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. 2018 Jun 22;9(1):2439.
doi: 10.1038/s41467-018-04823-3.

The Photosystem I Assembly Apparatus Consisting of Ycf3-Y3IP1 and Ycf4 Modules

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Free PMC article

The Photosystem I Assembly Apparatus Consisting of Ycf3-Y3IP1 and Ycf4 Modules

Sreedhar Nellaepalli et al. Nat Commun. .
Free PMC article

Abstract

In oxygenic photosynthesis, light energy is converted into redox energy by two photosystems (PSI and PSII). PSI forms one of the largest multiprotein complexes in thylakoid membranes consisting of a core complex, peripheral light-harvesting complexes (LHCIs) and cofactors. Although the high-resolution structure of the PSI-LHCI complex has been determined, the assembly process remains unclear due to the rapid nature of the assembly process. Here we show that two conserved chloroplast-encoded auxiliary factors, Ycf3 and Ycf4, form modules that mediate PSI assembly. The first module consists of the tetratricopeptide repeat protein Ycf3 and its interacting partner, Y3IP1, and mainly facilitates the assembly of reaction center subunits. The second module consists of oligomeric Ycf4 and facilitates the integration of peripheral PSI subunits and LHCIs into the PSI reaction center subcomplex. We reveal that these two modules are major mediators of the PSI-LHCI assembly process.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Ycf3–Y3IP1 transiently associates with newly synthesized PSI reaction center subunits. a Physical map of the vector containing ycf3-HA expression cassette inserted at the BamHI site downstream of the psbA gene, which consists of five exons (E1–E5), is shown. The host strain, Fud7, has an 8.2 kbp deletion starting from the downstream of the E1. The ycf3 cassette contains the coding sequence of ycf3 and HA tag flanked by P/5′-UTR of psaA and 3′-UTR of rbcL. Ycf3-HA contains HA tag inserted at C-terminus of Ycf3. E, X, and B depict restriction sites for EcoRI, XhoI, and BamHI, respectively. P9/P10 and P11/P12 represent pairs of primers used for PCR (Supplementary Fig. 1a). b The expression of Ycf3-HA was confirmed by immunoblotting. Total cellular proteins from control, ycf3-HA (clones 1–3), and ycf3-HA/Δycf3 mutants grown under photoheterotrophic condition were analyzed. The nitrocellulose membrane was stained with Ponceau for loading control. c Polypeptide compositions of the affinity-purified preparations from ycf3-HA and WT strains. Polypeptides separated by SDS-PAGE were visualized by staining with the fluorescence dye, Flamingo. Ycf3-HA and Y3IP1 were detected in the Ycf3-HA preparation. d Immunoblotting with antibodies against Ycf3, Y3IP1, Ycf4, and PSI polypeptides (PsaA, PsaC, PSAD, and PSAF). The purified PSI–LHCI was loaded as a reference. e Pulse-chase labeling of proteins with 35S. Cells were labeled for 10 min (P) and then were chased for 6 h (C). TM: the purified thylakoid membranes, Ycf3-HA: the affinity-purified Ycf3-HA
Fig. 2
Fig. 2
Separation of Ycf3-HA preparation on SDG ultracentrifugation. The purified Ycf3-HA was concentrated and subjected to SDG ultracentrifugation, and the resulting gradient was fractionated and was analyzed by immunoblot using anti-PsaA, Y3IP1, Ycf4, Ycf3, and PSAF antibodies. Ycf3 and Y3IP1 were fractionated in the upper region of the gradient, whereas PsaA and Ycf4 were detected at positions around 700 kDa. Separation of chlorophyll–protein complexes from the WT thylakoid extracts was shown on the top as a reference to show apparent molecular sizes on the gradient. LHCII-T: trimeric LHCII, LHCI-M: monomeric LHCII
Fig. 3
Fig. 3
