Roman Sobotka

PhD
Scientist
Academy of Sciences of the Cze... · Laboratory of Photosynthesis, Trebon

Publications

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    ABSTRACT: Type IV pilins are bacterial proteins that are small in size but have a broad range of functions, including motility, transformation competence and secretion. Although pilins vary in sequence, they possess a characteristic signal peptide that has to be removed by the prepilin peptidase PilD during pilin maturation. We generated a pilD (slr1120) null mutant of the cyanobacterium Synechocystis 6803 that accumulates an unprocessed form of the major pilin PilA1 (pPilA1) and its non-glycosylated derivative (NpPilA1). Notably, the pilD strain had aberrant membrane ultrastructure and did not grow photoautotrophically because the synthesis of Photosystem II subunits was abolished. However, other membrane components such as Photosystem I and ATP synthase were synthesised at levels comparable to the control strain. Proliferation of the pilD strain was rescued by elimination of the pilA1 gene, demonstrating that PilA1 prepilin inhibited the synthesis of Photosystem II. Furthermore, NpPilA1 co-immunoprecipitated with the SecY translocase and the YidC insertase, and both of these essential translocon components were degraded in the mutant. We propose that unprocessed prepilins inactivate an identical pool of translocons that function in the synthesis of both pilins and the core subunits of Photosystem II.
    Molecular Microbiology 07/2014; · 5.03 Impact Factor
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    ABSTRACT: Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.
    The Plant Cell 03/2014; · 9.25 Impact Factor
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    ABSTRACT: Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.
    The Plant Cell 03/2014; · 9.25 Impact Factor
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    ABSTRACT: Chromera velia is an alveolate alga associated with scleractinian corals. Here we present detailed work on chromatic adaptation in C. velia cultured under either blue or red light. Growth of C. velia under red light induced accumulation of a light harvesting antennae complex exhibiting unusual spectroscopic properties with red-shifted absorption and atypical 710 nm fluorescence emission at room temperature. Due to these characteristic features the complex was designated "Red-shifted chromera light harvesting complex" (Red-CLH complex). Its detailed biochemical survey is described in the accompanying paper (Bina et al. 2013, this issue). Here, we show that the accumulation of Red-CLH complex under red light represents a slow acclimation process (days) that is reversible with much faster kinetics (hours) under blue light. This chromatic adaptation allows C. velia to maintain all important parameters of photosynthesis constant under both light colors. We further demonstrated that the C. velia Red-CLH complex is assembled from a 17 kDa antenna protein and is functionally connected to photosystem II as it shows variability of chlorophyll fluorescence. Red-CLH also serves as an additional locus for non-photochemical quenching. Although overall rates of oxygen evolution and carbon fixation were similar for both blue and red light conditions, the presence of Red-CLH in C. velia cells increases the light harvesting potential of photosystem II, which manifested as a doubled oxygen evolution rate at illumination above 695 nm. This data demonstrates a remarkable long-term remodeling of C. velia light-harvesting system according to light quality and suggests physiological significance of 'red' antenna complexes.
    Biochimica et Biophysica Acta 01/2014; 1837:734-743. · 4.66 Impact Factor
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    ABSTRACT: The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulse-field gel electrophoresis and PCR, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function.
    Molecular Biology and Evolution 08/2013; · 14.31 Impact Factor
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    ABSTRACT: Magnesium-protoporphyrin IX monomethylester cyclase is one of the key enzymes of the bacteriochlorophyll biosynthesis pathway. There exist two fundamentally different forms of this enzyme. The oxygen-dependent form, encoded by the gene acsF, catalyzes the formation of the bacteriochlorophyll fifth ring using oxygen whereas the oxygen-independent form encoded by the gene bchE utilizes an oxygen atom extracted from water. The presence of acsF and bchE genes was surveyed in various phototrophic Proteobacteria using the available genomic data and newly designed degenerated primers. It was found that while the majority of purple non-sulfur bacteria contained both forms of the cyclase, the purple sulfur bacteria contained only the oxygen-independent form. All tested species of aerobic anoxygenic phototrophs contained acsF genes but some of them also retained the bchE gene. In contrast to bchE phylogeny, the acsF phylogeny was in good agreement with 16S inferred phylogeny. Moreover, the survey of the genome data documented that the acsF gene occupies a conserved position inside the photosynthesis gene cluster, whereas the bchE location in the genome varied largely between the species. This suggests that the oxygen-dependent cyclase was recruited by purple phototrophic bacteria very early during their evolution. The primary sequence and immunochemical similarity with its cyanobacterial counterparts suggests that acsF may have been acquired by Proteobacteria via horizontal gene transfer from Cyanobacteria. The acquisition of the gene allowed purple non-sulfur phototrophic bacteria to proliferate in the mildly oxygenated conditions of the Proterozoic era.
