Nejvíce citovaný článek - PubMed ID 24681617
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.
In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.
- Klíčová slova
- CP: Plants, Synechocystis, arginine metabolism, bilins, chlorophyll, genome-uncoupled-4, nitrogen homeostasis, tetrapyrrole biosynthesis,
- MeSH
- arginin metabolismus MeSH
- chlorofyl metabolismus MeSH
- dusík MeSH
- ornithin MeSH
- Synechocystis * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- arginin MeSH
- chlorofyl MeSH
- dusík MeSH
- ornithin MeSH
Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.
- MeSH
- elektronová kryomikroskopie MeSH
- fotosystém II (proteinový komplex) * metabolismus MeSH
- kyslík metabolismus MeSH
- mangan metabolismus MeSH
- sinice * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fotosystém II (proteinový komplex) * MeSH
- kyslík MeSH
- mangan MeSH
FtsH proteases are membrane-embedded proteolytic complexes important for protein quality control and regulation of various physiological processes in bacteria, mitochondria, and chloroplasts. Like most cyanobacteria, the model species Synechocystis sp. PCC 6803 contains four FtsH homologs, FtsH1-FtsH4. FtsH1-FtsH3 form two hetero-oligomeric complexes, FtsH1/3 and FtsH2/3, which play a pivotal role in acclimation to nutrient deficiency and photosystem II quality control, respectively. FtsH4 differs from the other three homologs by the formation of a homo-oligomeric complex, and together with Arabidopsis thaliana AtFtsH7/9 orthologs, it has been assigned to another phylogenetic group of unknown function. Our results exclude the possibility that Synechocystis FtsH4 structurally or functionally substitutes for the missing or non-functional FtsH2 subunit in the FtsH2/3 complex. Instead, we demonstrate that FtsH4 is involved in the biogenesis of photosystem II by dual regulation of high light-inducible proteins (Hlips). FtsH4 positively regulates expression of Hlips shortly after high light exposure but is also responsible for Hlip removal under conditions when their elevated levels are no longer needed. We provide experimental support for Hlips as proteolytic substrates of FtsH4. Fluorescent labeling of FtsH4 enabled us to assess its localization using advanced microscopic techniques. Results show that FtsH4 complexes are concentrated in well-defined membrane regions at the inner and outer periphery of the thylakoid system. Based on the identification of proteins that co-purified with the tagged FtsH4, we speculate that FtsH4 concentrates in special compartments in which the biogenesis of photosynthetic complexes takes place.
- Klíčová slova
- FtsH4, high light-inducible protein, photosystem II biogenesis, proteolysis, thylakoid,
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- chloroplasty metabolismus MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- fylogeneze MeSH
- metaloproteasy genetika metabolismus MeSH
- proteasy MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- Synechocystis * genetika metabolismus MeSH
- tylakoidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fotosystém II (proteinový komplex) MeSH
- FtsH4 protein, Arabidopsis MeSH Prohlížeč
- metaloproteasy MeSH
- proteasy MeSH
- proteiny huseníčku * 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.
- Klíčová slova
- CP47, High-light-inducible protein, Photosynthesis, Photosystem II,
- MeSH
- bakteriální proteiny metabolismus MeSH
- fotosyntéza MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- protein TNFSF14 metabolismus MeSH
- Synechocystis * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- fotosystém II (proteinový komplex) MeSH
- protein TNFSF14 MeSH
High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.
- Klíčová slova
- CP47, Chlorophyll, High-light-inducible proteins, Photosystem II, Synechocystis,
- MeSH
- bakteriální proteiny metabolismus MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- protein TNFSF14 metabolismus MeSH
- světlosběrné proteinové komplexy * metabolismus MeSH
- Synechocystis * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- fotosystém II (proteinový komplex) MeSH
- protein TNFSF14 MeSH
- světlosběrné proteinové komplexy * 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
- chlorofyl metabolismus MeSH
- feofytiny metabolismus MeSH
- fotosystém I (proteinový komplex) metabolismus MeSH
- fotosystém II (proteinový komplex) * metabolismus MeSH
- Synechocystis * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- feofytiny MeSH
- fotosystém I (proteinový komplex) MeSH
- fotosystém II (proteinový komplex) * MeSH
Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.
- MeSH
- chlorofyl metabolismus MeSH
- karotenoidy metabolismus MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- přenos energie MeSH
- proteiny chloroplastové chemie genetika metabolismus MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- sbalování proteinů MeSH
- Synechocystis genetika metabolismus MeSH
- vazba proteinů MeSH
- xanthofyly metabolismus MeSH
- zeaxanthiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorofyl MeSH
- ELIP2 protein, Arabidopsis MeSH Prohlížeč
- karotenoidy MeSH
- light-harvesting-like protein 3, Arabidopsis MeSH Prohlížeč
- proteiny chloroplastové MeSH
- proteiny huseníčku MeSH
- violaxanthin MeSH Prohlížeč
- xanthofyly MeSH
- zeaxanthiny 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.
- Klíčová slova
- chlorophyll, cyanobacteria, cyclase, microevolution, photosynthesis,
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- bakteriochlorofyly biosyntéza genetika MeSH
- delece genu * MeSH
- oxygenasy genetika metabolismus MeSH
- Prochlorococcus genetika metabolismus MeSH
- Synechocystis genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- bakteriochlorofyly MeSH
- oxygenasy MeSH
Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.
- Klíčová slova
- Affinity chromatography, FLAG-tag, Membrane protein complexes, Photosystems, Protein purification, Synechocystis 6803,
- Publikační typ
- časopisecké články MeSH
