Diatom Fucoxanthin Chlorophyll a/c-binding Protein (FCP) and Land Plant Light-harvesting Proteins Use a Similar Pathway for Thylakoid Membrane Insertion

The light-harvesting proteins in plastids of different lineages including algae and land plants represent a superfamily of chlorophyll-binding proteins that seem to be phylogenetically related, although some of the light-harvesting complex (LHC) proteins bind different carotenoids. LHCs can be divided into chlorophyll a/b-binding proteins found in green algae, euglenoids, and higher plants and into chlorophyll a/c-binding proteins of various algal taxa. LHC proteins from diatoms are named fucoxanthin-chlorophyll a/c-binding proteins (FCP). In contrast to chlorophyll a/b-binding proteins, there is no information so far about the way FCPs integrate into thylakoid membranes. The diatom FCP preproteins have a bipartite presequence that is necessary to enable transport into the four membrane-bound diatom plastids, but similar to chlorophyll a/b-binding proteins there is apparently no presequence present for targeting to the thylakoid membrane. By establishing an in vitro import assay for diatom thylakoids, we demonstrated that thylakoid integration of diatom FCP depends on the presence of stromal factors and GTP. This indicates that a pathway involving signal recognition particles (SRP) is involved in membrane integration just as shown for LHCs in higher plants. We also demonstrate integration of diatom FCP into thylakoids of higher plants and vice versa SRP-dependent targeting of LHCs from pea and Arabidopsis into diatom thylakoids. The similar SRP-dependent modes of thylakoid integration of land plant LHCs and FCPs support recent analyses indicating a common origin of chlorophyll a/b- and a/c-binding proteins. The light-harvesting proteins in plastids of different lineages including algae and land plants represent a superfamily of chlorophyll-binding proteins that seem to be phylogenetically related, although some of the light-harvesting complex (LHC) proteins bind different carotenoids. LHCs can be divided into chlorophyll a/b-binding proteins found in green algae, euglenoids, and higher plants and into chlorophyll a/c-binding proteins of various algal taxa. LHC proteins from diatoms are named fucoxanthin-chlorophyll a/c-binding proteins (FCP). In contrast to chlorophyll a/b-binding proteins, there is no information so far about the way FCPs integrate into thylakoid membranes. The diatom FCP preproteins have a bipartite presequence that is necessary to enable transport into the four membrane-bound diatom plastids, but similar to chlorophyll a/b-binding proteins there is apparently no presequence present for targeting to the thylakoid membrane. By establishing an in vitro import assay for diatom thylakoids, we demonstrated that thylakoid integration of diatom FCP depends on the presence of stromal factors and GTP. This indicates that a pathway involving signal recognition particles (SRP) is involved in membrane integration just as shown for LHCs in higher plants. We also demonstrate integration of diatom FCP into thylakoids of higher plants and vice versa SRP-dependent targeting of LHCs from pea and Arabidopsis into diatom thylakoids. The similar SRP-dependent modes of thylakoid integration of land plant LHCs and FCPs support recent analyses indicating a common origin of chlorophyll a/b- and a/c-binding proteins. light-harvesting complex chlorophyll a/b-binding protein early light-inducible protein endoplasmic reticulum fucoxanthin chlorophyll a/c-binding protein, Fcp, gene encoding FCP peridinin chlorophyll a-binding protein denaturing polyacrylamide gel electrophoresis signal recognition particle dithiothreitol Photosynthetic organisms possess protein complexes that harvest light energy and transfer it to the reaction centers. The light-harvesting complexes (LHC)1 from eukaryotic algae and land plants probably evolved from a common ancestor that is not present in modern cyanobacteria (1Durnford D.G. Deane J.A. Tan S. McFadden G.I. Gantt E. Green B.R. J. Mol. Evol. 1999; 48: 59-68Crossref PubMed Scopus (199) Google Scholar, 2Grossman A.R. Bhaya D. Apt K.E. Kehoe D.M. Annu. Rev. Genet. 