    Applied and Environmental Microbiology 02/2013; · 3.95 Impact Factor
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    Roman Sobotka
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    ABSTRACT: Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein-protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.
    Photosynthesis Research 02/2013; · 3.15 Impact Factor
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    ABSTRACT: Most photosynthetic eukaryotes synthesize both heme and chlorophyll via a common tetrapyrrole biosynthetic pathway starting from glutamate. This pathway was derived mainly from cyanobacterial predecessor of the plastid and differs from the heme synthesis of the plastid-lacking eukaryotes. Here, we show that the coral-associated alveolate Chromera velia, the closest known photosynthetic relative to Apicomplexa, possesses a tetrapyrrole pathway that is homologous to the unusual pathway of apicomplexan parasites. We also demonstrate that, unlike other eukaryotic phototrophs, Chromera synthesizes chlorophyll from glycine and succinyl-CoA rather than glutamate. Our data shed light on the evolution of the heme biosynthesis in parasitic Apicomplexa and photosynthesis-related biochemical processes in their ancestors.
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    ABSTRACT: Cyanobacteria acclimate to high-light conditions by adjusting photosystem stoichiometry through a decrease of photosystem I (PSI) abundance in thylakoid membranes. As PSI complexes bind the majority of chlorophyll (Chl) in cyanobacterial cells it is accepted that the mechanism controlling PSI level/synthesis is tightly associated with the Chl biosynthetic pathway. However, how Chl is distributed to photosystems under different light conditions remains unknown. Using radioactive labeling by 35S and by 14C combined with native/2D electrophoresis we assessed the synthesis and accumulation of photosynthetic complexes in parallel with the synthesis of Chl in Synechocystis cells acclimated to different light intensities. Although cells acclimated to higher irradiances (150 and 300 μE .m-2.s-1) exhibited markedly reduced PSI content when compared to cells grown at lower irradiances (10 and 40 μE .m-2.s-1) they grew much faster and synthesized significantly more Chl, as well as both photosystems. Interestingly, even under high irradiance almost all labeled de novo Chl was localized in the trimeric PSI, whereas only a weak Chl labeling in PSII was accompanied by the intensive 35S protein labeling, which was much stronger than in PSI. These results suggest that PSII subunits are mostly synthesized using 'recycled' Chl molecules previously released during PSII repair-driven protein degradation. In contrast, most of 'fresh' Chl is utilized for synthesis of PSI complexes likely to maintain a constant level of PSI during cell proliferation.
    Plant physiology 10/2012; · 6.56 Impact Factor
  • Jana Kopečná, Roman Sobotka, Josef Komenda
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    ABSTRACT: In most oxygenic phototrophs, including cyanobacteria, two independent enzymes catalyze the reduction of protochlorophyllide to chlorophyllide, which is the penultimate step in chlorophyll (Chl) biosynthesis. One is light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) and the second type is dark-operative protochlorophyllide oxidoreductase (DPOR). To clarify the roles of both enzymes, we assessed synthesis and accumulation of Chl-binding proteins in mutants of cyanobacterium Synechocystis PCC 6803 that either completely lack LPOR or possess low levels of the active enzyme due to its ectopic regulatable expression. The LPOR-less mutant grew photoautotrophically in moderate light and contained a maximum of 20 % of the wild-type (WT) Chl level. Both Photosystem II (PSII) and Photosystem I (PSI) were reduced to the same degree. Accumulation of PSII was mostly limited by the synthesis of antennae CP43 and especially CP47 as indicated by the accumulation of reaction center assembly complexes. The phenotype of the LPOR-less mutant was comparable to the strain lacking DPOR that also contained <25 % of the wild-type level of PSII and PSI when cultivated under light-activated heterotrophic growth conditions. However, in the latter case, we detected no reaction center assembly complexes, indicating that synthesis was almost completely inhibited for all Chl-proteins, including the D1 and D2 proteins.
