Although it is widely recognized that during state transitions a pool of LHCII shuttles between PSII and PSI, direct structural evidence for the physical association of this mobile antenna with PSI has been revealed only recently. During the last 2 years, three important papers have highlighted various possible models for this association. We demonstrated, for the first time, a novel structural role of the Cab/minor light- harvesting subunit, CP29, in State 1-to-State 2 transition in Chlamydomonas . A well-established primary function of the monomeric CP29 protein is to stabilize the binding of the outer antenna LHCII trimers with the PSII reaction centre core complex [61–63]. Through the combination of MS, phosphopeptide mapping, EM and single particle analysis of the LHCI–PSI supercomplexes isolated from the State 2-induced Chlamydomonas cells, we showed that CP29 dissociates from PSII and binds with the core domain of PSI  (see Fig. 2). This redistribution of CP29 was correlated with a quadruple phosporylation of unique Thr and Ser residues at its N-terminal domain . The binding site was in the vicinity of the PsaH subunit (see Fig. 4a), a region previously suggested to bind mobile LHCII during state transitions  (see above, under ‘Docking site for mobile LHCII’). The protein density assigned to phospho-CP29 in the projection map derived from EM and single particle analysis of State 2 LHCI–PSI was absent in the corresponding particles isolated from State 1-induced cells (compare Fig. 2a,d with Fig. 2b,e) [60,64]. We have obtained further structural evidence for the direct binding of CP29 to PSI by EM analysis of the LHCI–PSI particles isolated from the CP29 null mutant of Chlamydomonas induced to State 2 (J Kargul, J Nield, S Benson, A Kanno, J Minagawa & J Barber, unpublished results). When the expression of CP29 is silenced by the interference with double-stranded RNA, the additional protein density in the proximity of PsaH detected in the State 2 wild-type PSI particles is completely missing from the whole population of the LHCI–PSI supercomplex particles analysed (see Fig. 2c,f). Moreover, the mutant depleted of CP29 lacked any detectable 35 kDa phosphor-CP29 in the State 2 LHCI–PSI supercomplex, even though the phosphorylation of major LHCII remained unaffected. Thus, for the first time, direct structural evidence for association of the LHCII-like component with PSI was obtained in support of the previous indirect biochemical, spectroscopic and immunolocalization data. We postulate that under some conditions CP29 acts as a sole monomeric Cab protein that increases the absorption cross-section of PSI. Alternatively, under more extreme State 2 conditions, it provides a linker domain for binding the additional LHCII subunits associated with PSI . This latter possibility is not excluded by our work  because it is likely that such PSI–CP29–LHCII supercomplexes are too labile to be successfully purified by standard fractionation procedures in the presence of the dodecyl-maltoside detergent. Whether hyperphosphorylation of CP29 occurs in higher plants, and whether this phosphoprotein binds to the plant PSI in State 2, remains to be determined, although Tikkanen et al. have recently demonstrated the STN7 kinase-dependent phosphorylation of the Arabidopsis CP29 isoform Lhcb4.2 . Importantly, one phosphorylation site (Thr16) identified in our studies [60,65] under State 2 conditions is fully conserved between higher plant (Arabidopsis and maize) CP29 and its Chlamydomonas orthologue. In plants, the absence of CP29 prevents the assembly of LHCII–PSII supercomplexes , whereas inactivation of other minor light-harvesting components (CP26 and CP24) does not inhibit the formation of a basic structural unit of the LHCII–PSII supercomplex [63,66]. Therefore, it is feasible that in green algae, the large amplitude of state transitions may be, to some extent, caused by a substantial destabilization of the LHCII–PSII supercomplex upon dissociation of hyperphosphorylated CP29, triggering a large increase of the PSI antenna absorption cross-section as a result of the availability of an increased pool of mobile LHCII (see Fig. 3).
Figure 2. Minor Cab protein CP29 associates with LHCI–PSI in State 2. EM top-view projections of State 1 and State 2 LHCI–PSI supercomplexes of wild-type (A, B, D, E) and the CP29-less mutant (C, F) of Chlamydomonas reinhardtii viewed from the stromal side. (A) Projection of wild-type State 1 LHCI–PSI. (B), Projection of wild-type State 2 LHCI–PSI. (C), Projection of LHCI–PSI from the State 2-induced CP29-less mutant. (D–F), Modelling of the projection maps for the LHCI–PSI supercomplex isolated from wild-type (D, E) and CP29-less (F) C. reinhardtii cells placed in State 1 (D) and State 2 (E, F). Modelling is based on higher plant coordinates 1QZV.pdb for the higher plant LHCI–PSI  and 1RWT.pdb for the LHCII monomer . PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK (magenta), PsaG (purple), PsaI (orange), PsaL (cyan) and PsaH (white, arrowed in d and f). Chlorophylls are shown in yellow and for clarity were excluded from LHCI and CP29 subunits. The additional density observed in the State 2 LHCI–PSI supercomplex, which is able to accommodate an additional Cab subunit, is indicated with a white arrow in (B) and coloured in red in (E) and corresponds to phospho-CP29 (see the text). The detergent shell surrounding the particles is assigned as a wide outer contour (yellow) of ∼ 15 Å. Scale bar represents 50 Å. Data in (A) and (B) were taken from Kargul et al. .
