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Photosynthetica

, Volume 56, Issue 1, pp 300–305 | Cite as

Isolation of the cyanobacterial YFP-tagged photosystem I using GFP-Trap®

  • A. Strašková
  • J. Knoppová
  • J. Komenda
Brief Communication

Abstract

A strain of Synechocystis sp. PCC 6803 expressing the yellow fluorescent protein (YFP) fused to the C-terminus of the PsaF subunit of PSI has been constructed and used to isolate native PSI complexes employing the GFP-Trap®, an efficient immunoprecipitation system which recognizes the green fluorescent protein (GFP) and its variants. The protein analysis and spectroscopic characterization of the preparation revealed an isolate of trimeric and monomeric PSI complexes, which showed minimal unspecific contamination as demonstrated by comparison with the wild type control. Interestingly, we detected CP43 subunits of PSII and small amounts of PSII core complexes specifically pulled-down with the YFP-PSI, supporting the association of PSII assembly modules and intermediate assembly complexes with PSI, as observed in our previous studies. The results demonstrate that the GFP-Trap® system represents an excellent tool for studies of PSI biogenesis and interconnection of PSI and PSII assembly processes.

Additional key words

assembly factor pigment–protein complex two-dimensional electrophoresis 

Abbreviations

Chl

chlorophyll

CN

clear native

GFP

green fluorescent protein

PAGE

polyacrylamide gel electrophoresis

TM

thylakoid membrane

WT

wild type

YFP

yellow fluorescent protein

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References

  1. Aoki T., Takahashi Y., Koch K.S. et al.: Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting.–FEBS Lett. 384: 193–197, 1996.CrossRefPubMedGoogle Scholar
  2. Bečková M., Gardian Z., Yu J. et al.: Association of Psb28 and Psb27 proteins with PSII-PSI supercomplexes upon exposure of Synechocystis sp. PCC 6803 to high light.–Mol. Plant. 10: 62–72, 2017.CrossRefPubMedGoogle Scholar
  3. Boehm M., Nield J., Zhang P. et al.: Structural and mutational analysis of band 7 proteins in the cyanobacterium Synechocystis sp. strain PCC 6803.–J. Bacteriol. 191: 6425–6435, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Enoki S., Maki K., Inobe T. et al.: The equilibrium unfolding intermediate observed at pH 4 and its relationship with the kinetic folding intermediates in green fluorescent protein.–J. Mol. Biol. 361: 969–982, 2006.CrossRefPubMedGoogle Scholar
  5. Fromme P., Jordan P., Krauss N.: Structure of photosystem I.–BBA-Bioenergetics 1507: 5–31, 2001.CrossRefPubMedGoogle Scholar
  6. Fromme P., Witt H.T.: Improved isolation and crystallization of photosystem I for structural analysis.–BBA-Bioenergetics 1365: 175–184, 1998.CrossRefGoogle Scholar
  7. Gulis G., Narasimhulu K.V., Fox L.N. et al.: Purification of His6-tagged photosystem I from Chlamydomonas reinhardtii.–Photosynth. Res. 96: 51–60, 20CrossRefPubMedGoogle Scholar
  8. Hladík J., Šofrová D.: Does the trimeric form of the photosystem I reaction center of cyanobacteria in vivo exist?–Photosynth. Res. 29: 171–175, 1991.PubMedGoogle Scholar
  9. Jordan P., Fromme P., Witt H.T. et al.: Three-dimensional structure of cyanobacterial photosystem I at 2.5 angstrom resolution.–Nature 411: 909–917, 2001.CrossRefPubMedGoogle Scholar
  10. Knoppová J., Sobotka R., Tichý M. et al.: Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis.–Plant Cell 26: 1200–1212, 2014.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Komenda J., Barber J.: Comparison of psbO and psbH deletion mutants of Synechocystis PCC 6803 indicates that degradation of D1 protein is regulated by the QB site and dependent on protein synthesis.–Biochemistry 34: 9625–9631, 1995.CrossRefPubMedGoogle Scholar
