The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants.

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., Chlamydomonas reinhardtii). In this study, we biochemically characterized the cpSRP system of the charophyte Chaetosphaeridium globosum and the bryophyte Physcomitrella patens. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 (Kd ~10 μM) in Chaetosphaeridium globosum and Physcomitrella patens as well as relatively low affinity binding of cpSRP54 to cpSRP RNA (Kd ~1 μM) in Physcomitrella patens. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the Physcomitrella patens cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA

AQP5-1364A/C Polymorphism Affects AQP5 Promoter Methylation

The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin (AQP5)-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele—leading to lower AQP5 expression—has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that AQP5 promoter methylation differs between genotypes in specific types of immune cells.: AQP5 promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of AQP5-1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils (p = 0.0055) and a four-fold increase in monocytes (p = 0.0005) and lymphocytes (p = 0.0184) in septic patients and healthy controls as well. In addition, a decreased AQP5 promoter methylation was accompanied by an increased AQP5 expression in HL-60 (p = 0.0102) and REH cells (p = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the AQP5 promoter. Hence, AQP5 promoter methylation could depict a key mechanism in genotype-dependent expression

Determination of binding affinities between <i>Physcomitrella patens</i> cpSRP54 and various SRP RNAs using microscale thermophoresis.

<p>Binding affinities of <i>Physcomitrella patens</i> cpSRP54 complex formation with <i>Physcomitrella patens</i> cpSRP RNA (A), <i>E</i>. <i>coli</i> SRP RNA (B), <i>Ostreococcus tauri</i> cpSRP RNA (C), and the <i>Physcomitrella patens/E</i>. <i>coli</i> (Pp/Ec)-hybrid cpSRP RNA (D). The structure of used (cp)SRP RNAs are given in the left panels: (green) <i>Physcomitrella patens</i>, (orange) <i>E</i>. <i>coli</i>, (black) <i>Ostreococcus tauri</i> and (orange/green) <i>Physcomitrella patens/E</i>. <i>coli</i>-hybrid. Fluorescently labeled Pp-cpSRP54 was kept between 10 to 20 nM, while the indicated (cp)SRP RNAs were titrated in a micromolar excess (e.g., 90 μM Pp-cpSRP RNA, 5 μM Ec-SRP RNA, 10 μM Ot-cpSRP RNA and 5 μM Pp/Ec-hybrid cpSRP RNA). The difference in normalized fluorescence [‰] was plotted against the concentration of the SRP RNAs, and the binding affinity (K_d) was evaluated using the MO.Affinity Analysis Software (NanoTemper Technologies GmbH, Munich, Germany) (left panel) (right panel, raw MST-traces). All experiments were performed at least twice.</p

CpSRP components in various organisms of the green lineage.

<p>(A) The presence of a SRP54 subunit, cpSRP43 and cpSRP RNA in the indicated SRP systems is denoted by green dots, while the lack of a component is marked with a red dot. In addition, the ability or inability of (cp)SRP54 to bind one of these components is also represented by a green or red dot, respectively. The given K_d values were either described previously in Buskiewicz et al., 2005^(a) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166818#pone.0166818.ref022" target="_blank">22</a>] or were obtained in this work ^(b). (B) Comparison of different (cp)SRP RNA structures of <i>E</i>. <i>coli</i>, <i>Ostreococcus tauri</i> and <i>Physcomitrella patens</i>. All (cp)SRP RNAs are composed of an asymmetric loop, a symmetric loop and an apical loop region. <i>E</i>. <i>coli</i> and <i>Ostreococcus tauri</i> (cp)SRP RNA harbor a conserved tetraloop structure while this structure is elongated in <i>Physcomitrella patens</i> cpSRP RNA. (C) The cpSRP54 protein consists of an N-terminal NG-domain with GTPase activity and a C-terminal M-domain where the cpSRP43 binding motif is localized. <i>Arabidopsis thaliana</i> (At), <i>Physcomitrella patens</i> (Pp) and <i>Chaetosphaeridium globosum</i> (Cg) cpSRP54 harbor the conserved A(R/K)R cpSRP43-binding motif (see red boxes and alignment). The numbering of the amino acid sequence of Cg-54M refers to the used EST clone as described in materials and methods. <i>Chlamydomonas reinhardtii</i> cpSRP54 is not complexed with cpSRP43 because of a valine instead of an alanine in the cpSRP43-binding motif (see grey box and alignment). (D) CpSRP43 is composed of three chromodomains (CD1-CD3) and four ankyrin repeats (A1-A4). The cpSRP54 binding region is located in chromodomain 2 (CD2). CD2 of <i>Arabidopsis thaliana</i> (At), <i>Physcomitrella patens</i> (Pp) and <i>Chaetosphaeridium globosum</i> (Cg) forms two aromatic cages that recognize the A(R/K)R cpSRP43-binding motif (see purple boxes and alignment) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166818#pone.0166818.ref019" target="_blank">19</a>]. Residues forming the cage 1 and cage 2 regions in <i>Arabidopsis thaliana</i> cpSRP43 and the corresponding positions in <i>Physcomitrella patens</i>, <i>Chaetosphaeridium globosum</i> and <i>Chlamydomonas reinhardtii</i> cpSRP43 are highlighted with black and white circles. Residues in <i>Chaetosphaeridium globosum</i> and <i>Chlamydomonas reinhardtii</i> cpSRP43, which differ in these positions from <i>Arabidopsis thaliana</i> and <i>Physcomitrella patens</i> cpSRP43, are marked by gray boxes. Pro-255 in <i>Chlamydomonas reinhardtii</i> cpSRP43 that interferes with cpSRP54 binding is marked by a yellow box. Symbols display the degree of conservation: identical residues (asterisk), conserved substitution (colon), and semiconserved substitution (dot); TS, transit sequence.</p

Determination of binding affinities of cpSRP54/cpSRP43 complex formation from <i>Arabidopsis thaliana</i>, <i>Physcomitrella patens</i> and <i>Chaetosphaeridium globosum</i> using microscale thermophoresis.

