Nejvíce citovaný článek - PubMed ID 11856320
Absence of the psbH gene product destabilizes photosystem II complex and bicarbonate binding on its acceptor side in Synechocystis PCC 6803
Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.
- Klíčová slova
- Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
- MeSH
- chlorofyl a metabolismus MeSH
- chlorofyl metabolismus MeSH
- dimerizace MeSH
- ferrochelatasa chemie metabolismus MeSH
- fylogeneze MeSH
- karotenoidy metabolismus MeSH
- konformace proteinů MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Synechocystis enzymologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl a MeSH
- chlorofyl MeSH
- chlorophyll b MeSH Prohlížeč
- ferrochelatasa MeSH
- karotenoidy MeSH
- světlosběrné proteinové komplexy 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
- 2D gelová elektroforéza MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- chlorofyl metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- mutace MeSH
- ribozomy metabolismus MeSH
- světlosběrné proteinové komplexy genetika metabolismus MeSH
- Synechocystis genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- chlorofyl MeSH
- fosfoproteiny MeSH
- fotosystém II (proteinový komplex) MeSH
- photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
- photosystem II, psbH subunit MeSH Prohlížeč
- světlosběrné proteinové komplexy MeSH
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.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- fotosystém I (proteinový komplex) metabolismus MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- multiproteinové komplexy metabolismus MeSH
- mutace MeSH
- stabilita proteinů MeSH
- Synechocystis metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- fotosystém I (proteinový komplex) MeSH
- fotosystém II (proteinový komplex) MeSH
- multiproteinové komplexy MeSH
- photosystem II, chlorophyll binding protein, CP-43 MeSH Prohlížeč
The role of the Psb28 protein in the structure and function of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. The protein was localized in the membrane fraction and, whereas most of the protein was detected as an unassembled protein, a small portion was found in the PSII core complex lacking the CP43 antenna (RC47). The association of Psb28 with RC47 was further confirmed by preferential isolation of RC47 from the strain containing a histidine-tagged derivative of Psb28 using nickel-affinity chromatography. However, the affinity-purified fraction also contained a small amount of the unassembled PSII inner antenna CP47 bound to Psb28-histidine, indicating a structural relationship between Psb28 and CP47. A psb28 deletion mutant exhibited slower autotrophic growth than wild type, although the absence of Psb28 did not affect the functional properties of PSII. The mutant showed accelerated turnover of the D1 protein, faster PSII repair, and a decrease in the cellular content of PSI. Radioactive labeling revealed a limitation in the synthesis of both CP47 and the PSI subunits PsaA/PsaB in the absence of Psb28. The mutant cells contained a high level of magnesium protoporphyrin IX methylester, a decreased level of protochlorophyllide, and released large quantities of protoporphyrin IX into the medium, indicating inhibition of chlorophyll (Chl) biosynthesis at the cyclization step yielding the isocyclic ring E. Overall, our results show the importance of Psb28 for synthesis of Chls and/or apoproteins of Chl-binding proteins CP47 and PsaA/PsaB.
- MeSH
- bakteriální proteiny fyziologie MeSH
- delece genu MeSH
- fotosystém II (proteinový komplex) biosyntéza genetika metabolismus MeSH
- mutace MeSH
- světlosběrné proteinové komplexy nedostatek metabolismus MeSH
- Synechocystis genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- fotosystém II (proteinový komplex) MeSH
- photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
- světlosběrné proteinové komplexy MeSH
Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.
- MeSH
- buněčná membrána metabolismus MeSH
- chlorofyl chemie metabolismus MeSH
- chloroplasty metabolismus MeSH
- elektronová mikroskopie MeSH
- fenotyp MeSH
- fluorescenční spektrometrie metody MeSH
- fotosyntetická reakční centra (proteinové komplexy) chemie fyziologie MeSH
- fotosyntéza MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- intracelulární signální peptidy a proteiny genetika metabolismus fyziologie MeSH
- lyasy chemie MeSH
- mutace MeSH
- porfyriny chemie MeSH
- sinice metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- transportní proteiny genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- fotosyntetická reakční centra (proteinové komplexy) MeSH
- fotosystém II (proteinový komplex) MeSH
- intracelulární signální peptidy a proteiny MeSH
- lyasy MeSH
- magnesium chelatase MeSH Prohlížeč
- photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
- porfyriny MeSH
- světlosběrné proteinové komplexy MeSH
- transportní proteiny MeSH
The involvement of the PsbI protein in the assembly and repair of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of PSII complexes in the wild-type strain showed that the PsbI protein was present in dimeric and monomeric core complexes, core complexes lacking CP43, and in reaction center complexes containing D1, D2, and cytochrome b-559. In addition, immunoprecipitation experiments and the use of a histidine-tagged derivative of PsbI have revealed the presence in the thylakoid membrane of assembly complexes containing PsbI and either the precursor or mature forms of D1. Analysis of PSII assembly in the psbI deletion mutant and in strains lacking PsbI together with other PSII subunits showed that PsbI was not required for formation of PSII reaction center complexes or core complexes, although levels of unassembled D1 were reduced in its absence. However, loss of PsbI led to a dramatic destabilization of CP43 binding within monomeric and dimeric PSII core complexes. Despite the close structural relationship between D1 and PsbI in the PSII complex, PsbI turned over much slower than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI. Overall, our results suggest that PsbI is an early assembly partner for D1 and that it plays a functional role in stabilizing the binding of CP43 in the PSII holoenzyme.
