The biogenesis of Photosystem II is a complicated process requiring numerous auxiliary factors to assist in all steps of its assembly. The cyanobacterial protein Ycf39 forms a stress-induced complex with 2 small chlorophyll-binding, High-light-inducible proteins C and D (HliC and HliD), and has been reported to participate in the insertion of chlorophyll molecules into the central D1 subunit of Photosystem II. However, how this process is organized remains unknown. Here, we show that Ycf39 and both HliC and HliD can form distinct complexes with chlorophyll synthase (ChlG) in the model cyanobacterium Synechocystis sp. PCC 6803. We isolated and characterized ChlG complexes from various strains grown under different conditions and provide a mechanistic view of the docking of Ycf39 to ChlG via HliD and the structural role of HliC. In the absence of stress, chlorophyll is produced by the ChlG-HliD2-ChlG complex, which is stabilized by chlorophyll and zeaxanthin molecules bound to the HliD homodimer. The switch to high light leads to stress pressure and greatly elevated synthesis of HliC, resulting in the replacement of HliD homodimers with HliC-HliD heterodimers. Unlike HliD, HliC cannot interact directly with ChlG or Ycf39. Therefore, the original ChlG-HliD2-ChlG complex is converted into a ChlG-HliD-HliC hetero-trimer that presumably binds transiently to Ycf39 and the nascent D1 polypeptide. We speculate that this molecular machinery promotes the delivery of chlorophyll to D1 upon high-light-induced chlorophyll deficiency. The HliD homodimers formed under standard, nonstress growth conditions and attached to ChlG could serve as an emergency chlorophyll reserve.

• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Chlorophyll metabolism   MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Carbon-Oxygen Ligases * metabolism   genetics   MeSH
• Light * MeSH
• Light-Harvesting Protein Complexes MeSH
• Synechocystis * metabolism   radiation effects   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Chlorophyll MeSH
• chlorophyll synthetase MeSH Browser
• Photosystem II Protein Complex * MeSH
• high light-inducible protein, cyanobacteria MeSH Browser
• Carbon-Oxygen Ligases * MeSH
• Light-Harvesting Protein Complexes MeSH

The growth of plants, algae, and cyanobacteria relies on the catalytic activity of the oxygen-evolving PSII complex, which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for PSI in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.

• MeSH
• Chlorophyll metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Cyanobacteria * metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Chlorophyll MeSH
• Photosystem II Protein Complex * MeSH

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

• MeSH
• Chlorophyll metabolism   MeSH
• Pheophytins metabolism   MeSH
• Photosystem I Protein Complex metabolism   MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Pheophytins MeSH
• Photosystem I Protein Complex MeSH
• Photosystem II Protein Complex * MeSH

Assembly of photosystem II (PSII), a water-splitting catalyst in chloroplasts and cyanobacteria, requires numerous auxiliary proteins which promote individual steps of this sequential process and transiently associate with one or more assembly intermediate complexes. In this study, we focussed on the role of a PSII-associated protein encoded by the ssl1498 gene in the cyanobacterium Synechocystis sp. PCC 6803. The N-terminal domain of this protein, which is here called Psb34, is very similar to the N-terminus of HliA/B proteins belonging to a family of high-light-inducible proteins (Hlips). Psb34 was identified in both dimeric and monomeric PSII, as well as in a PSII monomer lacking CP43 and containing Psb28. When FLAG-tagged, the protein is co-purified with these three complexes and with the PSII auxiliary proteins Psb27 and Psb28. However, the preparation also contained the oxygen-evolving enhancers PsbO and PsbV and lacked HliA/B proteins even when isolated from high-light-treated cells. The data suggest that Psb34 competes with HliA/B for the same binding site and that it is one of the components involved in the final conversion of late PSII assembly intermediates into functional PSII complexes, possibly keeping them free of Hlips. Unlike HliA/B, Psb34 does bind to the CP47 assembly module before its incorporation into PSII. Analysis of strains lacking Psb34 indicates that Psb34 mediates the optimal equilibrium of HliA/B binding among individual PSII assembly intermediates containing CP47, allowing Hlip-mediated photoprotection at all stages of PSII assembly.

• Keywords
• CP47, High-light-inducible protein, Photosynthesis, Photosystem II,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 MeSH

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

• Keywords
• chlorophyll, cyanobacteria, cyclase, microevolution, photosynthesis,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bacteriochlorophylls biosynthesis   genetics   MeSH
• Gene Deletion * MeSH
• Oxygenases genetics   metabolism   MeSH
• Prochlorococcus genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacteriochlorophylls MeSH
• Oxygenases MeSH

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Mutation MeSH
• Ribosomes metabolism   MeSH
• Light-Harvesting Protein Complexes genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll MeSH
• Phosphoproteins MeSH
• Photosystem II Protein Complex MeSH
• photosystem II, chlorophyll-binding protein, CP-47 MeSH Browser
• photosystem II, psbH subunit MeSH Browser
• Light-Harvesting Protein Complexes MeSH