Abstract
In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-d-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233–2239; Boekema et al. 1999b, in Eur J Biochem 266:444–452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12–39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.
Similar content being viewed by others
Abbreviations
- ATP-ase:
-
ATP-synthase
- Chl:
-
Chlorophyll
- Cyt:
-
Cytochrome
- 2D:
-
Two-dimensional
- 3D:
-
Three-dimensional
- α-DDM:
-
n-Dodecyl-α-d-maltoside
- HPLC:
-
High-performance liquid chromatography
- LHC:
-
Light-harvesting complex
- lpBN-PAGE:
-
Large-pore blue native polyacrylamide gel electrophoresis
- MS:
-
Mass spectrometry
- OEC:
-
Oxygen-evolving complex
- PS:
-
Photosystem
- RC:
-
Reaction center
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- TEM:
-
Transmission electron microscopy
References
Albertsson PA (2001) A quantitative model of the domain structure of the photosynthetic membrane. Trends Plant Sci 6:349–354
Amunts A, Nelson N (2009) Plant photosystem I design in the light of evolution. Structure 17:37–650
Andersson B, Anderson JM (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach. Biochim Biophys Acta 593:427–440
Arnon DJ (1949) Copper enzymes in isolated chloroplasts, polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–14
Aro EM, Suorsa M, Rokka A, Allahverdiyeva Y, Paakkarinen V, Saleem A, Battchikova N, Rintamaki E (2005) Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes. J Exp Bot 56:347–356
Balsera M, Arellano JB, Revuelta JL, De Las Rivas J, Hermoso JA (2005) The 1.49 Å resolution crystal structure of PsbQ from photosystem II of Spinacia oleracea reveals a PPII structure in the N-terminal region. J Mol Biol 350:1051–1060
Barber J (1982) Influence of surface charges on thylakoid structure and function. Annu Rev Plant Physiol 33:261–295
Barera S, Pagliano C, Pape T, Saracco G, Barber J (2012) Characterization of PSII-LHCII supercomplexes isolated from pea thylakoid membrane by one-step treatment with α- and β-dodecyl-d-maltoside. Philos Trans R Soc Lond B Biol Sci 367:3389–3399
Boekema EJ, van Roon H, Calkoen F, Bassi R, Dekker JP (1999a) Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Biochemistry 38:2233–2239
Boekema EJ, Van Roon H, Van Breemen JF, Dekker JP (1999b) Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Eur J Biochem 266:444–452
Caffarri S, Croce R, Cattivelli L, Bassi R (2004) A look within LHCII: differential analysis of the Lhcb1-3 complexes building the major trimeric antenna complex of higher-plant photosynthesis. Biochemistry 43:9467–9476
Caffarri S, Kouřil R, Kereïche S, Boekema EJ, Croce R (2009) Functional architecture of higher plant photosystem II supercomplexes. EMBO J 28:3052–3063
Calderone V, Trabucco M, Vujicić A, Battistutta R, Giacometti GM, Andreucci F, Barbato R, Zanotti G (2003) Crystal structure of the PsbQ protein of photosystem II from higher plants. EMBO Rep 4:900–905
Daum B, Nicastro D, Austin JII, McIntosh R, Kühlbrandt W (2010) Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell 22:1299–1312
Dekker JP, Boekema EJ (2005) Supramolecular organization of thylakoid membrane proteins in green plants. Biochim Biophys Acta 1706:12–39
Dekker JP, Hassoldt A, Pettersson A, van Roon H, Groot ML, van Grondelle R (1995) On the nature of the F695 and F685 emission of photosystem II. In: Mathis P (ed) Photosynthesis: from light to biosphere. Kluwer Academic Publishers, Dordrecht, pp 53–56
Eshaghi S, Andersson B, Barber J (1999) Isolation of a highly active PSII-LHCII supercomplex from thylakoid membranes by a direct method. FEBS Lett 446:23–26
Gobets B, van Grondelle R (2001) Energy transfer and trapping in photosystem I. Biochim Biophys Acta 1507:80–99
Granvogl B, Reisinger V, Eichacker LA (2006) Mapping the proteome of thylakoid mem- branes by de novo sequencing of intermembrane peptide domains. Proteomics 6:3681–3695
Groth G, Pohl E (2001) The structure of the chloroplast F1-ATPase at 3.2 Å resolution. J Biol Chem 276:1345–1352
Hankamer B, Barber J, Boekema E (1997) Structure and membrane organization of photosystem II in green plants. Annu Rev Plant Physiol Plant Mol Biol 48:641–671
Hankamer B, Morris EP, Nield J, Gerle C, Barber J (2001) Three dimensional structure of the photosystem II core dimer of higher plants determined by electron microscopy. J Struct Biol 135:262–269
Hellmann U, Wernstedt C, Gonez J, Heldin CH (1995) Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem 224:451–455
Hemelrijk PW, Kwa SLS, van Grondelle R, Dekker JP (1992) Spectroscopic properties of LHCII, the main light-harvesting chlorophyll a/b protein complex from chloroplast membranes. Biochim Biophys Acta 1098:159–166
Ido K, Nield J, Fukao Y, Nishimura T, Sato F, Ifuku K (2014) Cross-linking evidence for multiple interactions of the PsbP and PsbQ proteins in a higher plant photosystem II supercomplex. J Biol Chem 289:20150–20157
Ifuku K, Nakatsu T, Kato H, Sato F (2004) Crystal structure of the PsbP protein of photosystem II from Nicotiana tabacum. EMBO Rep 5:362–367
Jackowski G, Kacprzak K, Jansson S (2001) Identification of Lhcb1/Lhcb2/Lhcb3 heterotrimers of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII). Biochim Biophys Acta 1504:340–345
Jansson S (1994) The light-harvesting chlorophyll a/b binding proteins. Biochim Biophys Acta 1184:1–19
Järvi S, Suorsa M, Paakkarinen V, Aro EM (2011) Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes. Biochem J 439:207–214
