The Cyanobacterial Homologue of HCF136/YCF48 Is a Component of an Early Photosystem II Assembly Complex and Is Important for Both the Efficient Assembly and Repair of Photosystem II in Synechocystis sp. PCC 6803

Abstract

The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair. The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair. The photosystem II (PSII) 2The abbreviations used are:PSIIphotosystem IIBN/PAGEblue native-PAGEChlchlorophyllRCCphotosystem II reaction center coreRC47photosystem II reaction center containing CP47HCFhigh chlorophyll fluorescenceWTwild typeTes2-{[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]amino}ethanesulfonic acidMes4-morpholineethanesulfonic acidPVDFpolyvinylidene difluoride. complex of plants, algae, and cyanobacteria is a multisubunit pigment-protein complex responsible for water oxidation during oxygenic photosynthesis (1Barber J. Biochem. Soc. Trans. 2006; 34: 619-631Crossref PubMed Scopus (132) Google Scholar). The complex consists of over 20 intrinsic and peripheral membrane proteins, which must be assembled in a highly coordinated manner to ensure proper functioning of the complex (2Aro E-M. Suorsa M. Rokka A. Allahverdiyeva Y. Paakkarinen V. Saleem A. Battchikova N. Rintamäki E. J. Exp. Bot. 2005; 56: 347-356Crossref PubMed Scopus (388) Google Scholar). At the heart of the complex is a heterodimer of the D1 (the psbA gene product) and related D2 (the psbD gene product) polypeptides, which binds most of the redox-active cofactors involved in PSII electron transfer (3Diner B.A. Rappaport F. Annu. Rev. Plant Biol. 2002; 53: 551-580Crossref PubMed Scopus (347) Google Scholar). The isolated PSII reaction center (RC) complex contains, in addition to D1 and D2, the intrinsic PsbI subunit and cytochrome b559, which bind to D1 and D2, respectively (4Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar). Surrounding the PSII RC complex are two chlorophyll-binding proteins, CP43 (the psbC gene product) and CP47 (the psbB gene product). D1 and CP43 also provide several amino acid ligands to the Mn4Ca cluster involved in the oxidation of water (5Ferreira K.N. Iverson T.N. 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Advances in Photosynthesis and Respiration, Photoprotection, Photoinhibition, Gene Regulation, and Environment. Springer, Dordrecht2006: 175-191Google Scholar). In most PSII-containing organisms, D1 is synthesized as a precursor protein (pD1) with a carboxyl-terminal extension (9Satoh K. Yamamoto Y. Photosynth. Res. 2007; 94: 203-215Crossref PubMed Scopus (34) Google Scholar). In the cyanobacterium Synechocystis sp. PCC 6803 this extension consists of 16 amino acid residues, which is removed in two steps by the CtpA endoproteinase: first by cleavage on the carboxyl side of residue Ala352, to generate i-D1, and then by cleavage after Ala344 to form mature D1 (10Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (196) Google Scholar, 11Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (142) Google Scholar, 12Inagaki N. Yamamoto Y. Satoh K. 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PubMed Scopus Google Scholar). in vivo that formation of the PSII RC complex in the of YCF48 B. FEBS Lett. 2002; PubMed Scopus Google Scholar). using YCF48 its interaction with a PSII assembly complex containing D2 and cytochrome b559 but a complex with PSII in vivo B. FEBS Lett. 2002; PubMed Scopus Google Scholar). YCF48 are in oxygenic the YCF48 is not for of PCC and is not for assembly of PSII J. J. Biol. 2002; PubMed Scopus Google Scholar). YCF48 an role in PSII assembly is and an to The of YCF48 in the cyanobacterium PCC 6803 is by the slr2034 gene is of the subunits of the two of cytochrome In this we the slr2034 gene product here as is involved in the assembly of cyanobacterial We show that YCF48 is a component of PSII RC-like assembly complexes of D2, cytochrome b559, and PsbI lacking CP47 and and that is for assembly of we provide that the unassembled precursor of D1 and are binding for this assembly and that YCF48 also functions in PSII repair. and of strains used in the were from the of Synechocystis sp. PCC 6803 Scopus Google to as wild type The strains were used in the the with the psbC gene by a M. Y. Photosynth. Res. PubMed Scopus Google the with the psbB gene by a Plant Biol. PubMed Scopus Google and the with the by a H.B. J. PubMed Scopus Google Scholar). The by the slr2034 gene with a the the were and on containing and of the by ycf48 strains were by of mutants lacking and using from and for the to were in in using an with of and used a chlorophyll of in addition and H.B. J. PubMed Scopus Google Scholar). for of strains in addition of were in and were using a time were used for of the and isolated from the Plant 2004; PubMed Scopus Google by and with of used for synthesis using and II were on the using the on two for two from the the used as a level to be to in The used to psbA gene The were to and and of fluorescence were in using a with an and the respectively M. I. Kuviková S. O. J. PubMed Scopus Google Scholar). The of in in containing using and as electron with the membrane of using a were with and for the to two were and during from to The of the from the the and and the PSII M. I. Kuviková S. O. J. PubMed Scopus Google and as a for PSII of and were by the using J. Barber J. Biochemistry. 