1995; 29: 231-288Crossref PubMed Scopus (236) Google Scholar). They form a superfamily of thylakoid membrane-intrinsic chlorophyll-carotenoid proteins. The LHCs include the chlorophyll a/b-binding proteins from green algae and land plants as well as the chlorophyll a/c-binding proteins from chromophytic algae and dinoflagellates that may bind different carotenoids like fucoxanthin (FCPs in diatoms, phaeophytes, and others) and peridinin (intrinsic PCPs, many dinoflagellates) (2Grossman A.R. Bhaya D. Apt K.E. Kehoe D.M. Annu. Rev. Genet. 1995; 29: 231-288Crossref PubMed Scopus (236) Google Scholar). Red algae only have chlorophyll a and use phycobilisomes as the major photosystem II antenna. The chlorophyll a-binding complexes isolated from the thylakoids of these organisms are also related to the LHCs of other eukaryotes (3Wolfe G.R. Cunningham Jr., F.X. Durnford D. Green B.R. Nature. 1994; 367: 566-568Crossref Scopus (136) Google Scholar, 4Tan S. Ducret A. Aebersold R. Gantt E. Photosynth. Res. 1997; 53: 129-140Crossref Google Scholar). To avoid confusion we will use the term “LHC protein” for the light harvesting complexes in general, and “CAB protein” will refer to the chlorophyll a/b-binding proteins of green algae and higher plants. The major PSII-associated chlorophyll a/b-binding proteins (LHCII) from higher plants have three α-helical, membrane-spanning regions as demonstrated by crystal structure analysis by Kühlbrandt and co-workers (5Kühlbrandt W. Wang D.N. Fujiyoshi Y. Nature. 1994; 367: 614-621Crossref PubMed Scopus (1542) Google Scholar). The first and the third helices are held together by ion pairs to ensure a compact complex to bring the carotenoids and the chlorophylls in close contact. Some CABs form trimeric homocomplexes (6Butler P.J.G. Kühlbrandt W. Proc. Natl. Acad. Sci. U. S. A. 1998; 85: 3797-3801Crossref Google Scholar). All genes encoding LHCs that are known so far reside in the nucleus. In plants, an N-terminal targeting signal (“transit peptide”) allows the transport of the preproteins into the plastid stroma (7Cline K. Henry R. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (188) Google Scholar) and is exchangeable with other stroma-targeting sequences from plants (8Lamppa G.K. Abad M.S. J. Cell Biol. 1987; 105: 2641-2648Crossref PubMed Scopus (38) Google Scholar). Targeting to the thylakoid membrane is accomplished by the mature protein itself. The third transmembrane domain is probably involved in targeting of CAB proteins to the thylakoids, whereas all three helices are needed for correct membrane insertion (9Auchincloss A.H. Alexander A. Kohorn B.D. J. Biol. Chem. 1992; 267: 10439-10446Abstract Full Text PDF PubMed Google Scholar). CAB proteins are inserted into the thylakoid membrane in a reaction requiring GTP and stromal factors (10Hoffman N.E. Franklin A.E. Plant Physiol. 1994; 105: 295-304Crossref PubMed Scopus (69) Google Scholar). The integration process of the LHCs from land plants into thylakoid membranes involves binding to a chloroplast signal recognition particle (SRP), which consists of at least two proteins named cpSRP54 and cpSRP43, and to a further protein, FtsY (11Li X. Henry R. Yuan J. Cline K. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3789-3793Crossref PubMed Scopus (160) Google Scholar, 12Schuenemann D. Gupta S. Persello-Cartieaux F. Klimyuk V.I. Jones J.D.G. Nussaume L. Hoffman N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10312-10316Crossref PubMed Scopus (165) Google Scholar, 13Tu C.J. Schuenemann D. Hoffman N.E. J. Biol. Chem. 1999; 274: 27219-27224Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). cpSRP54 and cpSRP43 are related to homologous proteins that are major factors of the cotranslational protein import pathway into the endoplasmic reticulum of eukaryotes (14Walter P. Johnson A.E. Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (719) Google Scholar). Overexpression and integration experiments in Escherichia coli show that integration does not depend on binding of chlorophylls and carotenoids to the LHC proteins (15Kohorn B.D. Auchincloss A.H. J. Biol. Chem. 1991; 266: 12048-12052Abstract Full Text PDF PubMed Google Scholar). Diatoms are representatives of the heterokont algae, which are assumed to have evolved by secondary endocytobiosis. This means that a eukaryotic host cell took up a photoautotrophic eukaryotic cell, probably an ancestor of modern red algae, followed by transformation of the endosymbiont into a plastid (16Delwiche C.F. Palmer J.D. Plant Syst. Evol. Suppl. 