Báo cáo khoa học: Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions doc

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions Joanna Kargul 1 , Maria V. Turkina 2 , Jon Nield 1 , Sam Benson 1 , Alexander V. Vener 2 and James Barber 1 1 Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK 2 Division of Cell Biology, Linko ¨ ping University, Sweden Excitation of the membrane-bound protein complexes photosystem I (PSI) and II (PSII) by light must be optimized to ensure the highest efficiency of photosyn- thetic electron transport. Redistribution of excitation energy between both photosystems as an immediate and dynamic response to changing illumination condi- tions occurs during the process termed ‘State transi- tions’, where State 1 is induced by excess PSI light and State 2 by excess PSII light [1]. State 1 to State 2 transition occurs in response to the reduction of the plastoquinone pool, triggering the activation of thyla- koid-bound kinases which in turn phosphorylate the mobile light-harvesting complex II (LHCII) antenna [2–5]. The phosphorylated LHCII is proposed to transfer physically from PSII to PSI to balance energy distribution between, and optimize the rate of electron transfer through, the two photosystems or induce cyclic electron flow around PSI [6–9]. Conversely, in PSI-favouring light, oxidation of plastoquinone occurs, leading to deactivation of LHCII-specific kinases and dephosphorylation of mobile LHCII by redox-inde- pendent phosphatases. As a consequence, LHCII deta- ches from PSI and functionally couples to PSII (State 2 to State 1 transition). Recent studies of the mutants that were blocked in State 1 revealed that thylakoid protein kinase Stt7 from green alga Chlamydomonas reinhardtii and its higher plant orthologue STN7 are required for phosphorylation of several LHCII poly- peptides [4,5], thus providing further evidence that pro- tein phosphorylation is essential for State transitions. Keywords Chlamydomonas; CP29; photosynthesis; protein phosphorylation; State transitions Correspondence J. Barber, Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK Fax: +44 20 7594 5267 Tel: +44 20 7594 5266 E-mail: j.barber@imperial.ac.uk (Received 17 June 2005, revised 29 July 2005, accepted 2 August 2005) doi:10.1111/j.1742-4658.2005.04894.x The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light- harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1- exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI–PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox- dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems. Abbreviations Chl, chlorophyll; DDM, b-dodecyl maltoside; EM, electron microscopy; IMAC, immobilized metal affinity chromatography; LHCII, light- harvesting complex II; PSI, photosystem I; PSII, photosystem II; S1 and S2, State 1 and State 2. FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS 4797 Although it is well established that State transitions are driven by the redox control of phosphorylation ⁄ dephosphorylation of a mobile pool of LHCII and that this mobile antenna system shuttles between PSI and PSII [6–9], little is known about the structural changes involved. Lunde et al. [11] showed that the PsaH protein of PSI was important in establishing State 2 and suggested that it could be the docking site for phosphorylated LHCII. This idea was recently reinforced by the 4.4 A ˚ X-ray structure of higher plant PSI [12], in which the PsaH protein was shown to be located at an exposed hydrophobic surface of PSI and to bind a chlorophyll (Chl) molecule which may aid energy transfer from phosphorylated LHCII to the PSI complex. The X-ray structure, however, lacked density indicative of binding of LHCII in this region. Indeed, to date, there is no direct structural evidence of how phosphorylated LHCII binds to PSI, although recent cross-linking and antisense studies have provided some evidence for binding of the LHCII antenna within the PsaH ⁄ I ⁄ O region of the PSI core in State 2 conditions [13,14]. In this study, we set out to characterize the physical association of phosphorylated LHCII to PSI in State 2 using biochemical analyses, electron microscopy and single particle image averaging of LHCI–PSI super- complexes isolated from the green alga C. reinhardtii. Compared with the LHCI–PSI supercomplex isolated from cells in State 1, we found an additional protein density in the isolated State 2 LHCI–PSI supercomplex in the vicinity of the PsaH protein region. This extra density seems to be due to the presence of a 35 kDa phosphoprotein which was shown by MS analyses to be the minor LHCII-like subunit, CP29. MS also revealed that CP29 in thylakoids isolated from algal cells, exposed to either State 2 or State 1 conditions, underwent multiple differential phosphorylation events. Therefore, our data indicate involvement of CP29 phosphorylation in State