Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase Elisabeth Schmidtmann, Ann-Christine König, Anne Orwat, Dario Leister, Markus Hartl, Iris Finkemeier  Molecular Plant  Volume 7, Issue 1, Pages 156-169 (January 2014) DOI: 10.1093/mp/sst144 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Amino Acid Sequence Alignment of Putative Mitochondrial Citrate Synthases from Selected Species. Arabidopsis thaliana (At_CS4: At2g44350.1, At_CS5: At3g60100), Oryza sativa (Os_CS: AF302906_1), Chlamydomonas reinhardtii (Cr_CS: XP_001702983), Sus scrofa (Ss_CS: NP_999441.1), and Homo sapiens (Hs_CS: NM_004077.2). The alignment was performed using Clustal O version 1.2.0. Highlighted in black are the amino acids of the catalytic triade which is conserved in all species. Cysteine residues are highlighted in gray. The position of cysteine residues from AT_CS4 are indicated on top of the sequence. Putative mitochondrial presequences are underlined as predicted with TargetP (Nielsen et al., 1997; Emanuelsson et al., 2000). Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Recombinant CS4 Protein Is an Active Citrate Synthase Enzyme. (A) Coomassie-stained SDS–PAGE of the Ni-NTA purified 6xHis-CS4 protein. (B) Enzyme activity of CS4 in dependence on pH. 100% are equal to the mean activity at pH 8 (mean ± SE, n = 3). (C) Enzyme activity of CS4 in dependence on temperature. 100% are equal to the mean activity at 20°C (mean ± SE, n = 3). (D) Lineweaver–Burk plot of CS4 activities depending on substrate concentrations at pH 8 and 20°C. Either oxaloacetate (OAA, white dots) or acetyl CoA (black dots) concentrations were varied while the other substrate was kept at a constant concentration of 200 μM (mean ± SE, n = 3). Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Arabidopsis Mitochondrial Citrate Synthase Forms High-Molecular-Weight Complexes In Vivo as well as In Vitro. (A) 2D-BN–PAGE of mitochondrial protein complexes solubilized with digitonin. (a) PonceauS staining after transfer of 2D-BN–PAGE onto a nitrocellulose membrane, (b) Western blot analysis using α-CS4 antibody. (B) Analysis of citrate synthase via size exclusion chromatography. The elution profile of the recombinant CS4 protein is shown as recorded at 280 nm. Recombinant CS4 (100 μg) and isolated mitochondria (180 μg) from Arabidopsis plants, respectively, were loaded onto a Superose 6 column. The eluted 1-ml fractions of recombinant CS4 or mitochondrial proteins (below) were analyzed via Western blot using αCS4-antibody (F5–F10). The arrow indicates the fraction containing the citrate synthase dimer. F5: >950kDa, F6: 460–950kDa, F7: 230–460kDa, F8: 110–230kDa, F9: 54–110kDa, F10: 26–54kDa. Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Cys108 and Cys325 Are Important for the Overall Citrate Synthase Activity. Activities of recombinant CS4 variants after incubation with (gray bars) or without (black bars) hydrogen peroxide. Recombinant proteins were purified under reducing conditions (+DTT). Citrate synthase activity was measured 30min after incubation with 400 μM H2O2 at room temperature (mean ± SE, n = 3). The percentage of inhibition after hydrogen peroxide treatment is indicated above the bars. Asterisks indicate significant differences (* p < 0.05, ** p < 0.005, *** p < 0.001). Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Thioredoxin (TRX)-Dependent Reduction of CS4. (A) TRX-dependent activation of recombinant citrate synthase variants. Recombinant proteins were purified under non-reducing conditions. Activity of citrate synthase was measured in the absence (black bars) or presence (gray bars) of E. coli TRX, NTR, and NADPH (mean ± SE, n = 6). The fold-change of activation after TRX treatment is indicated above the bars. Asterisks indicate significant differences (* p < 0.05, ** p < 0.005, *** p < 0.001). (B)In vitro interaction of mutated and inactive recombinant E. coli TRXi (TRX-Cys36Ser, mutated active site: CGPS) with CS4 variants. (a) CS4, (b) Cys108Ser, (c) Cys325Ser, (d) Cys365Ser. First lane without DTT (–DTT), second lane with DTT (+DTT), and third lane without DTT but with TRX–Cys36Ser (–DTT, +TRXi). Triangles indicate size of citrate synthase dimer. Asterisk indicates covalent interaction between CS4 variant with TRXi. Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Diagonal Redox 2D-SDS–PAGE to Analyze Disulfide Forma- tion in Recombinant CS4 Protein and Cys-Variants. (A) Western blots of diagonal 2D-SDS–PAGE of citrate synthase from Arabidopsis mitochondria (mt) with (a) and without (b) prior DTT treatment (100mM) in the first dimension. (B) Western blots of diagonal 2D-SDS–PAGE of recombinant CS4 proteins treated with (+DTT) or without (–DTT) prior DTT treatment (100mM) in the first dimension. (a, b) recombinant protein of CS4, (c, d) recombinant protein of Cys108Ser, (e, f) recombinant protein of Cys325Ser, (g, h) recombinant protein of Cys365Ser. (C) Western blots of diagonal 2D-SDS–PAGE of the non-reduced recombinant CS4 protein treated with E. coli TRX, NTR, and NADPH prior to the diagonal 2D-SDS–PAGE. For clarity of the identity of proteins, all gels were analyzed by Western blot using αCS4-antibody. The gel lanes were subjected to an SDS-gel electrophoresis in the second dimension under reducing conditions (R, 100mM DTT).Triangles indicate dimers of citrate synthase. Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Hypothetical Model on Redox Regulation of Arabidopsis Citrate Synthase. (A) Ribbon diagram of the predicted tertiary structure and positions of cysteine residues (spacefill) in Arabidopsis citrate synthase. The 3D structures were modeled with a Swiss PDB Viewer 4.0 and modified with Pymol Version 1.6. CS4 (At2g44350) was fitted to pig heart citrate synthase (3ENJ (left), 4CTS (right)). The cysteine residues are color-coded as follows: Cys108 (blue), Cys209 (green), Cys325 (red), Cys365 (yellow), Cys439 (orange), Asp461 for Cys467 (gray). (B) Scheme on redox regulation of Arabidopsis citrate synthase. The oxidized form of citrate synthase forms mixed disulfides. An intermolecular disulfide bridge between Cys325 (red) is formed. Additionally, Cys108 (blue) and Cys365 (yellow) can form an intramolecular disulfide bridge next to others. In the presence of TRX, the disulfide bonds are reduced and the active dimer which is independent on disulfides can be formed. Oxidants like hydrogen peroxide can again lead to an inactivation of citrate synthase enzyme. Molecular Plant 2014 7, 156-169DOI: (10.1093/mp/sst144) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions