-dependent ATP hydrolysis in control R. rubrumchromatophores (14.Gromet-Elhanan Z. Weiss S. Biochemistry. 1989; 28: 3645-3650Crossref Scopus (17) Google Scholar), as in β-less chromatophores reconstituted with WT RrF1β (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), were reported to be much more sensitive to inhibition by excess free Mg2+ or Mn2+ ions than their respective ATP synthesis activities. This difference in sensitivity provides the basis for the tight regulation of ATP hydrolysis in photosynthetic organisms, which enables them to limit the depletion of essential cellular ATP pools in the dark (2.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-839Google Scholar, 3.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 4.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, 5.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldshmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar). Chromatophores reconstituted with the RrF1β-T159S mutant retained this tight regulation more efficiently in presence of MgCl2 than in presence of MnCl2 (Fig. 4, compare A and B). Control chromatophores show no ATP synthesis in the presence of Ca2+, and their CaATPase, unlike the Mg- and Mn-dependent ATPase activities, is not coupled to proton-translocation. It is instead decoupled, since the presence of CaATP does not inhibit the light-induced, electron transport-triggered proton translocation (14.Gromet-Elhanan Z. Weiss S. Biochemistry. 1989; 28: 3645-3650Crossref Scopus (17) Google Scholar). It was therefore rather surprising that the β-T159S mutant, which restored the proton-coupled ATP synthesis as well as the Mg- and MnATPase activities as efficiently as the WT RrF1β (Figs. 3 and 4, A and B), did not restore at all the proton-decoupled CaATPase activity (Fig.4 C). β-T159S was in this specific assay similar to the completely inactive β-T159V and T159A mutants rather than to the WT RrF1β. These results revealed a clear difference between the ligands for Mg2+ and Mn2+, where threonine could be replaced by serine, and for Ca2+, where only threonine is active. Anions, such as sulfite, were found to stimulate the MgATPase activities of control (40.Webster G.D. Edwards P.A. Jackson J.B. FEBS Lett. 1977; 76: 29-35Crossref PubMed Scopus (34) Google Scholar) as well as β-less chromatophores reconstituted with the WT RrF1β (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The MgATPase activity restored by the RrF1β-T159S mutant was also stimulated by sulfite although somewhat less than that restored by the WT β subunit (Fig. 5). A similar effect was observed also with the MnATPase activity (TableI). The two other β-T159A and β-T159V mutants did not restore any activity even in the presence of 100 mm sulfite (Fig. 5).Table IComparison of ATP synthesis and hydrolysis activities restored in β-less chromatophores reconstituted with WT and RrF1β-T159 and β-E184 mutantsAssayed activitiesActivities restored by the following RrF1β subunitsWTT159SE184K or E184Qμmol/h per mg BchlATP synthesis340390<0.1MgATPase110124<0.1MgATPase + sulfite320205<0.1MnATPase11585<0.1MnATPase + sulfite350220<0.1CaATPase57<1<0.1Conditions for reconstitution of the β-less chromatophores were as described under "Experimental Procedures." Restored ATP synthesis was assayed as described in Fig. 3, with 5 mmMgCl2, and ATP hydrolysis as described in Fig. 4, with 2 mm MgCl2 or MnCl2 and 8 mmCaCl2. When stated, 100 mm sulfite were added. Open table in a new tab Conditions for reconstitution of the β-less chromatophores were as described under "Experimental Procedures." Restored ATP synthesis was assayed as described in Fig. 3, with 5 mmMgCl2, and ATP hydrolysis as described in Fig. 4, with 2 mm MgCl2 or MnCl2 and 8 mmCaCl2. When stated, 100 mm sulfite were added. The x-ray structure of MF1 showed a density for a water molecule, hydrogen-bonded to the equivalent MF1β-Glu188, at a distance of 4.4 Å from the γ-phosphate of the βTP (7.