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. 2018 Oct;178(2):583-595.
doi: 10.1104/pp.18.00749. Epub 2018 Aug 20.

Configuration of Ten Light-Harvesting Chlorophyll a/ b Complex I Subunits in Chlamydomonas reinhardtii Photosystem I

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Configuration of Ten Light-Harvesting Chlorophyll a/ b Complex I Subunits in Chlamydomonas reinhardtii Photosystem I

Shin-Ichiro Ozawa et al. Plant Physiol. .
Free PMC article

Abstract

In plants, the photosystem I (PSI) core complex stably associates with its light-harvesting chlorophyll a/b complex I (LHCI) to form the PSI-LHCI supercomplex. The vascular plant PSI core complex associates with four distinct LHCI subunits, whereas that of the green alga Chlamydomonas reinhardtii binds nine distinct LHCI subunits (LHCA1-LHCA9). The stoichiometry and configuration of these LHCI subunits in the PSI-LHCI supercomplex of C. reinhardtii remain controversial. Here, we determined the stoichiometry of the nine distinct LHCI subunits relative to PSI subunits through uniform labeling of total proteins using 14C. We separated the nine LHCI polypeptides by three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Our data revealed that the PSI-LHCI supercomplex contains two LHCA1 proteins and one of each of the other eight LHCI subunits. Subsequently, we identified their cross-linked products by immunodetection and mass spectrometry to determine the configuration of the 10 LHCI subunits within the PSI-LHCI supercomplex. Furthermore, analyses of PSI-LHCI complexes isolated from ΔLHCA2 and ΔLHCA5 mutants and oligomeric LHCI from a PSI-deficient (ΔpsaA/B) mutant provided supporting evidence for the LHCI subunit configuration. In conclusion, eight LHCI subunits bind to the PSI core at the site of PSAF subunit in two layers: LHCA1-LHCA8-LHCA7-LHCA3 from PSAG to PSAK, in the inner layer, and LHCA1-LHCA4-LHCA6-LHCA5 in the outer layer. The other two LHCI subunits, LHCA2 and LHCA9, bind PSAB between PSAG and PSAH, PSAG-LHCA9-LHCA2-PSAH. Our study provides new insights into the LHCI configuration linked to the PSI core.

Figures

Figure 1.
Figure 1.
Autoradiograms of 14C-labeled PSI-LHCI polypeptides. A, Lane a, Separation by High-Molarity-Tris SDS-PAGE at 55°C. LHCA2 was detected in LHCA7 and LHCA8 bands, indicated as LHCA7/2 and LHCA8/2, respectively. LHCA1 and LHCA9 were detected in the same band, indicated as LHCA1+9. Lane b, Separation by High-Molarity-Tris SDS-PAGE at 6°C. LHCA1 and LHCA9 were separated as two distinct bands. LHCA3 and LHCA5 as well as LHCA7 and LHCA2 were detected in the same bands, indicated as LHCA3+5 and LHCA7+2, respectively. B, Separation by 2D-SDS-PAGE. The separation profile by the MES-Tris system is shown to the right of the 2D profile.
Figure 2.
Figure 2.
Immunochemical identification of cross-linked products of PSI and LHCI subunits. Cross-linked products of PSI and LHCI subunits were separated and identified by immunoblotting using a battery of antibodies against each of the PSI and LHCI subunits. Two nitrocellulose membrane strips were incubated separately with each of the two antibodies indicated at the top of each pair of gels. Polypeptides before (−) and after (+) chemical cross-linking were subjected to SDS-PAGE. The chemical cross-linkers used were DSG (A and B), DSS (C–K), EDC (L–N), and DMS (O and P), as summarized in Table 2. Black arrowheads indicate cross-linked products, whereas white arrowheads correspond to non-cross-linked products. The cross-linked product identification numbers are as shown in Table 2. The bands assigned as 13 and 14 correspond to the cross-linked products of LHCA7/PSAN and LHCA8/PSAN, respectively.
Figure 3.
Figure 3.
LHCA2 position at the PSAH side of the PSI core. The PSI-LHCI structure from vascular plants (protein database identifier 4Y28) is shown as an overview (A) and a closeup of the cross-linked region (B), as viewed from the stromal side. The PSI core is depicted in gray, except PSAB (green) and PSAH (blue). The single light-harvesting layer of vascular plants is depicted in dark yellow, except for LHCA2 (red). Modeled C. reinhardtii LHCA2 (light blue) at the PSAH side of the core is in a position where the cross-link distance constraints are fulfilled (for details, see “Materials and Methods: Mapping Cross-Links to Molecular Structure”). The cross-linked peptide combinations marked with I, II, and III in Supplemental Table S2 are visualized as three different cross-links: LHCA2 with PSAB (I and II) and LHCA2 with PSAH (III). Cross-linked amino acids are highlighted in yellow, as well as their connection through the cross-linker. The cross-links were identified from LC-MS/MS data with Crosslinx.
Figure 4.
Figure 4.
A model of the LHCI subunit configuration in PSI-LHCI. The configuration of the subunits of PSI (green) and LHCI in PSI-LHCI is viewed from the stromal side. Outlines of subunits on the lumenal side are shown with dotted lines (PSAN and PSAH). The stromal extrinsic subunits, PSAC, PSAD, and PSAE, are omitted. The locations of the second copy of LHCA1 and PSAO are drawn with dashed lines. Letters and numbers represent the names of PSI and LHCI subunits, respectively.
Figure 5.
Figure 5.
LHCI and PSI proteins of ΔLHCA2 and ΔLHCA5 mutants. Total cellular proteins (cell; 1 μg of Chl), thylakoid proteins (TK; 0.5 μg of Chl), and PSI-LHCI proteins (PSI) from control (C), ΔLHCA2 (2), and ΔLHCA5 (5) strains were separated and detected immunochemically with antibodies against LHCI subunits and PSI subunits. The loading amount for the PSI-LHCI preparations was normalized to the signal intensity of PSAD. Although faint signals were detected by anti-LHCA2 antibody in proteins from the cell, TK, and PSI from the ΔLHCA2 mutant, they are ascribed to a cross reaction with LHCA8, which comigrates with LHCA2 under this electrophoretic condition.

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