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.
Highlights
We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp
70% of the overall Psb27 level in the wild-type strain was detected in the monomeric PSII complex [RCC(1), Fig. 1, left, band 4], approximately 20% was detected in large supercomplexes close to the edge of the gel, and the remaining Psb27 was found in the PSII dimer [RCC(2), band 3] and in a small complex with mobility similar to that of unassembled CP43 or CP47
To help localize Psb27, we exposed wild-type cells for 20 min to high irradiance (2,000 mmol photons m22 s21) in the presence of the protein synthesis inhibitor chloramphenicol to disassemble the large PSII complexes into smaller complexes. This treatment resulted in a partial reduction in the amount of RCC(2) and RCC (1) and increased levels of the PSII monomer lacking CP43 and unassembled forms of CP47 and CP43 (Fig. 1, right)
Summary
To gain more information about the location of Psb within PSII, we analyzed the membrane proteins of several strains of Synechocystis 6803 using twodimensional (2D) blue native (BN)/SDS-PAGE and identified Psb using an antibody raised to Psb from Synechocystis 6803. To help localize Psb, we exposed wild-type cells for 20 min to high irradiance (2,000 mmol photons m22 s21) in the presence of the protein synthesis inhibitor chloramphenicol to disassemble the large PSII complexes into smaller complexes This treatment resulted in a partial reduction in the amount of RCC(2) and RCC (1) and increased levels of the PSII monomer lacking CP43 (designated RC47) and unassembled forms of CP47 and CP43 (Fig. 1, right). To test the physiological importance of Psb for photoprotection and PSII repair, we assessed the sensitivity of a deletion mutant, DPsb (see Supplemental Fig. S9 online), to photoinhibition by following the time course of light-induced inhibition of oxygen evolution in cultures of the wild type and DPsb subjected to white light of 500 mmol photons m22 s21, either in the absence or presence of the protein synthesis inhibitor lincomycin. When we compared the distribution of the different types of PSII complex in the wild type and DPsb using 2D CN/SDS-PAGE, we noticed that DPsb exhibited a higher ratio of RCC(2)/RCC(1) (a value of 1–1.5 compared with 0.5–0.7 in the wild type) and that the level of large CP43-containing complexes was higher in the mutant, whereas unassembled CP43 was more abundant in the wild type (see Supplemental Fig. S10 online)
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