34: PubMed Scopus Google with the were and in containing and and were using the in F. I. F. A. Pakrasi H.B. B. B. 2002; PubMed Scopus Google of protein isolated were with and by blue in to Biochem. PubMed Scopus Google with the for and for were the The protein of the complexes were in the by in a containing J. J. J. Biochem. 2002; PubMed Scopus Google Scholar). The from the for in containing and on the of the two were in a in the were either by or were with and then with The used in the were in of the D1 the of the D2 from Synechocystis of the isolated CP43 from Synechocystis the isolated subunit of cytochrome b559 from Synechocystis of subunit of cytochrome b559 from Synechocystis and the from A. thaliana in J. J. PubMed Scopus Google Scholar). the or the membrane with to for or to a used for and of or were with the and were on a of using of the containing were in of in a for and then a of and The to and the and the were in and used for of of b559 and as J. M. Plant 2005; PubMed Scopus Google using the subunit of cytochrome b559 isolated from of the were with and after with the the to were by the containing and The by to and used for and Yeast split ubiquitin system used to membrane protein in N. A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, J. J. 2005; PubMed Scopus Google Scholar). slr2034 from Synechocystis using and the by and, the and of the either the form iD1, or the mature D1 as as psbD and were using the in in and the were the and of or the and of The containing the psbA of pD1 has been J. J. 2005; PubMed Scopus Google Scholar). The by F. were in the J. J. 2005; PubMed Scopus Google Scholar). protein synthesis by with and of gene by with as J. J. 2005; PubMed Scopus Google Scholar). or were with and in the from the and K. Advances in Photosynthesis Scholar). YCF48 a of PSII RC in Synechocystis A. YCF48 to as an assembly that is to PSII assembly but is not a component of assembled and core complexes B. 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In of the YCF48 protein in the most and The in low for of YCF48 also M. M. J. Plant 2007; PubMed Scopus Google but after to its to YCF48 in we that and from by the of YCF48 and that most or as protein The also that from and by of The is to cytochrome b559 and as a is to the D2 protein J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google Scholar). This PSII RC complexes and, in with the only unassembled YCF48 be in the Absence of YCF48 and of PSII RC the role of YCF48 in Synechocystis we an in which the slr2034 gene by a the of PCC J. J. Biol. 2002; PubMed Scopus Google the Synechocystis a of than the used The of the the of in the This by an in also a of chlorophyll in the of the of in the of an electron the fluorescence which is a of the of PSII and PSII showed a to decrease in the levels of PSII in the reduction in the levels of PSII by of which showed only the levels of and in the PSII complexes in the of electron transfer to the fluorescence that electron transfer on the side of PSII in and as the of provide the that a level of PSII with Besides to the PSII complexes in the of the of PSII complex were also in In of the D1 subunit in the and core and as by the of the with the to in the complex of and in an of of 20 of in the of electron of of in the of and of by in the of of were by the the in the from to with the containing of of in an of of 20 of in the of electron of of in the of and of by in the of of were by the the in the from to with the containing of of in a the PSII assembly in and the we with and the of unassembled PSII subunits as as assembled in the of PSII complex In this a the and the D1 protein in PSII complexes and RC complexes or unassembled D1 were in the which is with the of newly synthesized and other PSII proteins, PSII complexes the used for the In in the than of the PSII proteins, D2, and D1 were in the of In the of unassembled D1 protein only be in using a much and a M. M. J. Plant 2007; PubMed Scopus Google Scholar). of D1 be the form, the processed form, and mature D1 M. M. J. Plant 2007; PubMed Scopus Google Scholar). In in unassembled D1 after the and only the mature D1 in this during low the processed of D1 were in of that D1 synthesis in not by of psbA were in than not in the of PSII complexes in and were also by This to a of the complex in but not in the The YCF48 the binding of