1997; 11: 53-86Crossref Google Scholar, 17Cavalier-Smith T. Trends Plant Sci. 2000; 5: 174-182Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 18Kroth P.G. Strotmann H. Physiol. Plant. 1999; 107: 136-141Crossref Scopus (24) Google Scholar). This process resulted in plastids having four bounding membranes. It has been demonstrated that protein import into these so-called complex plastids depends on a bipartite presequence and occurs in at least two steps (19Lang M. Apt K.E. Kroth P.G. J. Biol. Chem. 1998; 273: 30973-30978Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Although diatoms and other heterokont algae represent an essential part of aquatic ecosystems, contributing significantly to the total oxygen and biomass production, little is known about the photosystems of these organisms. Recent genetic analyses of Fcp genes show that similar to genes encoding CAB proteins Fcp genes belong to a multigene family. Several different Fcp genes have already been found in diatoms, and it is likely that there are even more present (20Bhaya D. Grossman A.R. Nucleic Acids Res. 1993; 46: 71-78Google Scholar, 21Eppard M. Krumbein W.E. von Haeseler A. Rhiel E. Plant Biol. 2000; 2: 283-289Crossref Scopus (37) Google Scholar). So far there are no reports about functional aspects of FCPs with respect to integration into thylakoid membranes and cooperation with the diatom photosystems. Therefore, the characterization of integration characteristics of FCP proteins might reveal the history and the relationship of FCPs with other carotenoid-binding light-harvesting proteins. Here we present the first data on thylakoid membrane integration of a member of the FCP family using a respective protein from the diatom Odontella sinensis. We have established anin vitro thylakoid import assay from purified diatom plastids enabling the study of FCP integration. Our results suggest that FCPs are integrated mainly by an SRP-dependent integration mechanism as found for LHCs. Complementary integration experiments using LHC and FCP proteins as well as thylakoid membranes from land plant and diatom The Fcp gene from a of the diatom as and into the P.G. Plant Mol. Biol. 1995; PubMed Scopus Google Scholar). a more gene of the of the using the and The gene from pea in by Cline of K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). gene is the gene encoding chlorophyll a/b-binding protein of from Arabidopsis in a from of regions from Fcp by reaction using to the All by using the The of the signal in the FCP by the Fcp gene including the and gene but the for the signal and of the reaction by the the mature form of the the Fcp gene with the and the gene and of All Fcp with the for the Fcp form a of the the at from pea isolated (19Lang M. Apt K.E. Kroth P.G. J. Biol. Chem. 1998; 273: 30973-30978Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) from to import and of the plastids as to a of together with isolated to S. Hoffman N.E. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google The diatoms and as Kroth P.G. K. Strotmann H. 1998; Scopus (24) Google Scholar). isolated by of the in a and of and plastids by on a as Kroth P.G. K. Strotmann H. 1998; Scopus (24) Google Scholar). plastids for by oxygen Kroth P.G. K. Strotmann H. 1998; Scopus (24) Google Scholar). thylakoid the plastids by a and for in to the plastids a of of the thylakoids by at for of The on The thylakoids two three in to stromal and in the stroma in at a of of thylakoids by of the plastids with and of the thylakoids with thylakoids by thylakoids in including the at a of for The by and by in a of including encoding LHC and FCP proteins and in a as in the using The for the thylakoid integration experiments with pea diatom thylakoids similar for the different import experiments of thylakoids of and of reaction To SRP-dependent thylakoids in the stromal for different import as and GTP at of The integration by the protein and the at for Some of The by the on All divided into three The first part for analysis and as The and third and the thylakoids in on further The for as for the of thylakoids. All proteins by an on for of the with the thylakoids and for the The third