transitions and suggest that hyperphosphorylated CP29 may provide a functional link between a mobile LHCII antenna and the PSI core in State 2. Results Biochemical characterization of State 1 and State 2 LHCI–PSI supercomplexes It is well established that when Chlamydomonas is sub- jected to anaerobic conditions in the dark, the cells convert from State 1 to State 2 due to over-reduction of the redox pool linking PSI and PSII [10,15]. Using this procedure we were able to establish that this conversion occurs by monitoring their low-temperature Chl emission spectrum and comparing it with that of Chlamydomonas cells in State 1 induced by normal aerobic dark conditions. As shown in Fig. 1, in State 2 the yield of fluorescence from PSI (peaking at 715 nm), which is a measure of its absorption cross- section, was significantly higher than that from PSI of State 1 cells, based on normalization with the fluores- cence from PSII (peaking at 685 nm). This result confirmed the increase of functional light-harvesting antenna in PSI during State 1 to State 2 transition. Using previously optimized sucrose gradient frac- tionation of thylakoid membranes partially depleted from PSII and solubilized with 0.9% b-dodecyl malto- side (DDM) [16], we isolated LHCI–PSI complexes from State 1 (S1) and State 2 (S2)-induced Chlamydo- monas cells. Three Chl-containing fractions were obtained with the densest fractions corresponding to the LHCI–PSI supercomplexes (S1–F3 and S2–F3 frac- tions) [16]. The protein profiles of S1 and S2 thyla- koids (Thy) and also of the S1 ⁄ S2–F3 sucrose-gradient factions (Fig. 2A) were essentially identical (as judged by Coomassie Brilliant Blue staining) and similar to the S1 profiles reported previously [16]. Western blot- ting and spectroscopic analyses of S1–F3 and S2–F3 fractions confirmed the presence of PSI core subunits and the functionally coupled LHCI antenna which Fig. 1. State transitions in C. reinhardtii. We measured 77 K fluor- escence emission spectra measured from the psbD-His cells, induced to State 1 (solid) or State 2 (dotted). Note the resultant rel- ative change in fluorescence of PSI (715 nm) and PSII (685 nm) owing to relative changes in the absorption cross-section of each photosystem. Spectra were obtained from dark-adapted aerated cells (State 1) or from cells preadapted to anaerobic conditions in the dark (State 2). Phospho-CP29 and state transitions J. Kargul et al. 4798 FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS form the outer light-harvesting proteins of PSI (data not shown) [16]. These LHCI proteins are not phos- phorylated in State 1 and indeed, there is no reported evidence that they become phosphorylated in State 2 [17–19]. Nevertheless, as shown in Fig. 2B, significantly increased phosphorylation of several proteins was detected within intact S2 thylakoid membranes by western blotting with antiphosphothreonine serum. In S1 thylakoids, only the 10 kDa phosphoprotein was clearly detected under the conditions used, whereas in S2 thylakoids additional phosphorylated proteins were clearly distinguished in the range of 29–35 kDa (Fig. 2B). The latter proteins correspond to phosphor- ylated CP29, CP26 and unresolved major LHCII antenna polypeptides undergoing phosphorylation in S2 thylakoids [4,10]. In the case of the S2–F3 fraction, a single phosphorylated protein of  35 kDa was spe- cifically detected (arrowed in Fig. 2B), which was not present in the S1–F3 fraction. Although antiphospho- threonine serum readily interacted with the phospho- LHCII solubilized from S2 thylakoid membranes, it was less effective at detecting phospho-CP29 either in the thylakoid membrane or in S2 LHCI–PSI super- complex fractions (Fig. 2B). To identify the 35 kDa phosphoprotein in the S2–F3 fraction, the protein band was excised from the poly- acrylamide gel and digested with trypsin. The peptides were extracted after the procedure of tryptic in-gel digestion and subjected to tandem MS. Collision- induced fragmentation of peptide ions revealed sequences of four peptides ranging in length from 12 to 27 amino acids (Table 1, peptides 1–4). The blast database search [20] identified that all the subsequent peptides originate from the minor LHCII-like subunit CP29. The positions of the sequenced peptides in the sequence of the mature CP29 are indicated in Table 1. These data also confirmed recent findings [21] that the putative transit peptide of the nuclear-encoded CP29 in Chlamydomonas is not removed but processed by methionine excision and acetylation (peptide 1 in Table 1). However, we were unable to detect any phos- phopeptides from CP29, which could be explained by the frequently observed loss of the phosphorylated peptides during the in-gel digestion procedure and the following peptide extraction, as well as the suppressed ionization of the phosphorylated peptides in the pres- ence of