<p>Fluorescently labeled cpSRP43 (At-43 or Pp-43) or eGFP-Cg-43 was kept constant at 20 nM or 75 nM, respectively. The indicated cpSRP54 constructs were titrated in a micromolar excess (e.g., up to 5 μM (At-54), 100 μM (Pp-54) and 265 μM (Cg-54M)) ((A/B) <i>Arabidopsis thaliana</i>, (C) <i>Physcomitrella patens</i>, (D) <i>Chaetosphaeridium globosum</i>). The difference in normalized fluorescence [‰] was plotted against the concentration of the indicated cpSRP54 constructs (left panel) (right panel, raw MST-traces). For cpSRP complex formation analysis in <i>Chaetosphaeridium globosum</i>, the M-domain of cpSRP54 (cpSRP54M) was used. All experiments were performed at least twice, and the binding affinity (K_d) was evaluated using the MO.Affinity Analysis Software (NanoTemper Technologies GmbH, Munich, Germany).</p

Analysis of complex formation between cpSRP54, cpSRP RNA and cpSRP43 from <i>Physcomitrella patens</i> by gel filtration.

<p>Complex formation of <i>Physcomitrella patens</i> cpSRP54 (Pp-54) and a truncated cpSRP43 construct (Pp-43ΔCD1) as well as Pp-cpSRP RNA were analyzed using size exclusion chromatography with equimolar amounts of the indicated components: (green) Pp-54-His and Pp-cpSRP RNA, (brown) Pp-His-43ΔCD1 and Pp-cpSRP RNA, (blue) Pp-54-His, (red) Pp-His-43ΔCD1, (black, not depicted in the chromatogram) Pp-cpSRP RNA, (orange) Pp-54-His and Pp-His-43ΔCD1. Elution fractions in a range from 10 to 17 ml were separated using SDS-PAGE and detected using Coomassie staining. Elution fractions containing RNA were analyzed using polyacrylamide gels and detected using SYBR Safe DNA gel stain.</p

Interaction analysis between cpSRP54 and various cpSRP43 constructs of <i>Arabidopsis thaliana</i> and <i>Physcomitrella patens</i>.

<p>(A) Yeast two-hybrid interaction studies. For yeast two-hybrid assays, the yeast strain Y190 was co-transformed with pGBKT7 constructs encoding full-length cpSRP54 (54) and pACT2 constructs encoding cpSRP43 (43) or the indicated cpSRP43 mutants of <i>Arabidopsis thaliana</i> (At) and <i>Physcomitrella patens</i> (Pp). Co-transformed cells were dotted onto minimal media lacking Leu and Trp (-LT) to check for co-transformation, or lacking Leu, Trp and His (-LTH) to assess interaction. Negative controls were conducted with an empty vector (pGBKT7 or pACT2). (B) <i>In vitro</i> pull-down assays were performed with recombinant GST-cpSRP43 (At-, Pp-GST-43) constructs and His-tagged cpSRP54 proteins (At-His-54, Pp-54-His) as indicated, using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were separated using SDS-PAGE and detected using Coomassie staining. The asterisk (*) indicates the used His-tagged constructs.</p

Interaction analysis between cpSRP54M and various cpSRP43 constructs of <i>Chaetosphaeridium globosum</i>.

<p>(A) <i>In vitro</i> pull-down assays were performed as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166818#pone.0166818.ref015" target="_blank">15</a>]. Combinations of recombinant GST-cpSRP43 (At-, Cg-GST-43) or the mutant construct Cg-GST-43(V192P) and His-tagged cpSRP54M (At-, Cg-His-54M) proteins were analyzed as indicated using glutathione-sepharose. Control reactions were performed with recombinant GST. One-tenth of the loaded proteins (upper panel) and one-third of eluted proteins (lower panel) were analyzed by SDS-PAGE and Coomassie staining. The asterisk (*) indicates the used His-tagged constructs. (B) Protein-protein interactions between His-tagged <i>Chaetosphaeridium globosum</i> cpSRP54M (Cg-54M) and cpSRP43 (Cg-43) or cpSRP43(V192P) were analyzed by size exclusion chromatography using equimolar amounts of the indicated recombinant proteins: (green) Cg-His-43 and Cg-His-54M, (orange) Cg-His-43(V192P) and Cg-His-54M, (blue) Cg-His-43, (violet) Cg-His-43(V192P), and (red) Cg-His-54M. Elution fractions in a range from 8.5 to 14.5 ml were separated by SDS-PAGE and detected by Coomassie staining.</p