part of the import assay in different to the integration of proteins. to J. Biol. Chem. 1994; Full Text PDF PubMed Google the thylakoids with of for at and to two including The thylakoids by a To the of FCP three whereas in experiments all with the thylakoids in of to the the for To the membrane of FCP of and to of at for the with of as the membranes the to with The by at for by and for at and on PubMed Scopus Google Scholar). of the proteins, the in and in on a of integration the protein in the to from the diatom FCP protein by regions of the Fcp gene for different of the presequence by the signal domain necessary for cotranslational transport the chloroplast of the four diatom and the a domain The of the on the of of signal to the of von Nucleic Acids Res. PubMed Scopus Google Scholar) indicating a at and the mature form as found the plastids (20Bhaya D. Grossman A.R. Nucleic Acids Res. 1993; 46: 71-78Google Scholar). To the integration of FCPs into the thylakoid membranes from diatoms, we established an in vitro import assay for diatom thylakoids. and plastids isolated from the diatoms and purified by a as Kroth P.G. K. Strotmann H. 1998; Scopus (24) Google followed by of the The of the thylakoids by transport isolated thylakoids and of the presence of and and the of the using the S. H. M. J. PubMed Scopus Google Scholar). The as Strotmann H. 1995; Scopus Google Scholar). The thylakoid membranes to up a This to of the not for the of the thylakoids to up a are the for of the We found that of the plastids is with for but the thylakoids to be in a at least to avoid and of LHC proteins not membrane integration be by a in LHCs and FCPs that might be similar in FCPs as there is no presequence present for thylakoid targeting M. Krumbein W.E. von Haeseler A. Rhiel E. Plant Biol. 2000; 2: 283-289Crossref Scopus (37) Google Scholar). In study we of the and we the results with established to membrane-bound protein and To the that of the presequence needed for plastid import might be involved in the integration we integration of as well as of proteins show similar integration to J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) we different for proteins from thylakoid membranes at no FCP integration of stromal and that and have the to FCP protein from diatom thylakoid membranes for followed by and of FCP protein to the thylakoids and as The of protein by thylakoid membrane proteins by and analysis of the of the FCP using a that with only as experiments by of thylakoids and FCP protein of further only integration of of the membranes with and resulted in a of FCP protein of of protein In contrast with not to proteins To the we the integration of by steps including and of the membranes with resulted in a of whereas the steps only a further of the membrane protein steps with resulted in a of FCP probably to membrane by the of the resulted in a of membrane-bound protein on results steps with the to of proteins and to results for the of FCP integration in the In to the steps the of the FCPs into thylakoid membranes by the It has been demonstrated that some of the CAB proteins from higher plants are the and integrated into the thylakoid membrane S. Hoffman N.E. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In experiments a of thylakoids FCP integration on for to and integrated protein not to the results by with needed for FCP integration into diatom thylakoids by of and from with diatom thylakoids a of different proteins integration as shown for results also with thylakoid from diatoms and By using the with as we found a of FCPs only thylakoids and FCP proteins of FCP integration by of stromal and GTP In an of higher of FCP in the thylakoid membranes. of the of GTP show a GTP of FCP integration GTP of about to be for FCP integration. and resulted in results of FCP of from Odontella into thylakoids by thylakoids with FCP protein, stromal and GTP as with and on a The of proteins the by a show of with the of the reaction are integration to the that other known protein integration might also be involved in FCP integration. The is involved in the import reaction of the protein D. A. Plant Mol. Biol. 1998; PubMed Google Scholar) and depends on the presence of stromal factors and of of stromal to the integration assay not in an of FCP integration