this protein in the containing and also the of protein in the of unassembled subunit of cytochrome b559 and unassembled D2 subunit in the first of the of the of a D2-cytochrome b559 pre-complex, as identified in B. Plant 11: PubMed Scopus Google Scholar). The of this in Synechocystis by of subunits of cytochrome b559 and D2 from a using the subunit of cytochrome b559 In the of the complex is also by the of the cytochrome b559 in the of the of Synechocystis 6803 J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google Scholar). We also a of mutants in which the ycf48 gene in strains lacking the PSII or D2. In the lacking CP43 PSII assembly not formation of the complex and the D1 protein in this complex of ycf48 in a of D1 in the that only the of D2. This with the of D2 in the unassembled protein in the of the newly synthesized D1 in the the of the complex in this as the complex in the The has been to the RC complexes and with D2 and of D1 in and J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google Scholar). In the formation of RC complexes in as judged by and a role for YCF48 in the of RC complexes in this in the on the of D2, and cytochrome b559 in membrane isolated by a PSII RC complex of subunits has been to be assembled in this membrane E. B. F. B. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google which then to the to its YCF48 is with RC we for its in highly and J. M. Kuviková S. M. P.J. J. Biol. 2006; PubMed Scopus Google Scholar). the of YCF48 with the but not the membrane YCF48 with the of of PSII in that YCF48 the of pD1 and of the the of only of unassembled mature processed D1 be in the that D2 not in the J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google that YCF48 the of interaction of pD1 with D2. of CP43 and CP47 also in the and synthesis not to be by of ycf48 and Overall the a role for YCF48 in the of the precursor of D1 in YCF48 the to with two-hybrid analyses were using the split ubiquitin system N. A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, J. J. 2005; PubMed Scopus Google Scholar). This system the of membrane to be in by gene to in gene after of YCF48 and the pD1 precursor protein as to a lesser extent, the of mature in of in YCF48 the processed form of This that YCF48 binds to the extension of the pD1 In interaction with the D2 protein or proteins, as the subunit of photosystem or the J. J. 2005; PubMed Scopus Google Scholar). the CtpA which the of also not by that two not to during II The D1 the for of YCF48 to the PSII RC-like of an interaction of YCF48 with the precursor of but not mature the that the extension an role in the binding of YCF48 during formation of the PSII RC-like complexes. we a lacking the D1 extension and CP47. of its membrane complexes showed that the and complexes and YCF48 is that the YCF48 also with the mature D1 but this interaction is to be in by the two-hybrid The Absence of YCF48 PSII and the of the D1 the of YCF48 the of PSII we the time of of in and to of either in the or of the of in of only to of the level during the in the of this in of to with to that in the of from low after a high in The of PSII repair in also with the synthesis and degradation of the D1 as by This a of D1 in of in the only of the protein during the the also that D1 in the and that the D2 which on the in the of unassembled than in not be used for PSII assembly to the low of the D1 protein caused by the of the YCF48 that PSII repair and selective D1 also in the most of the of newly synthesized D1 of YCF48 in the of RC YCF48 is to be for formation of the PSII RC complex during assembly of PSII in Arabidopsis B. FEBS Lett. 2002; PubMed Scopus Google the of an PSII in a ycf48 of a role in PSII assembly in we that YCF48 is also not for PSII assembly in the cyanobacterium Synechocystis 6803. we provide a variety of to an role for YCF48 in PSII assembly the level of PSII RC assembly an we is that YCF48 a role in PSII assembly in cyanobacteria and In we show for the first time that YCF48 with PSII RC complexes in vivo and and M. M. J. Plant 2007; PubMed Scopus Google the of PSII RC complexes in a lacking CP47 and that complexes with to the of assembly not in the final PSII The that of assembly is The of YCF48 in the complex that YCF48 binding of CP47 to the PSII RC also that the of the RC not the most protein to in complexes not which is with binding of this assembly on in PSII assembly E. B. M. Plant 2006; PubMed Scopus Google Scholar, Pakrasi H.B. J. Biol. 2007; PubMed Scopus Google Scholar). in the of the RC complexes by is related to binding of YCF48 to the PSII subunits D1 D2, cytochrome b559, and and of PSII in the a on the of assembly of PSII in the The of PSII in the is of the of this and in J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google strains of unassembled D2, and CP47. the but of mature D1 in