nonphosphorylated ones [22]. Importantly, no peptides corresponding to CP29 were detected in the S1–F3 sample subjected to identical tandem MS analy- sis, even though all the proteins present in the region of 25–40 kDa were analysed by in-gel digestion fol- lowed by MS characterization. In order to investigate the status of CP29 phos- phorylation in the algal cells exposed to State 2 condi- tions, we subjected isolated thylakoid membranes to proteolytic ‘shaving’ and enriched the phosphopeptides by immobilized metal affinity chromatography (IMAC) using the procedure described previously [21]. Sequen- cing of the phosphopeptides obtained by nanospray quadrupole time-of-flight MS revealed four distinct phosphorylated peptides from the CP29 protein (Table 1, peptides 5–8). Identification and mapping of the three previously unknown phosphorylation sites in CP29 was achieved A B Fig. 2. Protein composition and phosphorylation of thylakoids and LHCI–PSI obtained from State 1 and State 2 C. reinhardtii cells. (A) Protein profiles of thylakoids (Thy) and LHCI–PSI (F3) complexes obtained from psbD-His State 1- (S1) and State 2 (S2)-induced cells. (B) Phosphory- lation of thylakoids and LHCI–PSI complexes isolated from psbD-His cells. Proteins were separated on SDS ⁄ PAGE at 5 lg of Chl per lane. Detection of phosphoproteins was performed with antiphosphothreonine serum as described previously [16]. Protein size markers are indica- ted on the left. The 35 and 10 kDa phosphobands are marked with arrows in (B). The 35 kDa protein was identified as CP29 by MS. Posi- tions of other proteins were identified by western blotting as in Kargul et al. [16]. J. Kargul et al. Phospho-CP29 and state transitions FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS 4799 by collision-induced dissociation of the corresponding peptide ions and examination of the resultant spectra for the presence of the signals produced by ‘neutral loss’ of phosphoric acid, which are characteristic of phosphorylated peptides [22–24]. Analysis of the spectra for the presence of N- and C-terminal frag- ments that contain phosphate and show neutral loss of phosphoric acid allowed unambiguous localization of the exact phosphorylation sites. The first (Fig. 3A) and second (Fig. 3B) peptides contained three threonine residues each, but only the third N-terminal threonine Table 1. The sequences of tryptic peptides from CP29 revealed by tandem MS. A single-letter amino acid code is used; Ac- designates N-terminal acetylation; the lower case ‘t’and‘s’ specify phosphor- ylated threonine and serine residues, correspondingly. Positions of the peptides in the sequence of the mature CP29 are indicated by corresponding amino acid numbers. The sequences of the peptides 1–4 were obtained after in-gel digestion of the putative phospho- protein from the State 2 LHCI–PSI supercomplex preparation. The sequences of the phosphorylated peptides 5–8 were obtained after phosphopeptide enrichment from State 2 thylakoid membranes (see Experimental procedures). No. Peptide sequence Amino acid numbers 1 Ac-VFKFPTPPGTQK 1–12 2 GFDPLGLSKPSEFVVIGVDENDQNAAK 71–97 3 GSVEAIVQATPDEVSSENR 101–119 4 LAPYSEVFGLAR 120–131 5 Ac-VFKFPtPPGTQK 1–12 6AGtTATKPAPK 14–24 7VAtSTGTR 30–37 8 NNKGsVEAIVQATPDEVSSENR 98–119 m/z 200 600 800 400 A AGtTATKPAPK b 234 5 7 9 1098 76 54 32 1 y Intensity (%) 10 20 40 30 568.7 519.7 y9* y9* 2+ y2 b4* b3 y8 b9* y7 y6 b7* y10* 2+ y4 b5* b2 y1 b3* y3 y5 C NNKGsVEAIVQATPDEVSSENR b 8910 12 13 12 11 10 9 8 6 5 4 2 y m/z Intensity (%) 20 40 60 200 1000 1400600 832.1 y13 y12 y11 y9 b10 799.4 y10 b9 b8 y8 y9 2+ b12 2+ y6 y5 a5 a5* y4 b10* 2+ y2 a5* 2+ B Intensity (%) 20 40 80 60 m/z 200 600 800 400 VAtSTGTR b 23 6 7 7654 3 21 y 443.7 y3 y4 y6* 2+ y7 b3* y1 y6 y7* y6* b7* y5 y5-H 2 0 b6* 394.7 y2-H 2 0 b2 a2 Fig. 3. MS sequencing of three phosphorylated peptides from CP29 in C. reinhardtii cells exposed to State 2 conditions. The b (N-terminal) and y (C-terminal) fragment ions are labelled and the peptide sequences shown. The lower case t and s in the sequences designate phosphorylated Thr and Ser residues, respectively. The sites of phosphorylation were localized according to the pattern of the fragment ions that do not contain phosphate and complimentary ions containing phosphate and satellite signals with the neutral loss of phosphoric acid (b and y ions marked with the asterisk). (A) Frag- mentation spectrum of the doubly protonated peptide ion with m ⁄ z ¼ 568.7. The pronounced doubly charged ion indicated at m ⁄ z ¼ 519.7 corresponds the neutral loss of phosphoric acid from the parent ion (568.7 · 2 ) 519.7 · 2 ¼ 98, which is the mass of H 3 PO 4 ). Thr3 in the peptide is phosphorylated: see, particularly, b3 ion with the phosphate, b3* after the neutral loss of H 3 PO 4 and complementary y8 ion without phosphate. (B) Fragmentation spec- trum of the doubly protonated peptide