the which be the also be involved To that the of FCP integration not to the presence of stromal factors with the thylakoid we an of the as well as the In integration to experiments In land plants proteins like and are into the thylakoids by a D. A. Plant Mol. Biol. 1998; PubMed Google Scholar). there is no FCP integration FCP integration in the light in the as well as the of which transmembrane not show The integration of the FCPs is not by the presence of the N-terminal presequence which is necessary for targeting of into the plastids in The FCP into the thylakoid membrane as well as the the mature form This is with of the higher plant LHCs S. E. H. E. R. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). steps as we found a of FCP to be with the thylakoid membrane even in the of stromal factors GTP. This might be to factors from the in vitro of proteins. a of the FCP protein in the integration assay resulted in FCP other known from higher plant thylakoids have been by it is that other so far might FCP integration. Therefore, we diatom thylakoids with to of FCP protein to protein that might be involved in protein we to proteins from the thylakoids by with In the of FCP protein as in not This might suggest that a part of the FCP proteins might be to integrate as for the of chloroplast from higher plants proteins S. Hoffman N.E. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, D. A. D. L. J. 1994; PubMed Scopus Google Scholar). To we FCP integration into a different of We protein with for the membranes with including we found of the the membrane To CAB and FCP integration in the we LHC proteins from pea and and FCP from Odontella with thylakoids isolated from pea shown in the integration characteristics of LHC from pea into diatom thylakoid membranes various are similar to the respective experiments with FCP integration the of GTP and stromal proteins resulted in a of FCP integration. results for LHC In experiments we show that integration of diatom FCP into thylakoid membranes from pea also depends on GTP and stromal factors of integration of FCP protein from Odontella into thylakoid membranes from protein with isolated thylakoids from pea as the thylakoids with as by The of proteins the in on by a show of with the of the reaction The present and stromal and GTP and stromal LHC proteins in all organisms so far have to at least two membranes inserted into the thylakoid membrane. In higher plants and in green and red algae, two plastid membranes have to be whereas in heterokont algae FCPs have to be four membranes to the stromal In N-terminal are for correct of FCPs and other plastid preproteins from diatoms have a bipartite The of these two have been vitro (19Lang M. Apt K.E. Kroth P.G. J. Biol. Chem. 1998; 273: 30973-30978Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and in E. L. J. M. R. A. R. P. in In plastids of higher plants have been for insertion of proteins into the thylakoid membrane targeting to the thylakoid D. A. Plant Mol. Biol. 1998; PubMed Google Scholar). proteins like the of the oxygen found to be by a homologous to the In contrast to the a allows the of more proteins Biol. Chem. 2000; Scopus Google Scholar). an N-terminal targeting domain is for which is found the for chloroplast import and the mature Some proteins that to be inserted into the thylakoid membrane use different of CAB proteins is on the presence of GTP and different stromal proteins, which are homologous to the transport at membranes. reports demonstrated that integration into thylakoid membranes R. Plant Mol. Biol. 1987; 10: PubMed Scopus Google but experiments demonstrated that GTP membrane insertion more (10Hoffman N.E. Franklin A.E. Plant Physiol. 1994; 105: 295-304Crossref PubMed Scopus (69) Google Scholar). proteins that are related to CAB proteins like early light-inducible proteins and are to integrate into the membrane the of proteins S. Hoffman N.E. