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Contrasted Effects of Inhibitors of Cytochromeb6 f Complex on State Transitions inChlamydomonas reinhardtii

2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês

10.1074/jbc.m010092200

1083-351X

Giovanni Finazzi, Francesca Zito, Romina Paola Barbagallo, Françis-André Wollman,

Algal biology and biofuel production

We have investigated the relationship between the occupancy of the Qo site in the cytochromeb6 f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochromeb6 f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b6 fthat relates the activation of the LHCII kinase to the occupancy of the Qo site and the movement of the Rieske protein. We have investigated the relationship between the occupancy of the Qo site in the cytochromeb6 f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochromeb6 f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b6 fthat relates the activation of the LHCII kinase to the occupancy of the Qo site and the movement of the Rieske protein. photosystem plastoquinone 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea Protein phosphorylation is a general mechanism for signal transduction that is present both in eucaryotes and procaryotes. It is usually triggered by the binding of an external signal molecule to a membrane located receptor, as in the case, for example, of the hormone-induced signal transduction pathway (1Gillman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). In other instances, however, membrane-bound receptors are not involved in the reception of external signals. This is the case of the short time chromatic adaptation phenomena, known as state transitions (2Bennett J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 281-311Crossref Scopus (231) Google Scholar, 3Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (723) Google Scholar), that occur in plants and in algae. In these organisms, changes in the quality of the absorbed light energy induce the phosphorylation and reversible migration of a fraction of the light harvesting proteins (LHCII) between the grana and the stroma domains of the thylakoids (4Bonaventura C. Myers J. Biochim. Biophys. Acta. 1969; 189: 366-383Crossref PubMed Scopus (504) Google Scholar). Following an illumination with light absorbed preferentially by photosystem II (PSII),1 LHCII is phosphorylated and becomes part of PSI antenna (State 1 to State 2 transition) (5Delosme R. Béal D. Joliot P. Biochim. Biophys. Acta. 1994; 1185: 56-64Crossref Scopus (47) Google Scholar, 6Delosme R. Olive J. Wollman F.-A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (160) Google Scholar). The illumination with PSI-absorbed light has the opposite effect: a dephosphorylation triggers the re-association of LHCII to PSII (State 2 to State 1 transition (5Delosme R. Béal D. Joliot P. Biochim. Biophys. Acta. 1994; 1185: 56-64Crossref Scopus (47) Google Scholar, 6Delosme R. Olive J. Wollman F.-A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (160) Google Scholar)). In vivo studies with the unicellular green alga Chlamydomonas reinhardtii have also demonstrated that state transitions are controlled by the intracellular demand for ATP: dark-adapted cells are locked in State 2 when the intracellular content in ATP is low, whereas they shift to a State 1 configuration when the ATP pool is restored (7Bulté L. Gans P. Rebéillé F. Wollman F.-A. Biochim. Biophys. Acta. 1990; 1020: 72-80Crossref Scopus (156) Google Scholar).The changes in the phosphorylation state of antenna proteins result from the combined actions of an LHCII kinase, the activation of which is redox-dependent (8Allen J.F. Bennett J. Steinback K.E. Arntzen C.J. Nature. 1981; 291: 25-29Crossref Scopus (513) Google Scholar), and a phosphatase that is considered permanently active (9Elich T.D. Edelmen M. Matoo A.K. FEBS Lett. 1997; 4111: 236-238Crossref Scopus (19) Google Scholar), although recent data have suggested the possibility of a regulation via its interaction with an immunophilin-like protein (10Fulgosi H. Vener A.V. Altchmied L. Hermann R.G. Andersson B. EMBO J. 1998; 17: 1577-1587Crossref PubMed Scopus (110) Google Scholar). The mechanism for kinase activation involves the reduction of the plastoquinone pool (3Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (723) Google Scholar, 11Horton P. Black M.T. Biochim. Biophys. Acta. 1981; 635: 53-62Crossref PubMed Scopus (125) Google Scholar) and requires the presence of cytochrome b6 fcomplexes (12Wollman F.