ion with m ⁄ z ¼ 443.7 (indica- ted). The ion originated after the neutral loss of phosphoric acid is indicated at m ⁄ z ¼ 394.7. Thr3 in the peptide is phosphorylated: see, particularly, y5 ion without phosphate and y6 with the phosphate plus b3* ion after the neutral loss of H 3 PO 4 . (C) Frag- mentation spectrum of the triply protonated phosphopeptide ion with m ⁄ z ¼ 832.1 and corresponding ‘neutral loss’ signal at m ⁄ z ¼ 799.4 (832.1 · 3 ) 799.4 · 3 ¼ 98). The peptide is phosphorylated at Ser5: see y11 to y13 fragments without phosphate and b8 to b10 ions with the phosphate. This pattern of fragment ions can only originate from the peptide in which Ser5 is phosphorylated. Phospho-CP29 and state transitions J. Kargul et al. 4800 FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS in each of these peptides was found to be phosphoryl- ated. These residues correspond to positions 16 and 32 in the sequence of the mature CP29 (Table 1). The third peptide contained one threonine and three serine residues (Fig. 3C). However, the fragmentation spec- trum (Fig. 3C) revealed that only the serine corres- ponding to position 102 in the amino acid sequence of CP29 was phosphorylated. All three newly identified phosphorylation sites are located in the long N-termi- nus of CP29 exposed to the stromal side of thylakoid membranes. These findings (Table 1, Fig. 3) are unique because there is no other report of any thylakoid pro- tein undergoing quadruple phosphorylation. To determine the extent of CP29 phosphorylation in State 1 we performed similar MS analyses of thylakoid membranes isolated from algal cells exposed to State 1 conditions. This study identified only two phosphoryl- ated peptides derived from CP29, which corresponded to phosphorylation of Thr6 and Thr32 (Table 1). The level of both phosphopeptide ions was significantly lower than in samples from the same amount of thyla- koids in State 2, probably accounting for the lack of detection of phospho-CP29 in State 1 thylakoids by antiphosphothreonine blotting (Fig. 2B). However, MS measurements that do not include labelling with stable isotopes are generally not quantitative and the exact levels of CP29 phosphorylation at positions 6 and 32 in State 1 and 2 conditions will be addressed in a sep- arate study. We did not find any phosphorylation of CP29 at residues 16 and 102 in State 1 thylakoid membranes and therefore, we conclude that phos- phorylation of these residues is specific to the State 2 condition. Single particle image averaging of State 1 and State 2 LHCI–PSI supercomplexes Both S1–F3 and S2–F3 sucrose density gradient frac- tions were analysed by electron microscopy of negat- ively stained particles followed by single-particle averaging. In the S1–F3 fraction, the population of the most structurally intact particles (3881 particles) cor- responded to LHCI–PSI supercomplexes described pre- viously for State 1 [16]. In the S2–F3 fraction, a novel population of larger LHCI–PSI supercomplexes (1675 particles) was identified. Top-view projection maps of the LHCI–PSI supercomplex isolated from State 1 and State 2 are compared in Fig. 4. The former (Fig. 4A) has maximum dimensions of 190 · 170 A ˚ (excluding detergent shell), whereas the State 2 supercomplex Fig. 4. Top-view projections of S1 and S2 LHCI–PSI supercomplexes of C. reinhardtii, as viewed from their stromal sides. (A) Pro- jection of State 1 LHCI–PSI, derived from an analysis of negatively stained particles by electron microscopy. (B) Projection of State 2 LHCI–PSI. (C,D) Outline (black) of the pea three-dimensional X-ray model 1qzv.pdb [12] emphasizing the monomeric PSI core and the four LHCI subunits overlaid onto projec- tions of State 1 and State 2 LHCI–PSI, respectively. Scale bar ¼ 50 A ˚ . J. Kargul et al. Phospho-CP29 and state transitions FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS 4801 (Fig. 4B) is larger with maximum dimensions of 190 · 205 A ˚ . This size difference is due to additional protein density (Fig. 4B). To gain further insight into the organization of Chlamydomonas LHCI–PSI super- complexes, the outline of the X-ray map of the recently published higher plant LHCI–PSI [12] was overlaid onto the S1 and S2 LHCI–PSI projections (Fig. 4C,D, respectively) using the crescent-like four- domain LHCI antenna as a visual reference for the fit- ting. In addition to the four Lhca subunits present within the X-ray model of the higher plant LHCI–PSI supercomplex, we were able to identify density which could accommodate two further LHC subunits in the S1 LHCI–PSI supercomplexes (Figs 4C and 5). Import- antly, in the S2 LHCI–PSI particles, the additional density compared with that of higher plant PSI, was larger than for State 1 particles, and the extra density corresponded to that expected for an additional LHC subunit (Fig. 5). As can be seen in Fig. 5, all the extra density