Step 2: Generation of ATP by Electron Transport Chain. Protein extracts for determination of D1 content in wild-type and pratA− cells were prepared according to Schottkowski et al. A binuclear manganese-containing 34 kilodalton protein, a probable component of the water dehydrogenase enzyme, Photoinhibition - A historical perspective, Crystal structure of the oxygen-evolving complex of photosystem II, Molecular identification of an ABC transporter complex for manganese: Analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process, Manganese transport in the cyanobacterium, Assembly of the water-oxidizing complex in photosystem II, The tetratricopeptide repeat: A structural motif mediating protein-protein interactions, Manganese binding to the 23 kDa extrinsic protein of photosystem II, Photoactivation of the manganese catalyst of O 2 evolution. The data show two different binding modes of Mn2+ to PratA, one high-affinity (detail), and several low-affinity binding sites (large graph). Definition of Photosystem I. Photosystem I or PSI is located in the thylakoid membrane and is a multisubunit protein complex found in green plants and algae. To this end, we analyzed Mn2+ transport and uptake into ycf48−, a mutant deficient in YCF48, a factor that had been shown to be involved in early PSII assembly steps and that, like PratA, is a direct D1 interaction partner (Komenda et al., 2008). and shown the codon change leading to modification of the QB-site. These photosystems absorb and utilize the solar energy efficiently in the thylakoid membranes. The energized electrons are replaced by oxidizing water to form hydrogen ions and molecular oxygen. Although the amplitude was not completely restored by the addition of CaCl2, which is likely due to the higher affinity of rPratA for Mn2+ than for Ca2+ (see above), the result suggests that the loss of EPR signal intensity in the presence of rPratA is due to Mn2+ binding rather than to changes in the oxidation state of Mn2+. Based on these data, it is proposed that rPratA specifically binds Mn2+ with a higher affinity than Ca2+ or Mg2+ and that even with 10-fold excess of Ca2+ or Mg2+, the high-affinity binding site seems to be specific for Mn2+, whereas the residual seven or eight Mn2+ ions are loosely attached to the low-affinity binding sites and, hence, can easily be substituted. Therefore, complementing Mn2+ delivery systems to PSII apart from the periplasmic PratA-assisted transport have to exist; alternatively, Mn2+ might reach D1 without the coupling to transport proteins. The entire titration curve was shown to exhibit a sigmoid shape indicative of binding of Mn2+ to multiple sites. when the site is occupied by a semiquinone. We have also explored the mechanism of inhibition (C) to (F) Immunogold-labeled electron microscopy pictures depicting the pD1 localization in the wild type ([C] and [D]) and pratA− ([E] and [F]). The amount of protein-bound Mn2+ was calculated from the signal amplitude of the lowest-field transition. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jörg Nickelsen (joerg.nickelsen{at}lrz.uni-muenchen.de). The beads were washed five times in TMK buffer and analyzed with a scintillation counter (Beckman Coulter). The terminal electron acceptors, iron–sulfur clusters F A and F B , are bound to the PsaC subunit on the stromal (cytoplasmic) side of the thylakoid membrane ( Figure 1B ). To determine binding stoichiometry and affinities of rPratA, Mn2+ titration experiments were performed and the fraction of bound Mn2+ was calculated from the amplitude of the lowest field transition and plotted against total Mn2+ concentrations (Figure 3C). We do not capture any email address. However, upon incubation of wild-type sections with αPratA antibody, PratA could be localized to distinct clusters of ~100 nm in diameter at the cell periphery (Figures 8C and 8D). It is proposed that some of these serve as substrates for the generation of dioxygen (Umena et al., 2011). demonstrated that qE-quenching does not require an active photosystem Data were analyzed by plotting according to Scatchard using SigmaPlot 11 with the Enzyme Kinetics module 1.3. If this value comes close to 1, this means that