-A. Lemaire C. Biochim. Biophys. Acta. 1988; 85: 85-94Crossref Scopus (98) Google Scholar, 13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar). The nature of the kinase is still obscure, even though its presence has been reported in partially purified preparations of cytochrome b6 fcomplexes (14Gal A. Hauska G. Herrmann R. Ohad I. J. Biol. Chem. 1990; 265: 19742-19749Abstract Full Text PDF PubMed Google Scholar). Although the molecular mechanism through which the redox state of the plastoquinone (PQ) pool is transduced to the kinase is not known, the implication of the quinol binding site, Qo, of the cytochrome b6 fcomplex has been demonstrated both in vivo with C. reinhardtii (13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar) and in vitro with thylakoid preparations from spinach (15Vener A.V. van Kan P.J. Gal A. Andersson B. Ohad I. J. Biol. Chem. 1995; 270: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar). In the latter case, Vener and colleagues (15Vener A.V. van Kan P.J. Gal A. Andersson B. Ohad I. J. Biol. Chem. 1995; 270: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar), have reported that the activation of the kinasein vitro could be obtained by a reversible acidification of the thylakoids that induces the reduction of ∼20% of the PQ pool. The activation was maintained even after reoxidation of the PQ pool, provided that a Qo-bound plastoquinol was retained per cytochrome b6 f complex (16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar).The same authors have explained the activation in terms of conformational changes of the Rieske subunit, whose flexibility has been recently demonstrated in cytochrome bc1(17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 18Zhang Z. Huang L. Shulmeister V. Chi Y. Kim K. Hung L. Crofts A. Berry E. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar) and b6 f (19Breyton C. J. Biol. Chem. 2000; 275: 13195-13201Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) complexes. The Rieske protein was shown to adopt at least two different positions: one close to the membrane surface, next to heme b1(the so called proximal position, Ref. 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar), another extending more in the lumen, next to heme c1 (respiratory,f) (the distal one, Ref. 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar). The existence of a third position, intermediate between the two, has also been suggested by Iwata and co-workers (17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar). According to the model proposed by Veneret al. (21Vener A.V. Ohad I. Andersson B. Curr. Opin. Plant Biol. 1998; 1: 217-223Crossref PubMed Scopus (122) Google Scholar), the Rieske subunit would be kinase-activating in its distal position, and inhibiting in the proximal one, due to some interaction with a putative transmembrane segment of the kinase. We have recently questioned this hypothesis and suggested that activation of the kinase was produced when the Rieske was in its proximal position (13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar).To further test the relationship between the movements of the Rieske protein and the activation of the LHCII kinase, we have studied the effects on state transitions of two Qo site inhibitors of electron transfer in the cytochromeb6 f complex. We have used stigmatellin, which blocks electron transfer in both thebc1 and b6 fcytochrome complex (22Frank K. Trebst A. Photochem. Photobiol. 1995; 61: 2-9Crossref PubMed Scopus (28) Google Scholar) by fixing the iron sulfur protein in its proximal conformation (17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 18Zhang Z. Huang L. Shulmeister V. Chi Y. Kim K. Hung L. Crofts A. Berry E. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 19Breyton C. J. Biol. Chem. 2000; 275: 13195-13201Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar). We have also used DBMIB, which inhibits cytochrome b6 f but notbc1 complexes (22Frank K. Trebst A. Photochem. Photobiol. 