The Psb27 assembly factor binds to the CP43 complex of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Native gel electrophoresis revealed that Psb27 was present mainly in monomeric PSII core complexes but also in smaller amounts in dimeric PSII core complexes, in large PSII supercomplexes, and in the unassembled protein fraction. We conclude from analysis of assembly mutants and isolated histidine-tagged PSII subcomplexes that Psb27 associates with the "unassembled" CP43 complex, as well as with larger complexes containing CP43, possibly in the vicinity of the large lumenal loop connecting transmembrane helices 5 and 6 of CP43. A functional role for Psb27 in the biogenesis of CP43 is supported by the decreased accumulation and enhanced fragmentation of unassembled CP43 after inactivation of the psb27 gene in a mutant lacking CP47. Unexpectedly, in strains unable to assemble PSII, a small amount of Psb27 comigrated with monomeric and trimeric PSI complexes upon native gel electrophoresis, and Psb27 could be copurified with histidine-tagged PSI isolated from the wild type. Yeast two-hybrid assays suggested an interaction of Psb27 with the PsaB protein of PSI. Pull-down experiments also supported an interaction between CP43 and PSI. Deletion of psb27 did not have drastic effects on PSII assembly and repair but did compromise short-term acclimation to high light. The tentative interaction of Psb27 and CP43 with PSI raises the possibility that PSI might play a previously unrecognized role in the biogenesis/repair of PSII.

Figure 1.

Localization of the Psb27 protein by 2D-BN/SDS-PAGE in membranes of Synechocystis wild-type (WT) cells cultivated under 40 μmol photons m⁻² s⁻¹ and then exposed to 2,000 μmol photons m⁻² s⁻¹ for 20 min in the presence of chloramphenicol (WT 20 min HL + CAP). Membrane proteins were separated by 2D electrophoresis, the gel was either stained by Sypro Orange (Stained gel) or blotted to a PVDF membrane (Blot), and CP43 and Psb27 were detected by specific antibodies. The identity of other designated proteins on the stained gel was verified by MS. Designation of complexes: RCCS1 and RCCS2, PSII supercomplexes; RCC(2) and RCC(1), dimeric and monomeric PSII core complexes, respectively; PSI(3) and PSI(1), trimeric and monomeric PSI; RC47, PSII core complex lacking CP43; u.CP43 and horizontal arrows, unassembled CP43; oblique arrows with numbers, Psb27-containing complexes. Small PSI subunits PsaD and PsaF are also designated by dots.

Figure 2.

Localization of the Psb27 protein in the membrane complexes of CP47-less (A) and CP43-less (B) mutants of Synechocystis. Membrane proteins from psbB deletion mutant, ΔCP47, and psbC deletion mutant, ΔCP43, were separated by 2D PAGE, blotted to a PVDF membrane, and CP43, CP47, D1, PsaD, and Psb27 proteins were detected by specific antibodies. Designation of complexes as in Fig. 1; RC47(1) and RC47(2), monomeric and dimeric form of the PSII core complex lacking CP43, respectively; RC* and RCa, RC complexes lacking both CP47 and CP43; u.CP47 and u.CP43, unassembled CP47 and CP43, u.CP43’ modified unassembled CP43; vertical arrows, Psb27-containing complexes related to PSI; iD1, D1 intermediate. [See online article for color version of this figure.]

Figure 3.

Presence of Psb27 in various PSII complexes isolated using His-tagged CP43 and CP47. PSII complexes CP47-His, CP43-His, RC47-His, and complete core complexes (PSII-His) were isolated by a combination of Ni-metal affinity and size exclusion chromatography. The complexes were analyzed by SDS-PAGE in an 18% gel. Wild-type thylakoid membranes (0.5 μg of Chl a) and final samples of CP47-His, RC47-His, CP43-His, and PSII-His (1 μg of Chl a) were loaded on the gels, and proteins were either stained by Coomassie Blue (Stained gel) or blotted onto a PVDF membrane (Blot) and probed with antibody against T. elongatus Psb27. The arrow designates a CP43 fragment.

Figure 4.

Accumulation of Psb27 in strains differing in the level of unassembled CP43 analyzed by Western blotting. Membranes from the wild type (WT), psbC deletion mutant ΔCP43, and psbB deletion mutant ΔCP47 were analyzed by denaturing SDS-PAGE, and D1 and Psb27 were detected using specific antibodies. Correct protein loading was shown by immunodetection with PsaD-specific antibody. One, 0.5, and 0.25 μg of Chl were loaded onto the gel for each sample.

Figure 5.

Accumulation of CP43 in the psbB deletion mutant ΔCP47 and the double mutant ΔCP47/ΔPsb27 analyzed by 2D BN/SDS-PAGE. Membranes of ΔCP47 (left) and ΔCP47/ΔPsb27 (right) were analyzed by 2D BN/SDS-PAGE in combination with immunoblotting. Top, Sypro Orange-stained gels. Bottom, Corresponding blots obtained using antibodies specific for CP43 and Psb27. Five micrograms of Chl was loaded for each sample. Designation of complexes as in Fig. 1; oblique arrows, fragment of CP43; vertical arrows, CP43-free complexes of Psb27 with PSI complexes; asterisks, cross-reactions of the anti Psb27 antibody; dots, small PSI subunits PsaD and PsaF. [See online article for color version of this figure.]

Figure 6.

Separation of different forms of His-tagged CP43 by 2D-CN/SDS-PAGE (A) and their absorption and 77 K fluorescence spectra (B). A, The CP43-His complex isolated by Ni-metal affinity chromatography was analyzed by 2D CN/SDS-PAGE, and separated proteins were identified by MS. The numbered spots with arrows are not specific to CP43-His and represent CbbL (1) and CbbS (2). The other proteins designated by arrows are specific for CP43-His and represent PSII subunits or their fragments (Table I). FP, Free pigments. B, Room temperature absorption and 77K Chl fluorescence spectra of His-tagged CP43 forms measured directly in green bands excised from the gel. [See online article for color version of this figure.]

Figure 7.

PSII repair (A) and degradation of the PSII proteins (B) in the wild type (WT) and ΔPsb27 strains under high irradiance. A, Autotrophic wild-type cells (left) and ΔPsb27 (right) were illuminated with 500 μmol photons m⁻² s⁻¹ of white light for 180 min without antibiotic (black symbols) or in the presence of 100 μg mL⁻¹ lincomycin (white symbols). During illumination, PSII oxygen-evolving activity was assayed in whole cells. The initial values of oxygen evolution were 672 ± 10 μmol O₂ mg Chl⁻¹ h⁻¹ and 708 ± 60 μmol O₂ mg Chl⁻¹ h⁻¹ for the wild type and ΔPsb27, respectively. B, Autotrophic cells of both strains were pulse-labeled with [³⁵S]Met/Cys, and then the chase of the label was followed at irradiance of 500 μmol photons m⁻² s⁻¹ of white light for 6 h. Thylakoids were isolated and run on an SDS-PAGE gel. The gel was stained (Gel stain), dried, and radioactive labeling of the proteins was visualized using a PhosphorImager (Autorad).

Figure 8.

Growth curves of the Synechocystis wild type (WT) and ΔPsb27 cells under 10 or 100 μmol photons m⁻² s⁻¹. Wild-type cells were grown autotrophically under 40 μmol photons m⁻² s⁻¹, diluted to the initial OD_750nm of 0.005, and transferred to microtitration plates exposed to the given irradiances; plotted values are means of 11 measurements ± sd.