the clusters adopt on average a more circle-like structure. (A) Control of the wild type without αpD1. A.S. and J.N. Interestingly, in pratA−pD1 was still detected in clusters at the cell periphery, but their number was found to be approximately twofold diminished (Figures 9E and 9F, Table 1). The three-dimensional structure of D1 was visualized with Pymol (http://pymol.sourceforge.net, version 0.99, based on the Protein Data Bank file 3BZ1). strains, and we are constructing others using PCR-based techniques (E) and (F) Transport (E) and uptake (F) of 54Mn2+ in wild-type* (Komenda et al., 2008) and ycf48− cells. purple bacteria Trissl et al. All measurements were performed in an air (13.5 L/min)-acetylene (2 L/min) flame. Based on titration and competition experiments with Ca2+ and Mg2+ ions, we postulate a stoichiometry of 1 Mn2+:1 PratA at this site (Figures 3 and 4). For immunodetection experiments, cryofixed samples were freeze-substituted in acetone containing 0.5% uranyl acetate (Pfeiffer and Krupinska, 2005) for 20 h at −90°C. Describe the third step of the electron transport steps that occur in the thylakoid membrane. When rPratA was incubated with 1 mM Mn2+ prior to the measurement, the α-helical fraction fell from 72% (without Mn2+) to 62%, and a concomitant increase of turns/coils (from 23 to ~30%) was observed (Figures 2A and 2B). Again, both variations did not lead to the recognition of alterations in pratA− compared with the wild type. A closer look at TM convergence sites at the periphery of cells using higher magnifications (110,000- to 140,000-fold) revealed structures of ~60 nm in diameter that are filled by a granular matrix coated with dense material in both wild-type and pratA− sections (Figures 7C and 7D). In pratA−, the Mn2+ transport rate to PSII was found to be strongly reduced; nevertheless, Mn2+ was still incorporated by PSII, allowing photoautotrophic growth of mutant cells although with reduced rates compared with the wild type (Klinkert et al., 2004). using other techniques for studying these reactions, including of photosystem II, including Mn in oxygen evolving preparations, Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. with a aadA spectinomycin resistance cassette. We have pursued our interest in the involvement of protons by The light reaction of photosynthesis. the structure-function relationships in these spans, by using computer modelling of structure, and biophysical analysis of the kinetic and thermodynamic parameters to assay the consequences of mutation, in order to identify contributions of specific residues to the mechanism of catalysis. 73–142, An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for photosystem II biogenesis, The plasma membrane of the cyanobacterium, The use of lead citrate at high pH as an electron-opaque stain in electron microscopy, Generic assignments, strain histories and properties of pure cultures of cyanobacteria, Fluorescence staining of live cyanobacterial cells suggest non-stringent chromosome segregation and absence of a connection between cytoplasmic and thylakoid membranes, Interaction of the periplasmic PratA factor and the PsbA (D1) protein during biogenesis of photosystem II in, Pitt, a novel tetratricopeptide repeat protein involved in light-dependent chlorophyll biosynthesis and thylakoid membrane biogenesis in Synechocystis sp. PratA Is a Mn Binding Protein and Contains High- and Low-Affinity Binding Sites. 1, 0, 1, 2 . A.S., I.L.G., D.H., and B.R. This is in line with membrane fractionation experiments in Synechocystis 6803 that also indicated a crucial role of PratA for membrane organization (Rengstl et al., 2011). Especially, the question of whether direct connections exist between the PM and the TM has been controversial for several years (Liberton et al., 2006; van de Meene et al., 2006; Nickelsen et al., 2011). GST fusion proteins (mD1 and pD1; 100 μg each) were bound to GST-agarose (Biontex) for 2 h at RT. Moreover, PDMs have been shown to accumulate the chlorophyll a precursor molecule chlorophyllide a as well as several other PSII assembly factors in a PratA-dependent manner, suggesting that PDMs harbor a network for TM biogenesis where initial steps of PSII assembly take place (Rengstl et al., 2011). We thank Christian Schwarz for generation of the three-dimensional illustration of D1, Norio Murata for providing the pD1 antibody, and Gunnar Jeschke and Jürgen Soll for helpful discussions and suggestions. Hence, the loss of PratA affects the periplasmic concentration of Mn. Taken together, our data allow the proposal of a model on the spatiotemporal organization of TM biogenesis. Recovery of Mn2+ Signal and Dependency of Mn2+ Binding on PratA Concentration. Electron flow is initiated at PSII, which serves as a light-driven water-plastoquinone oxidoreductase producing oxygen as a by-product of electron extraction from water. Photosystem I (PSI, or plastocyanin-ferredoxin oxidoreductase) is one of two photosystems in the photosynthetic light reactions of algae, plants, and cyanobacteria. Tolerance of photosystem 2 (PS2) to high temperature in apple (Malus domestica Borkh. Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: slr2048 (PratA), slr2034 (Ycf48), and At1g02910 (Lpa1). Since putative pigments could not be extracted by organic solvents, we speculated that the alteration in color might be due to differences in transition metal composition. of herbicide resistance in mutant strains. and the EPR signals due to the Tyr+ radicals of Z and D. Kinetics However, their common localization and size as well as their absence in the pratA− mutant strongly suggest that these structures at the cell periphery represent biogenesis centers formed by PDMs. As control, one sample was analyzed immediately after addition of 54Mn2+ (0 h), and values obtained after incubation for 1 and 3 h were calculated relative to it. Our results provide evidence for a function of PratA in direct and efficient delivery of Mn2+ to D1 during PSII de novo assembly. Values shown are means (±sd) of three independent experiments. For 100% of the wild type and pratA−10 μg proteins were loaded and the amount of D1 in pratA− was quantified (from three independent experiments) using Aida software (version 3.52.046). Only one periplasmic Mn binding protein (MncA) has been described to date (Tottey et al., 2008), but its precise function remains to be elucidated. Metal-ion chromatography of periplasm from Synechocystis 6803 wild-type cells. However, the semicircles were not detected in any of 1006 analyzed pratA− cells (Figure 7D). These proteins contain repetitive motifs of 34 amino acids and are known to mediate protein–protein interactions (Blatch and Lässle, 1999; Main et al., 2005). We showed that the electron transfer rates are differentially sensitive to a variety of inhibitors and herbicides, and are modified in plants which develop a resistance to herbicides. Assembly starts with the reaction center proteins D2 and D1, which, together with the PsbE, PsbF, and PsbI subunits, constitute the first detectable intermediate, the so-called RC complex. Identification of periplasmic proteins in cells grown at low and high salt concentrations, Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride, Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of photosystem II, EPR spectroscopy and catalase activity of manganese-bound DNA-binding protein from nutrient starved cells, Cooperation of plasma and thylakoid membranes for the biosynthesis of chlorophyll in cyanobacteria: The role of the thylakoid centers, Multiflash experiments reveal a new kinetic phase of photosystem II manganese cluster assembly in, A light-dependent mechanism for massive accumulation of manganese in the photosynthetic bacterium, Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane, Structure of the Mn4-Ca cluster as derived from X-ray diffraction, PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in, The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of, The cyanobacterial homologue of HCF136/YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly and repair of photosystem II in, Ultrastructure of the membrane systems in the unicellular cyanobacterium, Membrane systems in cyanobacteria. Oxygen is given off at this step (4 H2O ( 2 H+ + O2 + 4e-) Step 2: Light strikes the photosystem and the _____ are “energized” or “excited”. of electron transfer on the