Revisiting the supramolecular organization of photosystem II in Chlamydomonas reinhardtii

Abstract

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-D-maltoside (β-DM) or n-dodecyl-α-D-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.

FIGURE 1.

SDG ultracentrifugation of thylakoid membranes from C. reinhardtii strains. Thylakoids from WT (137C), ΔPSI (ΔPsaA), ΔPSII (Fud7), and ΔCyt b₆f (Fud6) cells grown under dim light conditions (<20 microeinsteins/m²/s) were solubilized with either β-DM or α-DM and subjected to a SDG centrifugation. Thylakoid membranes with 200 μg of Chl were loaded on the gradients. A1, dissociated LHCIIs; A2, PSII core dimer; A3, PSI-LHCI supercomplex; A2′, PSII-LHCII supercomplex.

FIGURE 2.

Immunoblot analysis of SDG fractions. Polypeptides in the SDG fractions from the α-DM-solubilized WT thylakoids were subjected to immunoblotting with antibodies specific to PSII core subunits (D1, CP47, PsbO), minor monomeric LHCIIs (CP26, CP29), major trimeric LHCII (LhcbM6), PSI subunits (PsaA/B, PsaF), and Cyt b₆.

FIGURE 3.

Gel filtration elution profile and polypeptide compositions of the PSII-LHCII supercomplexes in α-DM and β-DM. A, proteins in A2′ band of SDG shown in Fig. 1 were separated by gel filtration on a Superdex 200 PC 3.2/30 column. Elution profiles of PSII-LHCII supercomplexes in α-DM (black) and in β-DM (gray) were recorded by absorption at 280 nm. B, polypeptides in the A2′ fractions shown in Fig. 1 were analyzed by SDS-PAGE stained by Coomassie Brilliant Blue R-250. Samples were normalized to the amount of D1 protein. Type I, LHCII type I (LhcbM3, -4, -6, -8, -9); Type III, LHCII type III (LhcbM2, -7); Type IV, LHCII type IV (LhcbM1).

FIGURE 4.

Electron micrographs of PSII-LHCII supercomplexes in α-DM and β-DM. PSII-LHCII supercomplexes in α-DM (left) and β-DM (right) were loaded on glow-discharged carbon-coated copper grids and negatively stained with 2% uranyl acetate. Proteins are in white. The C₂S₂M₂L₂ particles are circled. Scale bar, 50 nm.

FIGURE 5.

Projection map of the C₂S₂M₂L₂ supercomplex. A, final projection map of C₂S₂M₂L₂ supercomplex at 16.8 Å resolution. B, fitting the C₂S₂M₂L₂ supercomplex projection map with the crystal structures of cyanobacterial dimeric PSII core and pea LHCII. Cyanobacterial dimeric PSII core at 2.9 Å resolution (3) (Protein Data Bank code 3BZ1/2) and pea LHCII at 2.5 Å resolution (6), (Protein Data Bank code 2BHW) are assigned to the C₂S₂M₂L₂ supercomplex. PSII subunits and LHCII are displayed in distinct colors: D1 (blue), D2 (cyan), CP43 (orange), CP47 (pink), LHCII trimer (green), and LHCII monomer (red). All projections are top-down views from the luminal side of the membrane. Scale bar, 10 nm.