was observed in the region adjacent to PsaH (highlighted in white in Fig. 5). Discussion The recent X-ray structure of the higher plant LHCI– PSI supercomplex revealed several unique features of the organization of the LHCI antennae and its bind- ing to the PSI core. First, the number of Lhca pro- teins constituting the higher plant LHCI appears to be lower than previously estimated from biochemical and spectroscopic studies. Four rather than eight Lhca subunits form the light-harvesting belt asymmet- rically located on the PsaG ⁄ J ⁄ K side of the core domain [12]. Second, the LHCI crescent is much more densely populated with Chl molecules than previously estimated, with 56 Chls bound within the peripheral LHCI antenna region and an additional 10 Chls pre- sent in the so-called ‘gap’ region, which are involved in energy transfer from the antenna to the reaction centre [12]. The crystal structure of the higher plant LHCI–PSI supercomplex prompted us to extend the modelling of the Chlamydomonas homologue visualized by electron microscopy [16]. We propose that in Chlamydomonas, the four major Lhca subunits of LHCI form a crescent positioned asymmetrically on the PsaG ⁄ J ⁄ K side of the core complex similar to the higher plant LHCI antenna. However, it is well established that the Chlamydomonas LHCI antenna complex comprises a larger number of Lhca proteins than in higher plants [25–27]. Therefore, as argued previously [16], the addi- tional density detected in the Chlamydomonas LHCI– PSI supercomplex particles from State 1 cells is likely to accommodate extra LHCI antenna subunits which are also retained in the supercomplex isolated from cells placed in State 2. According to modelling using the X-ray structure of higher plant PSI [12], we con- clude that S1 and S2 LHCI–PSI supercomplexes of Chlamydomonas contain six Lhca subunits (Fig. 5, red). The LHCI–PSI supercomplex, isolated from Chlamy- domonas cells in State 2 and present in the S2–F3 fraction of the sucrose density gradient, contained a single phosphoprotein with an apparent molecular mass of  35 kDa (Fig. 2B). Subsequent analyses by tandem MS identified this protein as CP29 whose well- established function is to aid the binding of LHCII to the PSII reaction centre core complex [28,29]. We there- fore suggest that the additional density observed in the S2 LHCI–PSI supercomplex in the vicinity of PsaH is indeed phosphorylated CP29, modelled in blue in Fig. 5 according to the X-ray structure of the LHCII protein [30]. In order to further test the hypothesis that phospho-CP29 plays a role in the binding of phospho- LHCII to facilitate the State 1 to State 2 transition, we Fig. 5. Detailed modelling of the projection map for the LHCI–PSI supercomplex isolated from C. reinhardtii cells placed in State 2. Modelling is based on higher plant coordinates 1qzv.pdb [12] with PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK (magenta), PsaG (purple), PsaI (orange), PsaL (cyan) and PsaH (white). The additional density observed in State 2 LHCI–PSI super- complex which is able to accommodate an additional LHC subunit is coloured blue and is suggested to be phospho-CP29 (see text). Chlorophylls are shown in yellow, but were excluded from the addi- tional density attributed to the LHCI and CP29 subunits. The deter- gent shell surrounding the particles in the hydrophobic membrane plane sits within any stain present and this shell is assigned here as an  15 A ˚ wide outer contour (yellow). Scale bar ¼ 50 A ˚ . Phospho-CP29 and state transitions J. Kargul et al. 4802 FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS conducted studies on a mutant of Chlamydomonas gen- erated by dsRNA antisense technology having an unde- tectable level of CP29 (A. Kanno and J. Minagawa, unpublished observations). We found that although this mutant was highly unstable with regards to CP29 suppression (experiments are currently being conducted to stabilize inhibition of CP29 expression; A. Kanno and J. Minagawa, unpublished observations), a prelim- inary mutant line depleted of CP29 did not contain the 35 kDa phosphoprotein in the purified State 2 LHCI– PSI complex even though thylakoid membranes from which it was isolated contained several phosphopro- teins including major LHCII (J. Kargul, J. Nield, S. Benson, A. Kanno, M. Turkina, A. Vener, J. Mina- gawa & J. Barber, unpublished observations). Conco- mitant with this finding, electron microscopy and single-particle analysis showed that density attributed to phospho-CP29 was absent in the LHCI–PSI particles isolated from the State 2-induced CP29 mutant cells (J. Kargul, J. Nield, S. Benson, A. Kanno, M. Turkina, A. Vener, J. Minagawa and J. Barber, unpublished observations). Although it is known that CP29 can undergo revers- ible N-terminal phosphorylation [21,31,32], it has not previously been shown to bind to PSI or be implicated