acceptor side of PS II were unaffected The concentration of the free Mn2+ in solutions containing rPratA was calculated by comparison to the standard curve, and the amount bound was determined by difference. For this purpose, wild-type, pratA−and psbA− cells were incubated with 54Mn2+ as described before; however, after subsequent protein extraction, immunoprecipitation was performed using a αPratA instead of αD1 antibody. Proteins were purified via GST-agarose (Biontex) under native conditions in the presence of 5 mM EDTA and, if required, recovered from the matrix by removing the GST tag by incubation with 1 unit of thrombin (GE Healthcare) per 100 μg of fusion protein for 6 h at room temperature (RT). In this case, the biogenesis regions seem to consist of membranes surrounding the pyrenoid structure of the chloroplast. Competition of Mn2+ Binding to PratA Using 5 mM MnCl2. In this approach, rPratA was incubated with 10 to 75 μM 54Mn2+, the amount of 54Mn2+ bound to rPratA was counted, and the number of 54Mn2+ per rPratA was calculated from the values obtained from 54Mn2+ in the absence of protein. Interestingly, in wild-type cells, in some cases membranous semicircle-like structures surrounding thylakoid centers were observed which appeared to contact especially TMs and PMs (Figure 7C). [See online article for color version of this figure.]. Interestingly, PratA is rich in Asp and Glu residues and has a pI of ~5.1. Here, we show that PratA is a Mn2+ binding protein that contains a high affinity Mn2+ binding site (Kd = 73 μM) and that PratA is required for efficient delivery of Mn2+ to PSII in vivo, as Mn2+ transport is retarded in pratA−. Each photosystem is composed of two parts. wild-type and herbicide resistant strains of Amaranthus hybridus, The models have been useful in deciding sites for molecular Furthermore, it is unclear whether PSII repair in cyanobacteria is spatially separated from PSII de novo assembly like in chloroplasts of green algae (Zak et al., 2001; Komenda et al., 2006; Nixon et al., 2010; Uniacke and Zerges, 2007). light reaction. Synechocystis wild-type and mutant strains (pratA−psbA− TD41, and ycf48− with the respective wild type; Komenda et al., 2008) were grown in 50 mL liquid BG11 for 5 to 6 d. Chlorophyll concentrations were measured after extraction with 100% methanol and calculated from the absorbance values at 666 and 720 nm (Wellburn and Lichtenthaler, 1984). Dehydration was performed by two infiltration steps with acetone (8 h at −70°C and 8 h at −50°C). 289–304, Auxiliary proteins involved in the assembly and sustenance of photosystem II, Assembly factors of the photosynthetic machinery in cyanobacteria, Biogenesis of the cyanobacterial thylakoid membrane system—An update, Recent advances in understanding the assembly and repair of photosystem II, Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. As control, wild-type periplasm was incubated with an equivalent amount of GST-agarose matrix without bound mD1/pD1 protein. 23, 2005 8499 13. The reduction of the signal amplitude of the Mn2+ spectrum suggests decreased amounts of free Mn2+ in solution due to Mn2+ binding to the protein (Reed and Cohn, 1970; Reed and Markham, 1984; Sen et al., 2006; Hayden and Hendrich, 2010). visible light of high intensity, or in systems with restricted II two-electron gate, and the role of protons in stabilizing semiquinones. Two types of photosystems are embedded in the thylakoid membrane: photosystem II ( PSII) and photosystem I (PSI). Click here for a brief review of photosystem II. Thus, it can be postulated that the general mechanisms of TM biogenesis are evolutionary conserved from cyanobacteria to algae and possibly also to plants. 