1995; 61: 2-9Crossref PubMed Scopus (28) Google Scholar), and develops contrasted interactions with the Rieske protein depending on its redox state (23Schoepp B. Brugna M. Riedel A. Nitschke W. Kramer D.M. FEBS Lett. 1999; 450: 245-250Crossref PubMed Scopus (52) Google Scholar). Remarkable differences were observed between the effects of the two inhibitors, indicative of the existence of a rather complex relationship between the occupancy of the Qo site and the activation of the LHCII kinase. We present here a structural hypothesis that could account for these observations. Protein phosphorylation is a general mechanism for signal transduction that is present both in eucaryotes and procaryotes. It is usually triggered by the binding of an external signal molecule to a membrane located receptor, as in the case, for example, of the hormone-induced signal transduction pathway (1Gillman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). In other instances, however, membrane-bound receptors are not involved in the reception of external signals. This is the case of the short time chromatic adaptation phenomena, known as state transitions (2Bennett J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 281-311Crossref Scopus (231) Google Scholar, 3Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (723) Google Scholar), that occur in plants and in algae. In these organisms, changes in the quality of the absorbed light energy induce the phosphorylation and reversible migration of a fraction of the light harvesting proteins (LHCII) between the grana and the stroma domains of the thylakoids (4Bonaventura C. Myers J. Biochim. Biophys. Acta. 1969; 189: 366-383Crossref PubMed Scopus (504) Google Scholar). Following an illumination with light absorbed preferentially by photosystem II (PSII),1 LHCII is phosphorylated and becomes part of PSI antenna (State 1 to State 2 transition) (5Delosme R. Béal D. Joliot P. Biochim. Biophys. Acta. 1994; 1185: 56-64Crossref Scopus (47) Google Scholar, 6Delosme R. Olive J. Wollman F.-A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (160) Google Scholar). The illumination with PSI-absorbed light has the opposite effect: a dephosphorylation triggers the re-association of LHCII to PSII (State 2 to State 1 transition (5Delosme R. Béal D. Joliot P. Biochim. Biophys. Acta. 1994; 1185: 56-64Crossref Scopus (47) Google Scholar, 6Delosme R. Olive J. Wollman F.-A. Biochim. Biophys. Acta. 1996; 1273: 150-158Crossref Scopus (160) Google Scholar)). In vivo studies with the unicellular green alga Chlamydomonas reinhardtii have also demonstrated that state transitions are controlled by the intracellular demand for ATP: dark-adapted cells are locked in State 2 when the intracellular content in ATP is low, whereas they shift to a State 1 configuration when the ATP pool is restored (7Bulté L. Gans P. Rebéillé F. Wollman F.-A. Biochim. Biophys. Acta. 1990; 1020: 72-80Crossref Scopus (156) Google Scholar). The changes in the phosphorylation state of antenna proteins result from the combined actions of an LHCII kinase, the activation of which is redox-dependent (8Allen J.F. Bennett J. Steinback K.E. Arntzen C.J. Nature. 1981; 291: 25-29Crossref Scopus (513) Google Scholar), and a phosphatase that is considered permanently active (9Elich T.D. Edelmen M. Matoo A.K. FEBS Lett. 1997; 4111: 236-238Crossref Scopus (19) Google Scholar), although recent data have suggested the possibility of a regulation via its interaction with an immunophilin-like protein (10Fulgosi H. Vener A.V. Altchmied L. Hermann R.G. Andersson B. EMBO J. 1998; 17: 1577-1587Crossref PubMed Scopus (110) Google Scholar). The mechanism for kinase activation involves the reduction of the plastoquinone pool (3Allen J.F. Biochim. Biophys. Acta. 1992; 1098: 275-335Crossref PubMed Scopus (723) Google Scholar, 11Horton P. Black M.T. Biochim. Biophys. Acta. 1981; 635: 53-62Crossref PubMed Scopus (125) Google Scholar) and requires the presence of cytochrome b6 fcomplexes (12Wollman F.-A. Lemaire C. Biochim. Biophys. Acta. 1988; 85: 85-94Crossref Scopus (98) Google Scholar, 13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar). The nature of the kinase is still obscure, even though its presence has been reported in partially purified preparations of cytochrome b6 fcomplexes (14Gal A. Hauska G. Herrmann R. Ohad I. J. Biol. Chem. 1990; 265: 19742-19749Abstract Full Text PDF PubMed Google Scholar). Although the molecular mechanism through which the redox state of the plastoquinone (PQ) pool is transduced to the kinase is not known, the implication of the quinol binding site, Qo, of the cytochrome b6 fcomplex has been demonstrated both in vivo with C. reinhardtii (13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar) and in vitro with thylakoid preparations from spinach (15Vener A.V. van Kan P.J. Gal A. Andersson B. Ohad I. J. Biol. Chem. 1995; 270: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar). In the latter case, Vener and colleagues (15Vener A.V. van Kan P.J. Gal A. Andersson B. Ohad I. J. Biol. Chem. 1995; 270: 25225-25232Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar), have reported that the activation of the kinasein vitro could be obtained by a reversible acidification of the thylakoids that induces the reduction of ∼20% of the PQ pool. The activation was maintained even after reoxidation of the PQ pool, provided that a Qo-bound plastoquinol was retained per cytochrome b6 f complex (16Vener A.V. van Kan P.J. Rich P.R. Ohad I. Andersson B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1585-1590Crossref PubMed Scopus (247) Google Scholar). The same authors have explained the activation in terms of conformational changes of the Rieske subunit, whose flexibility has been recently demonstrated in cytochrome bc1(17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 18Zhang Z. Huang L. Shulmeister V. Chi Y. Kim K. Hung L. Crofts A. Berry E. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar) and b6 f (19Breyton C. J. Biol. Chem. 2000; 275: 13195-13201Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) complexes. The Rieske protein was shown to adopt at least two different positions: one close to the membrane surface, next to heme b1(the so called proximal position, Ref. 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar), another extending more in the lumen, next to heme c1 (respiratory,f) (the distal one, Ref. 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar). The existence of a third position, intermediate between the two, has also been suggested by Iwata and co-workers (17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar). According to the model proposed by Veneret al. (21Vener A.V. Ohad I. Andersson B. Curr. Opin. Plant Biol. 1998; 1: 217-223Crossref PubMed Scopus (122) Google Scholar), the Rieske subunit would be kinase-activating in its distal position, and inhibiting in the proximal one, due to some interaction with a putative transmembrane segment of the kinase. We have recently questioned this hypothesis and suggested that activation of the kinase was produced when the Rieske was in its proximal position (13Zito F. Finazzi G. Delosme R. Nitschke W. Picot D. Wollman F.-A. EMBO J. 1999; 18: 2961-2969Crossref PubMed Scopus (222) Google Scholar). To further test the relationship between the movements of the Rieske protein and the activation of the LHCII kinase, we have studied the effects on state transitions of two Qo site inhibitors of electron transfer in the cytochromeb6 f complex. We have used stigmatellin, which blocks electron transfer in both thebc1 and b6 fcytochrome complex (22Frank K. Trebst A. Photochem. Photobiol. 1995; 61: 2-9Crossref PubMed Scopus (28) Google Scholar) by fixing the iron sulfur protein in its proximal conformation (17Iwata S. Lee J. Okada K. Lee J. Iwata M. Rasmussen B. Link T. Ramaswamy S. Jap B. Science. 1998; 281: 64-71Crossref PubMed Scopus (1054) Google Scholar, 18Zhang Z. Huang L. Shulmeister V. Chi Y. Kim K. Hung L. Crofts A. Berry E. Kim S. Nature. 1998; 392: 677-684Crossref PubMed Scopus (927) Google Scholar, 19Breyton C. J. Biol. Chem. 2000; 275: 13195-13201Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Crofts A.R. Berry E.A. Curr. Opin. Struct. Biol. 1998; 8: 501-509Crossref PubMed Scopus (96) Google Scholar). We have also used DBMIB, which inhibits cytochrome b6 f but notbc1 complexes (22Frank K. Trebst A. Photochem. Photobiol. 1995; 61: 2-9Crossref PubMed Scopus (28) Google Scholar), and develops contrasted interactions with the Rieske protein depending on its redox state (23Schoepp B. Brugna M. Riedel A. Nitschke W. Kramer D.M. FEBS Lett. 1999; 450: 245-250Crossref PubMed Scopus (52) Google Scholar). Remarkable differences were observed between the effects of the two inhibitors, indicative of the existence of a rather complex relationship between the occupancy of the Qo site and the activation of the LHCII kinase. We present here a structural hypothesis that could account for these observations. We thank Giorgio Forti (Milan) and Fabrice Rappaport (Paris) for stimulating discussions and critical reading of the manuscript.