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). This residue could therefore be appropriately positioned to activate the water molecule for an inline nucleophilic attack on the γ-phosphate during ATP hydrolysis. We have mutated the RrF1β-Glu184into glutamine (β-E184Q) and lysine (β-E184K), and both mutants were found to bind to the β-less chromatophores as efficiently as the WT RrF1β (data not shown). However, no restoration of any tested activity was obtained with either mutant when measured under conditions found optimal for WT RrF1β (Table I). There was also no restoration of any ATP synthesis or hydrolysis activity at higher concentrations of divalent cations, which enabled some restoration of activity in the RrF1β-E195Q and E195K mutants (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). These results demonstrate that the RrF1β-Glu184, although not required for assembly, is absolutely essential for all the above tested membrane-bound RrF0F1-activities. RrF1β-Thr159 and Glu184 are fully conserved in all sequenced F1 complexes. The equivalent MF1β-Thr163 was identified in the catalytic nucleotide binding site of the bovine MF1 crystal structure (7.Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar) as a ligand to Mg2+, and the MF1β-Glu188 was suggested to be directly involved in catalysis. Our analysis of the equivalent RrF1β-E184Q and E184K mutants yielded assembled but fully inactive enzymes (Table I). Similar results were reported for the equivalent EcF1β-E181 mutations (41.Senior A.E. Al-Shawi M.K. J. Biol. Chem. 1992; 267: 21471-21478Abstract Full Text PDF PubMed Google Scholar, 42.Park M.-Y. Omote H. Maeta M. Futain M. J. Biochem. 1994; 116: 1139-1145Crossref PubMed Scopus (46) Google Scholar), where even the β-E181D mutant had only 1.4% of the control MgATPase activity. Only the equivalent TF1β-E190D mutant (43.Amano T. Tozawa K. Yoshida M. Murakami H. FEBS Lett. 1994; 348: 93-98Crossref PubMed Scopus (56) Google Scholar) had 7% of the WT MgATPase activity. These results confirm that this residue is essential for catalysis in respiratory as well as photosynthetic F0F1-ATP synthases, but do not really clarify its function (see Ref. 6.Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar). Residues equivalent to RrF1β-Thr159 were mutated to serine in several respiratory systems (44.Mueller D.M. J. Biol. Chem. 1989; 264: 16552-16556Abstract Full Text PDF PubMed Google Scholar, 45.Omote H. Maeda M. Futai M. J. Biol. Chem. 1992; 267: 20571-20576Abstract Full Text PDF PubMed Google Scholar, 46.Jault J.-M. Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison W.S. J. Biol. Chem. 1996; 271: 28818-28824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and recently also in C. reinhardtii (24.Hu D. Strotmann H. Shavit N. Leu S. FEBS Lett. 1998; 421: 65-68Crossref PubMed Scopus (12) Google Scholar), and checked extensively on the MgATPase activity of isolated F1 or α3β3γ complexes. In all tested cases this mutation resulted in a 3–10-fold increase in the MgATPase activity. It also eliminated the stimulation of the MgATPase by oxyanions or alcohols, and reduced the sensitivity of the MgATPase activity to inhibition by azide. The three mutants RrF1β-T159S, T159A, and T159V have, however, been tested for their in vitro assembly into the β-less membrane-bound RrF0F1 as well as for their capacity to restore all the divalent cation-dependent ATP synthesis and hydrolysis activities of the assembled RrF0F1 complex. All three mutants did bind into the β-less chromatophores (Fig. 2), but only the assembled RrF0F1 containing the β-T159S was active. It did restore the WT rates and specific properties of ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, including the tight regulation of these ATPase activities, and their effective stimulation by sulfite. However, even this active mutant could not restore any CaATPase activity. Two unexpected results emerged from the present study with the RrF1β-T159S. One is the large difference between the membrane-bound activities and properties obtained here with the mutant, which were very similar to those obtained with WT RrF1β, and the much higher activities and different properties than the WT ones that were earlier observed in soluble F1 or α3β3γ complexes containing the equivalent threonine to serine mutants of CF1 (24.Hu D. Strotmann H. Shavit N. Leu S. FEBS Lett. 1998; 421: 65-68Crossref PubMed Scopus (12) Google Scholar) MF1(44.Mueller D.M. J. Biol. Chem. 1989; 264: 16552-16556Abstract Full Text PDF PubMed Google Scholar), and TF1 (46.Jault J.