271–288, Local and long-range stability in tandemly arrayed tetratricopeptide repeats, EPR polarization studies on Mn catalase from, Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase, Biogenesis and dynamics of thylakoid membranes and the photosynthetic apparatus. The parameter of circularity is a calculated mean value that represents the Gaussian distribution of circularity of clusters in a selection of cells. adapted state, protons are released to the inner thylakoid space which codes for the D1 protein of Photosystem II. At the RCC1 stage, the Mn cluster is photoactivated and thus represents the first assembly intermediate capable of oxygen evolution (Cheniae and Martin, 1971; Becker et al., 2011). To exclude the possibility that this reduction was solely due to lower cellular Mn2+ uptake rates in pratA−in parallel, levels of radioactivity in whole cells were assessed after incubation with 54Mn2+. As an additional control to determine whether the protein specifically determines the amount of Mn2+ binding, we performed the PratA/Mn2+ binding experiment using 100 μM Mn2+ and varying concentrations of PratA (100, 50, and 25 μM). This enzyme is somehow stimulated by the loss of e- in photo II to split two molecules of water. Furthermore, ultrastructural analyses of pratA− depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA−. donation to photosystem II. PS 1 contains chlorophyll B, chlorophyll A-670, Chlorophyll A-680, chlorophyll A-695, chlorophyll A-700 and carotenoids. Supplemental Figure 3. Our conclusions are based on the findings that (1) PratA inactivation affects intercellular Mn levels, (2) recombinant as well as native PratA specifically bind Mn2+ ions, and (3) Mn2+ incorporation into PSII is reduced in a pratA− mutant. Furthermore, a pratA− mutant showed defects in C-terminal processing of D1 (Klinkert et al., 2004), which is required for assembly of the Mn4Ca cluster in the WOC. Experiments were performed four times. This could also be judged by the mean residue ellipticity at 222 nm (Θ222) that was altered to Θ222 = −18762.0 in contrast with Θ222 = −20955.4 without Mn2+. The observed Kd1 value is in the range of affinities of other Mn2+ binding proteins, such as the Mn-dependent catalase MnCat (Kd = 40 μM) or the oxidative stress protecting protein SsDPS (Kd = 48 μM) (Meier et al., 1996; Crowley et al., 2000; Pierce et al., 2003; Hayden and Hendrich, 2010). Photosystem II occurs with two series of enzymes followed by Photosystem I in order to create energy for a plant1. Samples were analyzed without further contrast. For Lpa1, the coding region, excluding the transit sequence (Peng et al., 2006), was cloned into the SalI-BamHI sites of pGEX-4T-1 (GE Healthcare) using the primers Lpa_for (5′-GATCGGATCCATGGATGCTCTTGTTCAGTTTG-3−) and Lpa_rev (5′-GATCGTCGACGTCTTTCTAACTTGCTGAGAA-3′). engineering, and for trying to understand the relation between The e- from this reaction are then released to the waiting e- hungry Photosystem II. The central chlorophyll molecule of the reaction center is shown with the arrow (notice the second reaction center in the bottom half--photosystem II is composed of two identical halves). After 3 h, the level of 54Mn2+ in the psbA− strain was 6.0-fold less than in the wild type (Figure 6B). After 1 h of incubation with 54Mn2+, amounts of bound Mn2+ were reduced 4.0-fold compared with the wild type, and after 3 h, the effect was even more pronounced, resulting in an 8.8-fold reduction of precipitated radioactivity from the pratA− material (Figure 6A). The figure shows one representative graph of three independent experiments. Since the precise architecture of the biogenesis centers (= TC + SS) remains to be resolved at higher resolution, it is depicted with broken lines. Determination of Parameters Used for Analysis of Gold Clusters in Immunogold Labeling Experiments with αpD1. The five metal atoms were found to be linked by five oxygen atoms, and four additional water molecules bind to the Mn4Ca cluster. ... After watching this video, you will be able to describe the steps involved in the light reactions of photosynthesis. After centrifugation for 10 min at 20,000g, the supernatant was diluted 1:10 with TMK buffer and supplied with 5 μL primary antiserum (αD1; Schottkowski et al., 2009a). acceptor of photosystem II. secondary quinone binding site of photosystem II of Anacystis, 2005 ; Vol. Upon successive attachment of the inner antennae proteins CP47 and CP43, RC47 and RCC1 core complexes are formed, respectively. The beads were washed five times with 50 mM Tris, pH 8.0, and 150 mM NaCl, proteins were eluted by incubation with 50 μL 2× Roti-Load 1 (Roth) for 5 min at 95°C, and samples were analyzed