Affinity purification of Y3IP1-HA. a ΔY3IP1/CGL59 mutant (LMJ.RY0402.195677) from the CLiP contains the paromomycin resistance cassette (CIB1) inserted at the 7th Exon (E7) of CGL59/Y3IP1 (Cre06.g280650). Exons, introns, and untranslated regions are shown as red boxes, black lines, and blue boxes, respectively. This strain also contains second CIB1 cassette in CAH2 locus (Cre04.g223050). The mutant was complemented with cDNA of Y3IP1 (c-Y3IP1) or Y3IP1-HA (c-Y3IP1-HA). b The growth of CLiP host strain as control, ΔY3IP1, and three c-Y3IP1 clones (2, 3, and 4) and c-Y3IP1-HA clones (1,3, and 9), under the photoautotrophic condition in the light of 50 μmol photons m−2 s−1. c Total cell proteins from control, ΔY3IP1, c-Y3IP1, and c-Y3IP1-HA strains were analyzed by immunoblotting using antibodies against Y3IP1, Ycf3, PsaA, and PSAL. The nitrocellulose membrane was stained with Ponceau for loading control. d The polypeptide composition of the affinity-purified Y3IP1-HA and Ycf3-HA. Polypeptides separated by SDS-PAGE were visualized by staining with Flamingo. e Immunoblotting of the purified Y3IP1-HA and Ycf3-HA with antibodies against PsaA and Ycf4
Fig. 4
Fig. 4
HA-Ycf4 transiently associates with newly synthesized PSI core subunits. a Physical map of the vector containing HA-ycf4 expression cassette inserted at the BamHI site downstream of the psbA gene is shown. The ycf4 cassette contains the coding sequence of HA tag and ycf4 flanked by P/5′-UTR of psaA and 3′-UTR of rbcL. HA-Ycf4 contains HA tag inserted at N-terminus of Ycf4. E, X, and B depict restriction sites for EcoRI, XhoI, and BamHI, respectively. P9/P10 and P11/P12 represent pairs of primers used for PCR (Supplementary Fig. 3a). b The expression of HA-Ycf4 was confirmed by immunoblotting. Total cellular proteins from control, HA-ycf4 (clones 1–3), and HA-ycf4/Δycf4 (clones 1–3) mutants grown under photoheterotrophic condition were analyzed. The nitrocellulose membrane was stained with Ponceau for loading control. c Polypeptide compositions of the affinity-purified preparations from HA-ycf4 and WT strains. The purified PSI–LHCI was loaded as a reference. Polypeptides separated by SDS-PAGE were visualized by staining with Flamingo. d Polypeptide compositions detected by immunoblotting. e Estimation of enrichment of COP2 in the HA-Ycf4 preparation. Dilution series of thylakoid membranes (TM) corresponding to 1.0, 0.5, and 0.25 μg Chls per lane were loaded to estimate the abundance of D1, COP2, HA-Ycf4, and PSAD in the HA-Ycf4 preparation by immunoblotting. f Pulse-chase labeling of proteins with 35S. Cells were pulse-labeled for 10 min (P) and then were chased for 6 h (C). TM; the purified thylakoid membranes, HA-Ycf4; the affinity-purified HA-Ycf4 preparation
Fig. 5
Fig. 5
Ycf4 oligomer assists the assembly of PSI core and PSI–LHCI subcomplexes. a SDG ultracentrifugation separated three chlorophyll–protein complexes, A1, A2, and A3, from WT thylakoid membrane extracts (TM) while two PSI assembly intermediates, Y1 and Y2, from the HA-Ycf4 preparation (see also Supplementary Fig. 6). Y1 and Y2 are green, whereas Y3 is colorless. The positions of Y1 and Y2 correspond to those of A2 (PSII core) and A3 (PSI–LHCI), respectively. b Polypeptides of Y1, Y2, and Y3, after concentration, were separated by SDS-PAGE and stained with Flamingo. c Immunoblotting of Y1, Y2, and Y3 with antibodies against Ycf3, Ycf4, and various PSI proteins. d Immunoblotting of Y1, Y2, and Y3 with antibodies against nine LHCI proteins. Red frames indicate the polypeptides that are present in PSI–LHCI but absent in Y1 and Y2
Fig. 6
Fig. 6
Proposed model of PSI complex assembly. Newly synthesized Ycf3 associates with stable Y3IP1 to form a Ycf3–Y3IP1 module in the thylakoid membranes (stage I). This module mediates the assembly of newly synthesized PsaB and PsaA into a reaction center (RC) heterodimer (stage II). PPD1and PSA2 may assist PSI RC assembly from the lumenal side, whereas CGL71/PYG7 on the stromal side together with PSA3 may assists oxygen sensitive assembly steps. Oligomeric Ycf4 module stabilizes newly synthesized PSI RC subcomplex (stage II) and subsequently facilitates the integration of other PSI core subunits, i.e., PsaC, PSAD-F, H, I, J, L to the PSI RC subcomplex to form a PSI core subcomplex (stage III). Seven pre-existing LHCAs (except for LHCA2/9) are integrated to the core subcomplex to form a PSI–LHCI subcomplex on the Ycf4 oligomeric module (stage IV). Upon the integration of PSAG/K and LHCA2/9 to the PSI–LHCI subcomplex, the resulting mature PSI–LHCI supercomplex is released from the assembly apparatus (stage V). Y3IP1 and Ycf4 are reused for the subsequent PSI complex assembly while Ycf3 is replaced with newly synthesized one

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