- MeSH
- bakteriální proteiny metabolismus MeSH
- fotosyntetická reakční centra (proteinové komplexy) metabolismus MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- Synechocystis metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- fotosyntetická reakční centra (proteinové komplexy) MeSH
- fotosystém II (proteinový komplex) MeSH
- photosystem II, chlorophyll binding protein, CP-43 MeSH Prohlížeč
- PsbI protein, cyanobacteria MeSH Prohlížeč
The selective replacement of photodamaged D1 protein within the multisubunit photosystem II (PSII) complex is an important photoprotective mechanism in chloroplasts and cyanobacteria. FtsH proteases are involved at an early stage of D1 degradation, but it remains unclear how the damaged D1 subunit is recognized, degraded, and replaced. To test the role of the N-terminal region of D1 in PSII biogenesis and repair, we have constructed mutants of the cyanobacterium Synechocystis sp PCC 6803 that are truncated at the exposed N terminus. Removal of 5 or 10 residues blocked D1 synthesis, as assessed in radiolabeling experiments, whereas removal of 20 residues restored the ability to assemble oxygen-evolving dimeric PSII complexes but inhibited PSII repair at the level of D1 degradation. Overall, our results identify an important physiological role for the exposed N-terminal tail of D1 at an early step in selective D1 degradation. This finding has important implications for the recognition of damaged D1 and its synchronized replacement by a newly synthesized subunit.
- MeSH
- autotrofní procesy účinky léků účinky záření MeSH
- biologické modely MeSH
- dimerizace MeSH
- fluorescenční spektrometrie MeSH
- fotosystém II (proteinový komplex) chemie metabolismus MeSH
- linkomycin farmakologie MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mutantní proteiny metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- posttranslační úpravy proteinů * účinky léků účinky záření MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- světlo MeSH
- Synechocystis cytologie účinky léků metabolismus účinky záření MeSH
- tylakoidy účinky léků metabolismus účinky záření MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fotosystém II (proteinový komplex) MeSH
- linkomycin MeSH
- mutantní proteiny MeSH
- podjednotky proteinů MeSH
Cells of the psbH deletion mutant IC7 of the cyanobacterium Synechocystis PCC 6803 grown in the absence of glucose contain strongly reduced levels of chlorophyll when compared with cells grown in the presence of glucose, or compared with wild-type (WT) cells. Low-temperature fluorescence emission spectra revealed decreased content of both active PS II (Photosystem II) and PS I (Photosystem I) complexes. Analysis of thylakoid membrane complexes of IC7 by native electrophoresis showed a similar set of chlorophyll-proteins, namely a PS II core complex and trimeric and monomeric PS II complexes, as in WT. However, in contrast to WT, the (35)S-methionine protein labeling pattern of the mutant exhibited no preferential labeling of the D1 protein in the PS II core complexes, and the labeled D1 and D2 proteins accumulated predominantly in the PS II reaction center lacking CP47. The results show that in autotrophically grown cells of the psbH deletion mutant, selective D1 turnover is inhibited and synthesis of CP47 becomes a limiting step in the PS II assembly.
- MeSH
- delece genu * MeSH
- fosfoproteiny nedostatek genetika metabolismus MeSH
- fotosystém II (proteinový komplex) biosyntéza chemie genetika metabolismus MeSH
- glukosa metabolismus MeSH
- světlosběrné proteinové komplexy biosyntéza MeSH
- Synechocystis cytologie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfoproteiny MeSH
- fotosystém II (proteinový komplex) MeSH
- glukosa MeSH
- photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
- photosystem II, psbH subunit MeSH Prohlížeč
- světlosběrné proteinové komplexy MeSH
Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment-protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II.
- Publikační typ
- časopisecké články MeSH