  12. Komenda J., Knoppová J., Kopečná J. et al.: The Psb27 assembly factor binds to the CP43 complex of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.–Plant Physiol. 158: 476–486, 2012a.CrossRefPubMedGoogle Scholar
  13. Komenda J., Reisinger V., Müller B.C. et al.: Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803.–J. Biol. Chem. 279: 48620–48629, 2004.CrossRefPubMedGoogle Scholar
  14. Komenda J., Sobotka R., Nixon P.J.: Assembling and maintaining the photosystem II complex in chloroplasts and cyanobacteria.–Curr. Opin. Plant Biol. 15: 245–251, 2012b.CrossRefPubMedGoogle Scholar
  15. Kopečná J., Komenda J., Bučinská L. et al.: Long-term acclimation of the cyanobacterium Synechocystis sp. PCC 6803 to high light is accompanied by an enhanced production of chlorophyll that is preferentially channeled to trimeric photosystem I.–Plant Physiol. 160: 2239–2250, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kopečná J., Pilný J., Krynická V. et al.: Lack of phosphatidylglycerol inhibits chlorophyll biosynthesis at multiple sites and limits chlorophyllide reutilization in Synechocystis sp. strain PCC 6803.–Plant Physiol. 169: 1307–1317, 2015.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kubota H., Sakurai I., Katayama K. et al.: Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits.–BBABioenergetics 1797: 98–105, 2010.CrossRefGoogle Scholar
  18. Nickelsen J., Rengstl B.: Photosystem II assembly: from cyanobacteria to plants.–Annu. Rev. Plant Biol. 64: 609–635, 2013.CrossRefPubMedGoogle Scholar
  19. Ozawa S., Onishi T., Takahashi Y.: Identification and characterization of an assembly intermediate subcomplex of photosystem I in the green alga Chlamydomonas reinhardtii.–J. Biol. Chem. 285: 20072–20079, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Prodohl A., Ambill M., El-Mohsnawy E. et al.: Modular device for hydrogen production: Optimization of (individual) components.–In: Miyake J., Igarashi Y., Rogner M. (ed.): Biohydrogen III: Renewable Energy System by Biological Solar Energy Conversion. Pp. 171–179. Elsevier Science, Amsterdam 2004.CrossRefGoogle Scholar
  21. Shen G.Z., Boussiba S., Vermaas W.F.J.: Synechocystis sp. PCC 6803 strains lacking photosystem I and phycobilisome function.–Plant Cell 5: 1853–1863, 1993.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Tang H.D., Chitnis P.R.: Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I.–Indian J. Biochem. Bio. 37: 433–440, 2000.Google Scholar
  23. Watanabe M., Kubota H., Wada H. et al.: Novel supercomplex organization of photosystem I in Anabaena and Cyanophora paradoxa.–Plant Cell Physiol. 52: 162–168, 20CrossRefPubMedGoogle Scholar
  24. Wellburn A.R.: The spectral determination of chlorophyll a and chlorophhyll b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution.–J. Plant Physiol. 144: 307–313, 1994.CrossRefGoogle Scholar
  25. Witt I., Witt H.T., Gerken S. et al.: Crystallization of reaction center I of photosynthesis. Low-concentration crystallization of photoactive protein complexes from the cyanobacterium Synechococcus sp.–FEBS Lett. 221: 260–264, 1987.CrossRefGoogle Scholar
  26. Wollman F.A., Minai L., Nechushtai R.: The biogenesis and assembly of photosynthetic proteins in thylakoid membranes.–BBA-Bioenergetics 1411: 21–85, 1999.CrossRefPubMedGoogle Scholar

Copyright information

© The Institute of Experimental Botany 2018

Authors and Affiliations

  1. 1.Institute of Microbiology, Centre AlgatechOpatovický mlýnTřeboňCzech Republic

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