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Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy

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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.

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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

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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.

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Correspondence to Cristina Pagliano.

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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)

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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

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