Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Kita-ku, Tsushima-naka, Okayama 700-8530, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2010; 285(26):20072-9. DOI: 10.1074/jbc.M109.098954
Source: PubMed


Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4-9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

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    • "These two large subunits form a heterodimer that accounts for half of the molecular mass of the mature PSI complex and then PsaC, together with PsaD and PsaE, is integrated into the stromal side. PsaK and PsaG bind to the PSI core complex after the integration of LHCI in the late step of PSI assembly [47]. The assembly sequence of the other small peripheral and integral subunits remains unknown. "
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    ABSTRACT: Photosystem I, an integral membrane and multi-subunit complex, catalyzes the oxidation of plastocyanin and the reduction of ferredoxin by absorbed light energy. Photosystem I participates in photosynthetic acclimation processes by being involved in cyclic electron transfer and state transitions for sustaining efficient photosynthesis. The photosystem I complex is highly conserved from cyanobacteria to higher plants and contains the light-harvesting complex and the reaction center complex. The assembly of the photosystem I complex is highly complicated and involves the concerted assembly of multiple subunits and hundreds of cofactors. A suite of regulatory factors for the assembly of photosystem I subunits and cofactors have been identified that constitute an integrative network regulating PSI accumulation. This review aims to discuss recent findings in the field relating to how the photosystem I complex is assembled in oxygenic organisms. This article is a Special Issue entitled: Chloroplast biogenesis. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 01/2015; 1847(9). DOI:10.1016/j.bbabio.2014.12.011 · 5.35 Impact Factor
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    • "The protein composition of the PSI complexes purified from WT-His and Fl39 state 2 cells (bands B3 and B4 in the respective gradients in Figure 2a) was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 4a). The results confirm that both preparations contain PSI complexes, as indicated by the presence of PsaA and PsaB (~65 kDa), Lhca (20–27 kDa) and small core subunits (10–20 kDa; Bassi et al., 1992; Ozawa et al., 2010 "
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    ABSTRACT: State transitions represent a photoacclimation process that regulates the light-driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between Photosystem I(PSI) and II(PSII) by shuttling LHCII, the main light-harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas renihardtii where it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in state1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state2 is able to bind two LHCII trimers containing all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI-bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type II and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2. This article is protected by copyright. All rights reserved.
    The Plant Journal 02/2014; 78(2). DOI:10.1111/tpj.12459 · 5.97 Impact Factor
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    • "A higher-plant PSI complex seems to exist in a monomeric form made of 18 protein subunits (PsaA/B/C/D/E/F/G/H/I/J/K/L/N/O) including 4 LHCI (Lhca1-4) (see Fig. 7). Among them, PsaG/H/O/N are specific only for higher plants, and PsaO was not identified in the PSI structure revealed by X-ray crystallography [22] because PsaO is easily released [23]. LHCI subunits were bound to the PSI core at the side locations opposite to the binding sites of PsaH/L subunits. "
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    ABSTRACT: Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin + diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000 μmol mg Chl a− 1 · h− 1 to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17 K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.
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