with State transitions. CP29 in Chlamydomonas is unique because it is the only nuclear-encoded thyla- koid protein in which the transit chloroplast-targeting peptide is not removed but processed by excision of the N-terminal methionine, followed by acetylation and phosphorylation of Thr6 [21]. It has been proposed that it is the functional importance of this phosphorylation site which leads to retention of the transit peptide in the mature protein [21]. Importantly, our MS analyses identified three novel phosphorylation sites, in addition to Thr6, within the N-terminal domain of CP29 in Chlamydomonas exposed to State 2 conditions. We also found that phosphorylation of these sites is dynamically regulated by redox conditions in the photosynthetic membranes. Phosphorylation of CP29 in State 2 is more pronounced and two of the newly found modification sites are exclusively phos- phorylated only under conditions associated with the State 1 to State 2 transition. It is feasible that under State 2 conditions, these additional phosphorylations perturb the electrostatic properties of CP29 and trigger a conformational change leading to dissociation of this protein from PSII and its subsequent attachment to PSI. In conclusion, our results suggest that phospho- CP29, possibly in a multiphosphorylated form, strongly associates with PSI in State 2, adjacent to the PsaH protein. The absence of the mobile pool of LHCII in our State 2 LHCI–PSI supercomplex prepar- ation is likely to be a consequence of its weak interac- tion with PSI compared with phospho-CP29, and its displacement following DDM treatment [13]. Our data suggest that the functional role of the phospho-CP29 bound to LHCI–PSI is to act as a docking site for the mobile phospho-LHCII, as depicted in Fig. 6. The extent of LHCII binding to LHCI–PSI will depend on the degree of excitation imbalance between PSI and PSII. Therefore, in Chlamydomonas, it seems that CP29 may functionally couple LHCII to PSI as well as to PSII, with the former occurring under State 2 con- ditions. Previously, we estimated that the LHCI–PSI supercomplex in State 1 binds about 214 Chls [16] and if CP29 binds 14 Chls, as does each monomer of LHCII [30], then CP29 alone would increase the absorption cross section of the State 2 LHCI–PSI Fig. 6. Diagrammatic representation of how phospho-CP29 could tightly associate with PSI in State 2 and therefore facilitate the binding of mobile LHCII in order to regulate the absorption cross-section of PSI. J. Kargul et al. Phospho-CP29 and state transitions FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS 4803 supercomplex by  7% compared with its State 1 counterpart. This increase in antenna size would be enhanced by the functional association of phospho- LHCII, which in the case of Chlamydomonas can be very extensive compared with higher plants [4–6]. Whe- ther hyperphosphorylation of CP29 occurs in higher plants and whether this phosphoprotein associates with PSI in State 2 has yet to be determined. Importantly, one phosphorylation site identified in this study exclu- sively in State 2 thylakoid membranes (Thr16) is fully conserved between higher plant (Arabidopsis and maize) CP29 and its Chlamydomonas counterpart. Experimental procedures Culturing and State transitions C. reinhardtii psbD-His cells [33] were grown to mid-log phase photoheterotrophically using a Tris ⁄ acetate ⁄ phos- phate medium as described previously [16]. The cells were placed in either State 1 by aerobic dark incubation for 2 h or in State 2 by anaerobic dark incubation (bubbling with nitrogen) for 20 min in the presence of 40 mm NaF to inhi- bit phosphatase activity, as described previously [15]. The ability of the cells to carry out State transitions was checked by monitoring room fluorescence yield changes in response to illumination by light preferentially absorbed by PSII, light 2 (Balzer BG18 filter, Milan, Italy) or light pref- erentially absorbed by PSI, light 1 (Schott RG695 filter, Mainz, Germany). The room temperature fluorescence emission was monitored at > 650 nm using a Waltz chloro- phyll fluorimeter (PAM-101; Effeltrich, Germany). State transitions were also monitored by recording chlorophyll fluorescence spectra at 77 K using a Perkin–Elmer LS50 luminescence spectrophotometer (Beaconsfield, UK) with an excitation wavelength of 435 nm. Biochemical isolation and characterization Using a procedure reported previously [16], thylakoid mem- branes were isolated from cells that had been placed in either State 1 or State 2 using the dark aerobic ⁄ anaerobic procedures [15]. In the case of cells in State 2, 40 mm NaF was present in order to prevent dephosphorylation of phos- phoproteins. LHCI–PSI supercomplexes were isolated from thylakoids (0.8 mgÆmL )1 Chl) by solubilization with 0.9% DDM followed by sucrose density gradient centrifugation as detailed in Kargul et