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Figure 4. Models for association of the mobile LHCII antenna with type LHCI–PSI supercomplex during state transitions, as derived from EM and X-ray crystallography. (A) EM top-view projection of LHCI–PSI supercomplexes of State 2-induced Chlamydomonas reinhardtii viewed from the stromal side (data taken from Kargul et al. ). Modelling is based on coordinates 1QZV.pdb for the higher plant LHCI–PSI  and 1RWT.pdb  for CP29. The scale bar represents 50 Å. Association of the CP29 minor LHCII protein (blue) with the LHCI–PSI supercomplex close to the PsaH core subunit (white) in State 2-induced thylakoids of Chlamydomonas may provide an anchor for transient binding of mobile LHCII trimer or may be the sole monomeric light-harvesting subunit increasing absorption cross-section of PSI. PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK (magenta), PsaG (purple), PsaI (orange), PsaL (cyan). Chlorophylls are shown in yellow and for clarity were excluded from LHCI and CP29 subunits. (B) Overlay of the EM top-view projection of the Arabidopsis LHCII–LHCI–PSI supercomplex with the X-ray structures of plant LHCI–PSI  and trimeric LHCII  (data taken from Kouril et al. ). Positions of the LHCI subunits Lhca1–4 and the small core subunits are indicated. Additional protein density detected in the vicinity of the PsaH/L/O domain is postulated to accommodate a single LHCII trimer [68,69]. Note the additional protein densities (pink) between the PSI core and the postulated LHCII trimer, which may accommodate additional linker proteins or small core subunits. (C, D) X-ray crystallography-derived modelling of the possible association of the LHCII trimer (coordinates 2BHW.pdb ) with LHCI–PSI (coordinates 2O01.pdb ) under State 2 conditions (images taken from Amunts et al. ). (C), Top view of the putative LHCII–LHCI–PSI supercomplex from the stromal side of the membrane. The proposed LHCII–PSI interaction site viewed from the stromal side and depicted in (D) is formed between LHCII (red) and the PSI core domain composed of PsaH (magenta), PsaL (cyan), PsaA (orange) and PsaK (brown). Note the postulated excitonic coupling between two chlorophyll molecules of the LHCII monomer (blue) and two chlorophyll molecules co-ordinated by the PsaA reaction centre subunit (green).
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Figure 3. Role of CP29 in state transitions and in thermal energy dissipation. (A) Schematic representation of the subunit organization in the Chlamydomonas LHCII–PSII supercomplex taken from Turkina et al. . (B) Proposed mechanism for the regulation of light harvesting in Chlamydomonas by sequential phosphorylation of the CP29 linker protein . The open and closed circles mark the phosphorylation sites identified in cells induced to State 2 or exposed to high irradiance, respectively. Quadruple phosphorylation of CP29 upon State 1-to-State 2 transition causes detachment of phospho-CP29–LHCII from PSII and its docking onto PSI in the vicinity of the PsaH core subunit, as proposed previously [60,65]. High light illumination induces phosphorylation of CP29 at seven residues, leading to the dissociation of phospho-CP29–LHCII from PSII. This detachment may promote thermal energy dissipation within the LHCII trimers. In green plants, phosphorylated TSP9 protein may perform a similar role as a linker subunit, shuttling between PSII and PSI during state transitions, as proposed previously .
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Subsequent to our discovery of the novel function of CP29 in algal state transitions, Takahashi et al. demonstrated, through biochemical analysis, that under State 2 conditions, another minor light-harvesting component, CP26, together with the major LHCII subunit (LhcbM5) and CP29 may also become displaced from PSII and associate with PSI . However, it is important to emphasize that the direct structural evidence for such putative binding of LhcbM5 and CP26 remains to be established, as does the precise mapping of the phosphorylation sites postulated for both proteins based on immunodetection with anti-phosphothreonine serum .
The physical association of the major LHCII trimer with PSI in State 2 has been recently demonstrated in A. thaliana. Kouril et al. used digitonin to solubilize thylakoid membranes in the State 2 conformation followed by EM and single particle analysis of the resultant mildly solubilized protein complexes . This type of analysis allowed, for the first time, visualization of the higher plant LHCI–PSI supercomplex and the associated LHCII trimer. By constructing the 2D projection map of the State 2 PSI supercomplex at 15 Å, the authors propose that a single LHCII trimer binds asymmetrically to the PSI core domain containing PsaH/L/O/P subunits on the PsaA/H/L/K side of the complex [68,69] (Fig. 4b). This interpretation is in line with the crystallographic modelling of Amunts et al. who also postulate binding of a single LHCII trimer on the PsaK side near the PsaH subunit  (see Fig. 4c,d). The LHCII–PSI interaction site is suggested to be composed of PsaH, PsaL, PsaA and PsaK core subunits  (Fig. 4d). Importantly, Amunts et al. emphasize that only one LHCII monomer within the LHCII trimer is likely to be excitonically coupled with the PSI reaction centre under State 2 conditions (Fig. 4d), suggesting that binding of a single LHCII monomer is also feasible in higher plants . It is tempting to speculate whether CP29 is, in fact, the monomeric subunit and therefore functions to anchor the mobile LHCII trimer not only in green alga but also in higher plants. Lunde et al. determined a 33% relative increase of the antenna size of PSI in intact leaves of Arabidopsis upon State 1-to-State 2 transition . Bearing in mind that each LHCII monomer binds 14 chlorophyll molecules , and that the absolute number of chlorophyll molecules functionally associated with LHCI–PSI in State 1 has been measured as ∼ 160–200  and assigned as 168 in the latest 3.4 Å crystal structure of LHCI–PSI , the increase in the antenna size of PSI in State 2 corresponds to one to four LHCII monomers (or one LHCII-like monomer, such as CP29, and a single LHCII trimer). Notably, in the recently modified overlay projection map of the Arabidopsis LHCII–LHCI–PSI supercomplex, Jensen et al. have modelled in two additional protein densities, the largest one being at the interface between the LHCII trimer and the PsaH/L/K side of the PSI core  (see Fig. 4). The identity of these protein densities remains to be established.