Kirchhoff H, Hall C, Wood M, Herbstová M, Tsabari O, Nevo R, Charuvi D, Shimoni E, Reich Z (2011) Dynamic control of protein diffusion within the granal thylakoid lumen. Proc Natl Acad Sci USA 108:20248–20253
Kouřil R, Wientjes E, Bultema JB, Croce R, Boekema EJ (2013) High-light vs. low-light: effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana. Biochim Biophys Acta 1827:411–419
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Liu ZF, Yan HC, Wang KB, Kuang TY, Zhang JP, Gui LL, An XM, Chang WR (2004) Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428:287–292
Mullet JE (1983) The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts. J Biol Chem 258:9941–9948
Nanba O, Satoh K (1987) Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559. Proc Natl Acad Sci USA 84:109–112
Nevo R, Chuartzman SG, Tsabari O, Reich Z (2009) Architecture and plasticity of thylakoid membrane networks. In: Wada H, Murata N (eds) Lipids in photosynthesis. Springer, Berlin, pp 295–328
Nield J, Barber J (2006) Refinement of the structural model for the photosystem II supercomplex of higher plants. Biochim Biophys Acta 1757:353–361
Nield J, Orlova EV, Morris EP, Gowen B, van Heel M, Barber J (2000) 3D map of the plant photosystem two supercomplex obtained by cryoelectron microscopy and single particle analysis. Nat Struct Biol 7:44–47
Pagliano C, Barera S, Chimirri F, Saracco G, Barber J (2012) Comparison of the α and β isomeric forms of the detergent n-dodecyl-d-maltoside for solubilizing photosynthetic complexes from pea thylakoid membranes. Biochim Biophys Acta 1817:1506–1515
Pagliano C, Saracco G, Barber J (2013) Structural, functional and auxiliary proteins of photosystem II. Photosynth Res 116:167–188
Pagliano C, Nield J, Marsano F, Pape T, Barera S, Saracco G, Barber J (2014) Proteomic characterization and three-dimensional electron microscopy study of PSII-LHCII supercomplexes from higher plants. Biochim Biophys Acta 1837:1454–1462
Pan X, Li M, Wan T, Wang L, Jia C, Hou Z, Zhao X, Zhang J, Chang W (2011) Structural insights into energy regulation of light-harvesting complex CP29 from spinach. Nat Struct Mol Biol 18:309–316
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612
Rhee KH, Morris EP, Barber J, Kühlbrandt W (1998) Three-dimensional structure of the plant photosystem II reaction centre at 8 Å resolution. Nature 396:283–286
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858
Standfuss J, van Scheltinga ACT, Lamborghini M, Kühlbrandt W (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea light harvesting complex at 2.5 Å resolution. EMBO J 24:918–928
Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, Rees I, Ludtke SJ (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157:38–46
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
van Grondelle R, Dekker JP, Gillbro T, Sundstrom V (1994) Energy transfer and trapping in photosynthesis. Biochim Biophys Acta 1187:1–65
van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116:17–24
van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000) Single-particle electron cryo-microscopy: towards atomic resolution. Q Rev Biophys 33:307–369
Yakushevska AE, Jensen PE, Keegstra W, van Roon H, Scheller HV, Boekema EJ, Dekker JP (2001) Supermolecular organization of photosystem II and its associated light-harvesting antenna in Arabidopsis thaliana. Eur J Biochem 268:6020–6028
Acknowledgments
This work was supported by the Italian Ministry of Education, University and Research, “Futuro in Ricerca 2013” program RBFR1334SB to CP. We kindly thank Dr Alessandro Di Blasio (Politecnico di Torino, Italy) for helping in the glow discharge treatment of grids used for TEM analysis.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
(a) Silver-stained second-dimension SDS-PAGE of the entire lpBN-PAGE lane of band α5. Labels on the left indicate the molecular weight marker positions (Bio-Rad precision plus). (b) Western blot with the antibody against PsbP, performed on the second dimension of the entire lpBN-PAGE lane of band α5 (TIFF 17370 kb)
Fig. S2
The single-particle image classification as a visual whole; the entire 7171 particle dataset (see Table 1) is shown as 600 characteristic views (2D averaged projections), revealing not only the views of the major sub-populations chosen as being representative of the megacomplex observed (see Fig. 6; Table 1), but also the remaining tilted orientations more difficult to ascribe. The scale bar represents 50 nm (TIFF 4147 kb)
Table S1
List of integral PSII core subunits, extrinsic polypeptides, and LHCII proteins identified by LC–MS/MS present in the PSII-LHCII mc region of band α5, shown in Fig. 3c. Table reports: sequences of peptides obtained by LC–MS/MS (second column) with their corresponding precursor ion mass (third column); for each identified protein (first column), the calculated molecular mass (MW, fourth column), the accession number and the database in which the protein was found (fifth column), and the percentage of residue identities with P. sativum, when available, or the homolog Arabidopsis thaliana (sixth column). Underlined amino acid residues (second column) indicate modifications such as carbamidomethylation of cysteine (C), oxidation of methionine (M), deamidation of asparagine, and glutamine (N, Q) (XLSX 13 kb)
Rights and permissions
About this article
Cite this article
Albanese, P., Nield, J., Tabares, J.A.M. et al. Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy. Photosynth Res 130, 19–31 (2016). https://doi.org/10.1007/s11120-016-0216-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-016-0216-3