    Planta 09/2012; · 3.38 Impact Factor
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    ABSTRACT: A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.
    RNA 08/2012; 18(10):1846-61. · 5.09 Impact Factor
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    ABSTRACT: The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.
    Journal of Biological Chemistry 06/2012; 287(33):27823-33. · 4.65 Impact Factor
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    ABSTRACT: Ycf34 is a hypothetical chloroplast open reading frame that is present in the chloroplast genomes of several non-green algae. Ycf34 homologues are also encoded in all sequenced genomes of cyanobacteria. To evaluate the role of Ycf34 we have constructed and analysed a cyanobacterial mutant strain. Inactivation of ycf34 in Synechocystis sp. PCC 6803 showed no obvious phenotype under normal light intensity growth conditions. However, when the cells were grown under low light intensity they contained less and smaller phycobilisome antennae and showed a strongly retarded growth, suggesting an essential role of the Ycf34 polypeptide under light limiting conditions. Northern blot analysis revealed a very weak expression of the phycocyanin operon in the ycf34 mutant under light limiting growth in contrast to the wild type and to normal light conditions. Oxygen evolution and P(700) measurements showed impaired electron flow between photosystem II and photosystem I under these conditions which suggest that the impaired antenna size is most likely due to a highly reduced plastoquinone pool which triggers regulation on a transcriptional level. Using a FLAG-tagged Ycf34 we found that this protein is tightly bound to the thylakoid membranes. UV-vis and Mössbauer spectroscopy of the recombinant Ycf34 protein demonstrate the presence of an iron-sulphur cluster. Since Ycf34 lacks homology to known iron-sulphur cluster containing proteins, it might constitute a new type of iron-sulphur protein implicated in redox signalling or in optimising the photosynthetic electron transport chain.
    Biochimica et Biophysica Acta 06/2012; 1817(11):2016-26. · 4.66 Impact Factor
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    ABSTRACT: Heme is an iron-coordinated porphyrin that is universally essential as a protein cofactor for fundamental cellular processes, such as electron transport in the respiratory chain, oxidative stress response, or redox reactions in various metabolic pathways. Parasitic kinetoplastid flagellates represent a rare example of organisms that depend on oxidative metabolism but are heme auxotrophs. Here, we show that heme is fully dispensable for the survival of Phytomonas serpens, a plant parasite. Seeking to understand the metabolism of this heme-free eukaryote, we searched for heme-containing proteins in its de novo sequenced genome and examined several cellular processes for which heme has so far been considered indispensable. We found that P. serpens lacks most of the known hemoproteins and does not require heme for electron transport in the respiratory chain, protection against oxidative stress, or desaturation of fatty acids. Although heme is still required for the synthesis of ergosterol, its precursor, lanosterol, is instead incorporated into the membranes of P. serpens grown in the absence of heme. In conclusion, P. serpens is a flagellate with unique metabolic adaptations that allow it to bypass all requirements for heme.
    Proceedings of the National Academy of Sciences 03/2012; 109(10):3808-13. · 9.81 Impact Factor
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    Josef Komenda, Roman Sobotka, Peter J Nixon
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    ABSTRACT: Plants, algae and cyanobacteria grow because of their ability to use sunlight to extract electrons from water. This vital reaction is catalysed by the Photosystem II (PSII) complex, a large multi-subunit pigment-protein complex embedded in the thylakoid membrane. Recent results show that assembly of PSII occurs in a step-wise fashion in defined regions of the membrane system, involves conserved auxiliary factors and is closely coupled to chlorophyll biosynthesis. PSII is also repaired following damage by light. FtsH proteases play an important role in selectively removing damaged proteins from the complex, both in chloroplasts and cyanobacteria, whilst undamaged subunits and pigments are recycled. The chloroplastic Deg proteases play a supplementary role in PSII repair.
    Current opinion in plant biology 02/2012; 15(3):245-51. · 10.33 Impact Factor
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    ABSTRACT: Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates – e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching. Copyright: ß 2012 Kaň a et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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    ABSTRACT: Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates - e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching.
    PLoS ONE 01/2012; 7(1):e29700. · 3.53 Impact Factor
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    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.
    Plant physiology 11/2011; 158(1):476-86. · 6.56 Impact Factor

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