Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

Faculty of Science University of South Bohemia 37005 České Budějovice Czech Republic

Laboratory of Photosynthesis Centre Algatech Institute of Microbiology Academy of Sciences 37981 Třeboň Czech Republic

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Armbruster U, Zühlke J, Rengstl B, Kreller R, Makarenko E, Rühle T, Schünemann D, Jahns P, Weisshaar B, Nickelsen J, Leister D (2010) The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly. Plant Cell 22: 3439–3460 PubMed PMC

Bečková M, Gardian Z, Yu J, Koník P, Nixon PJ, Komenda J (2017) Association of Psb28 and Psb27 proteins with PSII-PSI supercomplexes upon exposure of PubMed

Boehm M, Romero E, Reisinger V, Yu J, Komenda J, Eichacker LA, Dekker JP, Nixon PJ (2011) Investigating the early stages of photosystem II assembly in PubMed PMC

Boehm M, Yu J, Reisinger V, Bečková M, Eichacker LA, Schlodder E, Komenda J, Nixon PJ (2012) Subunit composition of CP43-less photosystem II complexes of PubMed PMC

Chidgey JW, Linhartová M, Komenda J, Jackson PJ, Dickman MJ, Canniffe DP, Koník P, Pilný J, Hunter CN, Sobotka R (2014) A cyanobacterial chlorophyll synthase-HliD complex associates with the Ycf39 protein and the YidC/Alb3 insertase. Plant Cell 26: 1267–1279 PubMed PMC

Crawford TS, Eaton-Rye JJ, Summerfield TC (2016) Mutation of Gly195 of the ChlH subunit of Mg-chelatase reduces chlorophyll and further disrupts PS II assembly in a Ycf48-deficient strain of PubMed PMC

D’Haene SE, Sobotka R, Bučinská L, Dekker JP, Komenda J (2015) Interaction of the PsbH subunit with a chlorophyll bound to histidine 114 of CP47 is responsible for the red 77K fluorescence of Photosystem II. Biochim Biophys Acta 1847: 1327–1334 PubMed

Di Pietro F, Brandi A, Dzeladini N, Fabbretti A, Carzaniga T, Piersimoni L, Pon CL, Giuliodori AM (2013) Role of the ribosome-associated protein PY in the cold-shock response of PubMed PMC

Dobáková M, Sobotka R, Tichý M, Komenda J (2009) Psb28 protein is involved in the biogenesis of the photosystem II inner antenna CP47 (PsbB) in the cyanobacterium PubMed PMC

Dobáková M, Tichý M, Komenda J (2007) Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium PubMed PMC

Eaton-Rye JJ, Vermaas WFJ (1991) Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium PubMed

Fleischmann TT, Scharff LB, Alkatib S, Hasdorf S, Schöttler MA, Bock R (2011) Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution. Plant Cell 23: 3137–3155 PubMed PMC

Frauenfeld J, Gumbart J, Sluis EO, Funes S, Gartmann M, Beatrix B, Mielke T, Berninghausen O, Becker T, Schulten K, Beckmann R (2011) Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Nat Struct Mol Biol 18: 614–621 PubMed PMC

Galmozzi CV, Florencio FJ, Muro-Pastor MI (2016) The cyanobacterial ribosomal-associated protein LrtA Is involved in post-stress survival in PubMed PMC

Heinz S, Liauw P, Nickelsen J, Nowaczyk M (2016) Analysis of photosystem II biogenesis in cyanobacteria. Biochim Biophys Acta 1857: 274–287 PubMed

Hennon SW, Soman R, Zhu L, Dalbey RE (2015) YidC/Alb3/Oxa1 family of insertases. J Biol Chem 290: 14866–14874 PubMed PMC

Hollingshead S, Kopečná J, Armstrong DR, Bučinská L, Jackson PJ, Chen GE, Dickman MJ, Williamson MP, Sobotka R, Hunter CN (2016) Synthesis of chlorophyll-binding proteins in a fully segregated Δ PubMed PMC

Hollingshead S, Kopecná J, Jackson PJ, Canniffe DP, Davison PA, Dickman MJ, Sobotka R, Hunter CN (2012) Conserved chloroplast open-reading frame PubMed PMC