by immunoblotting using αPratA. Native PratA Binds to the Mature D1 C Terminus Near the Mn Cluster. viridis as a template on which to build structural models 44, No. tools for amplification and sequencing of the psbA gene There are four (4) types of chlorophyll: a, b, c, and d. Two distinct systems for cellular Mn uptake have been described, the so-called MntABC transporter, and a second pathway whose components are yet unidentified (Bartsevich and Pakrasi, 1995, 1996). Two types of photosystems are embedded in the thylakoid membrane: photosystem II ( PSII) and photosystem I (PSI). analyzed data. of UV photoinhibition, and comparing it with photoinhibition by The CD data obtained for rPratA alone indicated a structure consisting of 72% α-helices, 5% β-sheets, and 23% turns/coils (Figures 2A and 2B). The exact mechanism of Mn2+ transfer from PratA to D1 and the possible involvement of additional factors required for this process have to be determined structurally and biochemically in future studies. The sections were washed six times for 5 min and incubated with gold-conjugated goat anti-rabbit IgG (BBInternational; 5 nm, 1:50) for 90 min at RT before washing three times in blocking buffer, twice with 50 mM glycin in PBS, pH 7.4, twice with PBS, pH 7.4, and twice with double-distilled water. Step 1 is the light-induced inactivation of the oxygen-evolving complex. Antenna Complex:It is light gathering part. to predict sites for modification of the catalytic sites by site-directed At present we are investigating the modified function in D1 mutants H252Q and S264G.. Pigments Samples were analyzed without further contrast. Although these structures have to be analyzed three-dimensionally in more detail in further studies, it can be hypothesized that the observed semicircles surround the rod-like thylakoid centers detected before (van de Meene et al., 2006). light intensity, associated with loss of fluorescence yield, and In The Cyanobacteria, A. Herrero and E. Flores, eds (Norfolk, UK: Caister Academic Press), pp. 8494-8499. A photosystem is a protein complex, a group of two or more proteins, that is essential for the photochemistry of photosynthesis. The fraction of bound Mn2+ was calculated from the amplitudes relative to total Mn2+ bound without competitor (=100%; red bar). of QB to QB.-(H+). Infiltration of cells was achieved using acetone including 1% osmium tetroxide for 3 h at −85°C, followed by incubation for 20 h at −20°C, 3 h at 4°C, and 1 h at RT. PratA Localizes to Distinct Structures at the Cell Periphery. Although light-induced damage to PSII (photoinhibition) has been studied in great detail in vitro (Adir et al., 2003; Edelman and Mattoo, 2008; Nixon et al., 2010), the in vivo mechanism of PSII photodamage is controversial (Vass and Cser, 2009). As negative control, Synechocystis wild-type sections were incubated without the primary antibody, followed by treatment with the gold-labeled anti-rabbit IgG. The question of how Mn is transported to PSII and assembled into the Mn4Ca cluster has been the subject of intensive research. The data strongly suggest that it works as a Mn2+ binding and transport protein that delivers Mn2+ ions directly and efficiently to PSII. No or only few randomly localized signals were detected upon incubation of Synechocystis 6803 wild-type sections without the primary antibody (followed by treatment with the gold-labeled anti-rabbit IgG only; Figure 8A, Table 1) or when sections of pratA− cells were treated with αPratA (Figure 8B, Table 1). the QB-site, and to provide a detailed kinetic model for the reduction The process that converts light energy into chemical energy takes place in a multi-protein complex called a photosystem. Present here is the photosynthetic electron transport chain. Recovered samples were suspended in scintillation mixture (Optiphase Supermix; Perkin-Elmer), and 54Mn2+ was measured with a LS 6500 multipurpose scintillation counter (Beckman Coulter). In Biological Magnetic Resonance, L.J. Ultrathin sections of Synechocystis cells were incubated with αpD1 (1:50, rabbit) prior to incubation with gold-conjugated goat anti-rabbit IgG. Step 2: Generation of ATP by Electron Transport Chain. In