al. [16]. This procedure produced three Chl-containing bands, F1–F3, where F3 consists of the LHCI–PSI supercomplex as shown previously [16]. Protein analyses were conducted using SDS ⁄ PAGE, immunoblotting with antiphosphothreonine serum (Zymed Laboratories Inc., South San Francisco, CA, USA) [16] and by tandem MS (see below). Mass spectroscopy For protein identification, the procedures of in-gel digestion and peptide extraction were made as described previously [22]. Phosphorylated peptides were obtained after treatment of the isolated thylakoids by trypsin, conversion of the released peptides to methyl esters by methanolic HCl and following enrichment of the phosphopeptides by IMAC as described earlier [21]. Electrospray ionization tandem MS was performed on a hybrid spectrometer Q-STAR Pulsar I (Applied Biosystems, Foster City, CA, USA) equipped with a nano-electrospray ion source (MDS Protana, Odense, Denmark). Collision-induced dissociation of selected pre- cursor ions was performed with manual control of collision energy during spectrum acquisition. Electron microscopy and densitometry All samples were stained with 2% (w ⁄ v) uranyl acetate and imaged using a Philips CM100 electron microscope (FEI Company, Eindhoven, the Netherlands) at a calibrated mag- nification of · 50 850. Micrographs, which displayed no dis- cernible drift or astigmatism, were digitized using a Leafscan 45 densitometer at a step size of 10 lm and transferred to a networked cluster of Linux-based PC workstations. Image processing The densitometry resulted in a sampling frequency of 1.97 A ˚ per pixel on the specimen scale. All subsequent pro- cessing was performed using the imagic-v software environ- ment [34,35]. The first minima of the micrographs’ power spectra were measured to be in the range of 20.5–21.8 A ˚ . No correction was made for the contrast transfer function. Datasets consisting of 10 933 and 5195 particle images for State 1 LHCI–PSI (S1–F3) and State 2 LHCI–PSI (S2–F3) samples, respectively, were compiled by interactively select- ing all possible single particles from the micrographs. Mul- tivariate statistical analyses and reference-free alignments identified a number of subpopulations within each dataset [34,35]. Each of these subpopulations was extracted from the total data set and treated de novo, gaining initial two- dimensional class averages and then iterative refinement fol- lowed in order to obtain improved class averages. Standard molecular modelling programs were used to visualize the protein data bank file, 1qzv.pdb, of the higher plant LHCI– PSI structure [12]. The views obtained were subsequently overlaid onto the improved two-dimensional class averages by visual inspection. Acknowledgements We thank Jun Minagawa (Hokkaido University, Japan) for donating the Chlamydomonas psbD-His Phospho-CP29 and state transitions J. Kargul et al. 4804 FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS strain and preliminary CP29 mutant line. For financial support, JB acknowledges the Biotechnology and Bio- logical Sciences Research Council (BBSRC). AV is grateful for support by grants from the Swedish Research Council for Environment, Agriculture and Space Planning (Formas), Nordiskt Kontaktorgan fo ¨ r Jordbruksforskning (NKJ) and Graduate Research School in Genomics and Bioinformatics (FGB). JN is a Royal Society University Research Fellow. References 1 Bonaventura C & Myers J (1969) Fluorescence and oxygen evolution from Chlorella pyrenoidosa. Biochim Biophys Acta 184, 366–383. 2 Horton P & Black MT (1980) Activation of adenosine 5¢ triphosphate-induced quenching of pea chlorophyll fluorescence by reduced plastoquinone. The basis of state I–state II transitions in chloroplasts. FEBS Lett 119, 141–144. 3 Allen JF, Bennett J, Steinback KE & Arntzen CJ (1981) Chloroplast protein phosphorylation couples plastoqui- none redox state to distribution of excitation energy between photosystems. Nature 291, 25–29. 4 Depe ` ge N, Bellafiore S & Rochaix J-D (2003) Role of chloroplast protein kinase STT7 in LHCII phosphoryla- tion and state transition in Chlamydomonas. Science 299, 1572–1575. 5 Bellafiore S, Barneche F, Peltier G & Rochaix J-D (2005) State transitions and light adaptation require chloroplast thylakoid protein kinase STN7. Nature 433, 892–895. 6 Haldrup A, Jensen PE, Lunde C & Scheller HV (2001) Balance of power: a view of the mechanism of photosynthetic state transitions. Trends Plant Sci 6, 301–305. 7 Kruse O (2001) Light-induced short-term adaptation mechanisms under redox control in the PS II–LH CII supercomplex: LHC II state transitions and PS II repair cycle. Naturwissenschaften 88, 284–292. 8 Zer H & Ohad I (2003) Light, redox state, thylakoid- protein phosphorylation and signaling gene expression. Trends Biochem Sci 28, 467–470. 9 Allen JF (2003) State transitions – a question of bal- ance. Science 299, 1530–1532. 