Knoppová J, Sobotka R, Tichý M, Yu J, Koník P, Halada P, Nixon PJ, Komenda J (2014) Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in PubMed PMC

Komenda J. (2005) Autotrophic cells of the PubMed

Komenda J, Lupínková L, Kopecký J (2002) Absence of the PubMed

Komenda J, Reisinger V, Müller BC, Dobáková M, Granvogl B, Eichacker LA (2004) Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in PubMed

Komenda J, Sobotka R, Nixon PJ (2012) Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria. Curr Opin Plant Biol 15: 245–251 PubMed

Kopecná J, Komenda J, Bucinská L, Sobotka R (2012) Long-term acclimation of the cyanobacterium PubMed PMC

Kopečná J, Pilný J, Krynická V, Tomčala A, Kis M, Gombos Z, Komenda J, Sobotka R (2015) Lack of phosphatidylglycerol inhibits chlorophyll biosynthesis at multiple sites and limits chlorophyllide reutilization in the cyanobacterium PubMed PMC

Kopf M, Klähn S, Scholz I, Matthiessen JK, Hess WR, Voß B (2014) Comparative analysis of the primary transcriptome of PubMed PMC

Levey T, Westhoff P, Meierhoff K (2014) Expression of a nuclear-encoded psbH gene complements the plastidic RNA processing defect in the PSII mutant hcf107 in PubMed

Linhartová M, Bučinská L, Halada P, Ječmen T, Setlík J, Komenda J, Sobotka R (2014) Accumulation of the Type IV prepilin triggers degradation of SecY and YidC and inhibits synthesis of Photosystem II proteins in the cyanobacterium PubMed

Mitschke J, Georg J, Scholz I, Sharma CM, Dienst D, Bantscheff J, Voss B, Steglich C, Wilde A, Vogel J, Hess WR (2011) An experimentally anchored map of transcriptional start sites in the model cyanobacterium PubMed PMC

Nixon PJ, Michoux F, Yu J, Boehm M, Komenda J (2010) Recent advances in understanding the assembly and repair of photosystem II. Ann Bot 106: 1–16 PubMed PMC

Pilný J, Kopečná J, Noda J, Sobotka R (2015) Detection and quantification of heme and chlorophyll precursors using a High Performance Liquid Chromatography (HPLC) system equipped with two fluorescence detectors. Bio Protoc 5: e1390

Porra RJ, Thompson WA, Kriedmann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975: 384–394

Prinz A, Behrens C, Rapoport TA, Hartmann E, Kalies KU (2000) Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA. EMBO J 19: 1900–1906 PubMed PMC

Promnares K, Komenda J, Bumba L, Nebesářová J, Vácha F, Tichý M (2006) Cyanobacterial small chlorophyll-binding protein ScpD (HliB) is located on the periphery of photosystem II in the vicinity of PsbH and CP47 subunits. J Biol Chem 281: 32705–32713 PubMed

Rengstl B, Knoppová J, Komenda J, Nickelsen J (2013) Characterization of a PubMed

Rengstl B, Oster U, Stengel A, Nickelsen J (2011) An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis. J Biol Chem 286: 21944–21951 PubMed PMC

Sachelaru I, Petriman NA, Kudva R, Kuhn P, Welte T, Knapp B, Drepper F, Warscheid B, Koch HG (2013) YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein. J Biol Chem 288: 16295–16307 PubMed PMC

Seitl I, Wickles S, Beckmann R, Kuhn A, Kiefer D (2014) The C-terminal regions of YidC from PubMed

Slavov N, Semrau S, Airoldi E, Budnik B, van Oudenaarden A (2015) Differential stoichiometry among core ribosomal proteins. Cell Reports 13: 865–873 PubMed PMC

Sobotka R. (2014) Making proteins green; biosynthesis of chlorophyll-binding proteins in cyanobacteria. Photosynth Res 119: 223–232 PubMed

Sobotka R, Komenda J, Bumba L, Tichý M (2005) Photosystem II assembly in CP47 mutant of PubMed

Umena Y, Kawakami K, Shen JR, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473: 55–60 PubMed

Vavilin D, Brune DC, Vermaas W (2005) PubMed

Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6: 1215–1225 PubMed

Xue S, Barna M (2012) Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol 13: 355–369 PubMed PMC

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