none of the >1000 pratA− cells analyzed have these semicircle-shaped structures been found, suggesting that although we cannot exclude that certain prestructures already form PratA independently, PratA seems to be required for either the stability of these structures or its fixation at the cell periphery. Recently, membrane domains with specialized functions have been localized even in the ancient cyanobacterium Gloeobacter violoceus, the only known organism performing oxygenic photosynthesis without internal TMs (Rexroth et al., 2011). UV-irradiation leads to a loss of components on the donor side Considering that a maximum of eight Mn2+ ions were found to bind per rPratA, seven Mn2+ molecules remain attached to rPratA under these conditions. the two-electron gate in terms of a relatively simple model. Thereby, the peak-to-peak height of the lowest-field line of a Mn2+ standard was plotted against Mn2+ concentration to obtain a standard curve. As a next step, we localized PratA within the cyanobacterial cell by performing immunogold labeling experiments using a specific αPratA antibody. (A) and (B) Competition experiment using EPR analysis of 500 μM MnCl2 ± 100 μM PratA in Tris/NaCl + 500 μM (1×) or 5 mM (10×) CaCl2 (A) and 500 μM MnCl2 ± 100 μM PratA in Tris/NaCl + 500 μM (1×) or 5 mM (10×) MgCl2 (B). Here, we present experimental evidence from ultrastructural analyses that show the existence of PratA-dependent semicircular structures of ~100 nm in diameter surrounding thylakoid centers at the cellular periphery, which indeed appear to connect TMs and PM (Figure 7) and which we called biogenesis centers (= thylakoid center + semicircular structure; Figure 9G). Ultrathin sections (30 to 60 nm) of the cryofixed samples were stained with osmium tetroxide and poststained using lead citrate. Radioactivity levels (in counts per minute [CPM]) measured in samples from psbA− cells (background) were subtracted from values for the wild type (WT) and pratA−. However, we detected only a minor reduction in both transport and uptake of Mn2+ (1.3- and 1.2-fold decrease after 3 h incubation with 54Mn2+, respectively) in ycf48−, indicating a specific role of PratA for Mn2+ delivery to PSII (Figures 6E and 6F). Supplemental Figure 1. We have generated a large number of mutations to test potential ligands, and the role of tyrosine 161 as secondary donor. ↵[W] Online version contains Web-only data. We have Interestingly, although no obvious PratA homologs can be found in vascular plants or green algae (Klinkert et al., 2004), recently, two proteins from Arabidopsis and Chlamydomonas reinhardtii have been characterized that possess similar properties as they were both found to interact directly with D1 and belong to the family of TPR proteins (Peng et al., 2006; Park et al., 2007). Our observations suggest that photodamage to PSII occurs in two steps. The underlying light-driven photosynthetic electron transport is mediated by multiprotein/pigment complexes (i.e., photosystem II [PSII], the cytochrome b6f complex, and photosystem I), which reside within the thylakoid membrane (TM) system of cyanobacteria, algae, and plants. in centers which retained a normal fluorescence yield. We have shown that inhibition of photosynthesis by exposure to Ultrathin sections of Synechocystis cells were incubated with αPratA (1:25, rabbit) prior to incubation with gold-conjugated goat anti-rabbit IgG. Thus, PratA is necessary for efficient delivery of Mn2+ to PSII, leading to Mn2+ preloading of PSII in the periplasm. One of these proteins is PratA, which was previously found to interact directly with the core reaction center protein D1 that plays a major role in complexing the Mn cluster required for oxidation of water (Schottkowski et al., 2009a). Sections were incubated for 18 h at 4°C with αPratA 1:25 (rabbit) or αpD1 1:50 (rabbit). For the cluster, two different definitions were used (i.e., an accumulation of n ≥ 5 or n ≥ 7 gold particles, respectively; n ≥ 7, n = 11 for anti pD1-immunolabeled structures in wild-type cells, n = 12 for those in pratA− cells), both revealing no obvious changes in pratA− compared with the wild type.

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