10 Fleishmann MM, Ravanel S, Delosme R, Olive J, Zito F, Wollman F-A & Rochaix J-D (1999) Isolation and characterization of photoautotrophic mutants of Chla- mydomonas reinhardtii deficient in state transition. J Biol Chem 274, 30987–30994. 11 Lunde C, Jensen PE, Haldrup A, Knoetzel J & Scheller HV (2000) The PSI-H subunit of photosystem I is essen- tial for state transitions in plant photosynthesis. Nature 408, 613–615. 12 Ben-Shem A, Frolow F & Nelson N (2003) Crystal structure of plant photosystem I. Nature 426, 630–635. 13 Zhang S & Scheller HV (2004) Light-harvesting complex II binds to several small subunits of photosystem I. J Biol Chem 279, 3180–3187. 14 Jensen PE, Haldrup A, Zhang S & Scheller HV (2004) The PSI-O subunit of plant photosystem I is involved in balancing the excitation pressure between the two photosystems. J Biol Chem 279, 24212–24217. 15 Bulte ´ L, Gans P, Rebe ´ ille F & Wollman F-A (1990) ATP control on state transitions in vivo in Chlamydomo- nas reinhardtii. Biochim Biophys Acta 1020, 72–80. 16 Kargul J, Nield J & Barber J (2003) Three-dimensional reconstruction of a light-harvesting complex I–photosys- tem I (LHCI–PSI) supercomplex from the green alga Chlamydomonas reinhardtii. J Biol Chem 278, 16135– 16141. 17 Bennett J (1991) Protein-phosphorylation in green plant chloroplasts. Annu Rev Plant Physiol Plant Mol Biol 42, 281–311. 18 Croce R, Morosinotto T, Castelletti S, Breton J & Bassi R (2002) The Lhca antenna complexes of higher plants photosystem I. Biochim Biophys Acta 1556, 29–40. 19 Storf S, Stauber EJ, Hippler M & Schmid VHR (2004) Proteomic analysis of the photosystem I light-harvesting antenna in tomato (Lycopersicon esculentum). Biochem- istry 43, 9214–9224. 20 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 2, 3389–3402. 21 Turkina MV, Villarejo A & Vener AV (2004) The tran- sit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation. FEBS Lett 564, 104–108. 22 Carlberg I, Hansson M, Kieselbach T, Schroder WP, Andersson B & Vener AV (2003) A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes. Proc Natl Acad Sci USA 100, 757–762. 23 Aro EM, Rokka A & Vener AV (2004) Determination of phosphoproteins in higher plant thylakoids. Methods Mol Biol 274, 271–286. 24 Hunter AP & Games DE (1994) Chromatographic and mass spectrometric methods for the identification of phosphorylation sites in phosphoproteins. Rapid Com- mun Mass Spectrom 8, 559–570. 25 Stauber EJ, Fink A, Markert C, Kruse O, Johan- ningmeier U & Hippler M (2003) Proteomics of Chlamydomonas reinhardtii light-harvesting proteins. Eukaryot Cell 2, 978–994. 26 Tokutsu R, Teramoto H, Takahashi Y, Ono TA & Minagawa J (2004) The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein J. Kargul et al. Phospho-CP29 and state transitions FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS 4805 composition, gene structures and phylogenic implica- tions. Plant Cell Physiol 45, 138–145. 27 Takahashi Y, Yasui TA, Stauber EJ & Hippler M (2004) Comparison of the subunit compositions of the PSI–LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii. Biochemistry 43, 7816–7823. 28 Hankamer B, Barber J & Boekema EJ (1997) Structure and membrane organization of photosystem II in green plants. Annu Rev Plant Physiol Plant Mol Biol 48, 641–671. 29 Yakushevska AE, Keegstra W, Boekema EJ, Dekker JP, Andersson J, Jansson S, Ruban AV & Horton P (2003) The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes. Biochemistry 42, 608–613. 30 Liu Z, Yan H, Wang K, Kuang T-Y, Zhang J, Gui L, An X & Cheng W (2004) Crystal structure of spinach major light-harvesting complex at 2.72 A ˚ resolution. Nature 428, 287–292. 31 Testi MG, Croce R, Polverino-De Laureto P & Bassi R (1996) A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29. FEBS Lett 399, 245–250. 32 Hansson M & Vener AV (2003) Identification of three previously unknown in vivo protein phosphorylation sites in thylakoid membranes of Arabidopsis thaliana. Mol Cell Proteomics 2, 550–559. 33 Sugiura M, Inoue Y & Minagawa J (1998) Rapid and discrete isolation of oxygen-evolving His-tagged photo- system II core complex from Chlamydomonas reinhardtii by Ni 2+ affinity column chromatography. FEBS Lett 426, 140–144. 34 van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M et al. (2000) Single particle electron cryo-microscopy: towards atomic resolution. Q Rev Biophys 33, 307–369. 35 Ruprecht J & Nield J (2001) Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D-reconstruc- tion. Prog Biophys Mol Biol 75, 121–164. Phospho-CP29 and state transitions J. Kargul et al. 4806 FEBS Journal 272 (2005) 4797–4806 ª 2005 FEBS . Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions Joanna Kargul 1 , Maria V. Turkina 2 ,. immobilized metal affinity chromatography; LHCII, light- harvesting complex II; PSI, photosystem I; PSII, photosystem II; S1 and S2, State 1 and State 2. FEBS