the unassembled protein that D1 protein is synthesized but its for the assembly of RC complexes is an in psbA in the with in to the in the of D1 interaction of YCF48 with pD1 and a lesser from two-hybrid analyses and is with the of the ycf48 The of YCF48 not only in of pD1 but also led to a decrease in the level of unassembled D1 protein in low in the lacking CP47 and in the lacking cytochrome b559 and also to D2 results that YCF48 with pD1 its with D2 to its and In this YCF48 is of which also unassembled pD1 but with the assembly M. M. J. Plant 2007; PubMed Scopus Google Scholar). we were not to the complex of pD1 and YCF48 by protein or by experiments, a and interaction pD1 and our of the lacking CP47 showed that the is not the for binding of YCF48 to the PSII RC-like complexes. is that interaction of the newly synthesized precursor D1 with other assembly B. F. M. S. J. J. Biol. 2004; PubMed Scopus Google or PSII subunits absent in PsbI M. M. J. Plant 2007; PubMed Scopus Google is for interaction of YCF48 with other of is also that the interaction YCF48 and the D1 protein is for by the two-hybrid only the extension (or is is and in the of the mature In A. thaliana YCF48 to with the D2-cytochrome b559 than with D1 B. FEBS Lett. 2002; PubMed Scopus Google Scholar). the in the of D2 and pD1 in Arabidopsis not the of D2 or pD1 in the YCF48 PSII on our results we that the complex isolated from Arabidopsis not the D2-cytochrome b559 but the PSII RC complex containing pD1 of an be in with our of the Synechocystis 6803 In the of a D2-cytochrome b559 assembly has been B. Plant 11: PubMed Scopus Google Scholar). We been to show that a is also to form in as by in the using strains lacking either the YCF48 or the D1 protein PSII RC assembly complexes to be to form from and D2-cytochrome b559 we this the operation of assembly of showed a of YCF48 that is not assembled in of the YCF48 in mutants showed in the of assembled the of this protein is with the level in the lacking the The level of chlorophyll in the and the of CP43 and, CP47 in the indicate that the of YCF48 also other chlorophyll-binding B. FEBS Lett. 2002; PubMed Scopus Google an of the of YCF48 on the of but that the and CP47 bind we the that YCF48 is or involved in the of chlorophyll chlorophyll-binding the synthesis of D1 the synthesis of other chlorophyll-binding proteins, the of YCF48 on D1 and assembly be much in with other chlorophyll-binding two-hybrid analyses to provide for an interaction YCF48 and other chlorophyll-binding proteins, the of this type of be Yeast not chlorophyll and lacking chlorophyll be in a to with in the YCF48 for PSII a role in the assembly of PSII we for the first time that YCF48 functions in the PSII repair cycle and is for of PSII after has been that YCF48 to be involved in the stabilization of pD1 in the the most for D1 replacement in the ycf48 is the of pD1 for selective D1 The of YCF48 in PSII repair an for the of PSII in the Arabidopsis of PSII and in Synechocystis by E. B. F. B. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google on the of PSII subunits in membrane isolated by the of PSII RC-like complexes in the membrane This led to the that the PSII RC assembled in the membrane then the to its assembly the PSII E. B. F. B. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). In the D1 in the membrane that D1 to here during PSII assembly and PSII repair. We here that YCF48 is in the membrane and, in is of two PSII RC assembly complexes and on the of YCF48 a for and in the role for YCF48 be to in the of PSII and RC complexes from the membrane to the is the role of the membrane in PSII assembly and repair. The processed form of iD1, is in our with PSII RC complexes M. M. J. Plant 2007; PubMed Scopus Google Scholar, J. V. B. M. Granvogl B. J. Biol. 2004; PubMed Scopus Google but of of D1 in E. J. Plant 2002; Scopus Google Scholar). the selective degradation of D1 during PSII repair most in the which is in and the involved in D1 been to the membrane E. B. F. B. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, J. M. Kuviková S. M. P.J. J. Biol. 2006; PubMed Scopus Google Scholar). the PSII repair in the is the of the CtpA in the membrane E. B. F. B. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). CtpA has not been in this in analyses F. I. F. A. Pakrasi H.B. B. B. 2002; PubMed Scopus Google and is the used in is or related as the and the in the of the membrane in Synechocystis using E. A. Ohad I. J. 2007; PubMed Scopus Google we the that assembly and repair of PSII in of the than in the and that the isolated by membrane in addition to that the membrane in Synechocystis is than Battchikova N. B. 2006; PubMed Scopus Google Scholar). This in the membrane system in be to the in the membrane system of We are to J. M. and E. for W. and Pakrasi for PSII and as as and for the of with