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). It is these different integration are to CAB and proteins. The of thylakoid membrane insertion of LHC proteins so far has been for CAB proteins of higher plants In study we have the FCP proteins the insertion pathway as CAB proteins and process might be a for LHC proteins from all of photoautotrophic To the for integration of FCP from a diatom in a homologous we have established a to functional thylakoids from isolated diatom In contrast to thylakoids of higher plant plastids in diatoms, no are found of thylakoid The thylakoids from diatoms, by the to up to to up the plastids but to avoid of the thylakoids. thylakoid from diatoms resulted in thylakoid membranes that transport but no have been demonstrated M. 1998; PubMed Scopus Google Scholar). plastids from diatoms the as for from higher plants to avoid and to functional which has been demonstrated by oxygen Kroth P.G. K. Strotmann H. 1998; Scopus (24) Google Scholar). Therefore, for thylakoid we the of the plastids in a to a the to the thylakoids. of the of thylakoids from purified plastids is the to stromal for integration It to be to the of FCP integrated into the thylakoid membrane. to FCP protein, but the to be in a of of the protein using with and of the resulted in results that by the the analysis of on protein Our integration experiments a similar membrane integration for FCPs and CAB proteins. of FCP on stromal factors and on the presence of GTP. in the GTP show a on GTP and resulted in a of FCP integration at of GTP. This is in with integration experiments of CAB proteins into land plant thylakoids (10Hoffman N.E. Franklin A.E. Plant Physiol. 1994; 105: 295-304Crossref PubMed Scopus (69) Google Scholar). of on insertion in the and we found a of FCP protein together with the thylakoid membranes even in the of GTP and stromal proteins. The even of the thylakoids with to proteins involved in protein in membranes like from a of FCP and The membrane with we have been demonstrated to be in proteins J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). It to all of the FCPs in of the thylakoids the integration reaction that in a of FCPs might be the other CAB integration into diatom thylakoids and vice versa also resulted in a of FCP protein in the of This that so far we are not to FCP integration and FCP integration of FCPs be in contrast to CAB proteins, which GTP and proteins. LHC proteins to have evolved from and of light-inducible proteins, an protein Bhaya D. Grossman A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). The that can integrate into the thylakoid membrane as well as by the complex S. Hoffman N.E. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) indicates that these proteins might be to insertion to be for might have for insertion of the ancestor of present LHC proteins. integration the of CAB proteins. of the integration of CABs from more green algae might further into analyses have shown that FCP proteins are more related to LHC proteins from red algae and to proteins from dinoflagellates to CAB proteins (1Durnford D.G. Deane J.A. Tan S. McFadden G.I. Gantt E. Green B.R. J. Mol. Evol. 1999; 48: 59-68Crossref PubMed Scopus (199) Google which the origin of heterokont plastids from red algal (16Delwiche C.F. Palmer J.D. Plant Syst. Evol. Suppl. 1997; 11: 53-86Crossref Google Scholar). of red and green algae to the land from a common ancestor is to have early in the of We demonstrated that the diatom FCP can integrate into pea thylakoids as well as pea LHC into diatom thylakoids. This is it that protein use the apparently import This in organisms as diatoms and land plants indicates that the SRP-dependent integration of light-harvesting proteins may have been early in the of plastids and has This is as which are to be related to the of plastids, apparently not have light-harvesting proteins, but phycobilisomes Although it is that the ancestor of plastids from modern indicates that the light-harvesting proteins as well as the needed might have evolved endocytobiosis. The genetic history of the transfer of Fcp genes secondary from the endosymbiont to the of the host cell is also FCPs seem to be it is likely that might have been to the and to be in a secondary from the of the endosymbiont to the of the gene of the related algae L. D. M. S. J. Mol. Evol. 1996; PubMed Scopus Google Scholar) signal and domain of the FCP and analyses of light-harvesting proteins from and in suggest a gene transfer J.A. M. W. Durnford D.G. McFadden G.I. 2000; PubMed Scopus Google Scholar). We are to and of for and support a by M. in We are also to Cline of and for genes of higher plant LHCs. We also of for the