-M. Dou C. Grodsky N.B. Matsui T. Yoshida M. Allison W.S. J. Biol. Chem. 1996; 271: 28818-28824Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The second unexpected result is the inability of the RrF1β-T159S mutant to restore the proton-decoupled CaATPase, while effectively restoring the proton-coupled Mg- and Mn- ATPases. The capacity to restore CaATPase has not been tested with any other equivalent mutant, but an opposite effect of restoration of Ca- but not MgATPase activity was earlier observed with the RrF1β-E195G mutant (18.Nathanson L. Gromet-Elhanan Z. J. Biol. Chem. 1998; 273: 10933-10938Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). These results indicate that the ligands for Ca2+ and Mg2+ must be different, since only in the case of Ca2+ the threonine cannot be replaced by serine (Fig. 4 C). This difference in ligands might lead to a different conformational state of the catalytic site occupied by Ca2+ as compared with Mg2+ or Mn2+. It can explain the inability of the Ca2+-occupied catalytic sites to carry out ATP synthesis and proton-coupled ATP hydrolysis, by Ca-induced changes in the interactions between the β and γ subunits that may be involved in linking catalysis to proton translocation. Omote et al. (47.Omote H. Sambonnatsu N. Saito K. Sambong Y. Iwamoto-Kihara A. Yanagida T. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7780-7784Crossref PubMed Scopus (125) Google Scholar) have recently reported that they observed similar rotation and torque generation in engineered WT EcF1-α3β3γ and an uncoupled mutant EcF1-α3β3γM23K. This mutant is unable to translocate protons through F0 but has about 60% of the WT EcF1-MgATPase activity. These properties are very similar to those recorded with the proton-decoupled membrane-bound and the soluble RrF1-CaATPase activity. It would therefore be most interesting to test whether rotation can occur also during CaATPase as well as MgATPase activity. Although CaATPase activity has been tested very thoroughly in various photosynthetic F1-ATPases (2.Gromet-Elhanan Z. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 807-839Google Scholar, 3.McCarty R.E. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reaction. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 439-451Google Scholar, 4.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, 5.Strotmann H. Shavit N. Leu S. Rochaix J.D. Goldshmidt-Clermont M. Merchant S. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 477-500Google Scholar), however, it has not been studied in detail in respiratory bacterial F1-ATPases, whereas a direct full rotation of γ has not been demonstrated as yet with any engineered photosynthetic F1-α3β3γ. Incubation of the recombinant WT RrF1α and β monomers (23.Du Z. Gromet-Elhanan Z. Eur. J. Biochem. 1999; 263: 430-437Crossref PubMed Scopus (13) Google Scholar) with a recombinant spinach CF1γ subunit (48.Miki J. Maeda M. Mukohata Y. Futai M. FEBS Lett. 1998; 232: 221-226Crossref Scopus (120) Google Scholar, 49.Sokolov M. Lu L. Tucker W. Gegenheimer P.A. Richter M.L. J. Biol. Chem. 1999; 274: 13824-13829Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) has recently been found to result in assembly of a very active hybrid RrF1α3β3/γc, which has both Ca- and MgATPase activities. 2Z. Du, W. C. Tucker, M. L. Richter, and Z. Gromet-Elhanan, manuscript in preparation.This hybrid can be engineered to be used in direct rotation assays, and could thus enable the examination of rotation in presence of CaATP as well as MgATP. We thank Sara Weiss for help in preparing the various treated chromatophores and assaying the ratios of the released α and β subunits.
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