Volume 6, Issue 5, Pages 1503-1517 (September 2013) Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation  Anna Filonova, Paul Haemsch, Christin Gebauer, Wolfram Weisheit, Volker Wagner  Molecular Plant  Volume 6, Issue 5, Pages 1503-1517 (September 2013) DOI: 10.1093/mp/sst048 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Recombinant CrPDI2 Is a Redox-Active Protein and Has Refolding Activity. (A) The structure of CrPDI2. The domain architecture was derived from a BLASTp of the protein at the NCBI website (www.ncbi.nlm.nih.gov/BLAST/ ). The thick black lines show the position of the peptides used for antibody production. (B) Expression of the recombinant CrPDI2 in E. coli. Proteins were separated by SDS–PAGE and stained with Coomassie Brilliant Blue (lanes 1 and 2) or analyzed with anti-CrPDI2 peptide antibodies (lane 3). 80 μg of total crude extract proteins of the CrPDI2-overexpressing E. coli strain 2 h after IPTG induction were applied to lane 1. Lane 2 represents 6 μg purified 6xHis-tagged CrPDI2 and lane 3 0.125 μg crude extract proteins from the CrPDI2-overexpressing E. coli strain. The arrowhead indicates the position of the recombinant CrPDI2. After purification, recombinant CrPDI2 was dialyzed and applied to the enzymatic measurements. (C) Reduction of redox-active disulfides in CrPDI2 and E. coli thioredoxin by rat liver thioredoxin reductase. 10 μM purified recombinant CrPDI2 ( ), NADPH and thioredoxin reductase in reaction buffer as control ( ), and 20 μM E. coli thioredoxin ( ) were used in separate assays. Each reaction was started by the addition of 67nM thioredoxin reductase. Data represent the mean ± SD (n = 3). (D) Reduction of insulin by 4 μM purified recombinant CrPDI2 ( ). 1 mM DTT ( ) and 1 μM E. coli thioredoxin ( ) were used as negative and positive controls, respectively. Data represent the mean ± SD (n = 3). (E) Oxidative refolding activity was determined by an RNase A refolding assay. Recombinant CrPDI2 was tested in a redox buffer for the ability to refold scrambled RNase A. Scrambled RNase A from bovine pancreas was prepared by overnight incubation with DTT and guanidinium chloride. After desalting, the renaturation of 8 μM denatured RNase A was examined in the presence of glutathione, glutathione disulfide, and 3 μM recombinant CrPDI2 ( ). The hydrolysis rate of 4.5mM cCMP by native RNase A ( ) was set to 100%. Denatured RNase A without CrPDI2 was used as a control for non-catalyzed renaturation ( ). Data represent the mean ± SD (n = 3). Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Redox Conditions Do Not Change the Electrophoretic Mobility of CrPDI2. (A) Cells were grown under a LD cycle, then put under LL conditions and harvested at LL25, LL29, LL33, LL37, LL41, and LL45. Proteins from crude extracts (50 μg) were separated by SDS–PAGE and immunodetected with anti-CrPDI2 peptide antibodies. Representative Ponceau-stained protein bands are shown as loading controls. (B) Protein mobility shift assay. Cells were harvested at LL25 and LL41 and crude extracts were prepared under reducing (14mM DTT) and oxidizing (5mM H2O2) conditions. Proteins from crude extracts (50 μg) were separated by SDS–PAGE and immunodetected with anti-CrPDI2 peptide antibodies. Representative Ponceau-stained protein bands are shown as loading control. Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Analysis of the Circadian Abundance of Heparin-Bound CrPDI2. (A) Western blot analysis of proteins from crude extracts and heparin-bound proteins from C. reinhardtii with the CrPDI2 specific peptide antibodies. Cells were entrained for 3 d to a 12:12 h light/dark cycle, released into constant conditions of low light at LL0 and harvested at LL25 and LL41. Proteins from crude extracts were subjected to heparin affinity chromatography followed by a dialysis and a TCA/acetone precipitation according to the procedure described in the ‘Methods’ section. Proteins (40 μg) from crude extracts (CE) and heparin-bound proteins (heparin-bound) were separated by SDS–PAGE and immunodetected with anti-CrPDI2 peptide antibodies. The arrowhead indicates the CrPDI2-specific signals. Representative Ponceau-stained protein bands are shown as loading controls. (B) Quantification of spot volumes for the CrPDI2-immunoblotts of crude extracts and heparin-bound proteins. The results shown are the average percentage (whereas the average spot volume of the stronger signal was set to 100%) and the standard error of the mean (S.E.M.) of the spot volumes of three independent experiments. Spot volumes were determined using the ImageMaster-2D software (GE Healthcare). (C) The recombinant CrPDI2 binds to heparin. Recombinant CrPDI2 from E. coli was isolated, dialyzed, and 300 μg were applied to heparin affinity chromatography. Two microliter aliquots of the flow through (FT) and of the heparin column eluate fractions were diluted with SDS sample buffer, separated by SDS–PAGE, and immunodetected with anti-CrPDI2 peptide antibodies. Fraction numbers 8 and 9 represent the protein fraction eluted under low-salt conditions (15mM NaCl); fraction numbers 13 and 14 represent the high-salt eluates (1 M NaCl). Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Complex Formation of CrPDI2 during Day and Night Phases. Cells were grown under a LD cycle, then put under LL and harvested at LL25 and LL41. Three mg of proteins from crude extracts were separated on a linear 6–14% sucrose density gradient along with a molecular mass standard. The gradients were centrifuged at 74 100 g for 16 h at 4°C. Fraction numbering starts with the high percentage (14%). Aliquots of the fractions were separated by SDS–PAGE and immunodetected with anti-CrPDI2 peptide antibodies. Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 The Co-IP Column Is Specific for CrPDI2. 50 μg of proteins from crude extracts (CE) and 8 μl aliquots of the flow through (FT), the washing step (Wash), and the eluates from the anti-CrPDI2 antibody-loaded column (A) and the control column (B) were denatured with sample buffer, separated by SDS–PAGE and immunoblotted with the anti-CrPDI2 peptide antibodies. Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Overexpression of CrPDI2 in C. reinhardtii. (A) The overexpression construct used for CrPDI2 is shown. Hsp70a-rbcs2 indicates the truncated tandem promoter along with the first intron of rbcs2 that bears an enhancer. The tagged crpdi2 cDNA bears the crpdi2 ORF and the entire 3’UTR. (B, C) Different amounts of proteins from a crude extract (WT; 50, 100, and 150 μg per lane) of wild-type harvested at LL41 were separated by SDS–PAGE and used for Western blot analysis with anti-CrPDI2 antibodies along with proteins of crude extracts (50 μg per lane) from CrPDI2-ox11 and CrPDI2-ox14 harvested at LL41, respectively. The positions of CrPDI2 are indicated by arrowheads. Representative Ponceau-stained protein bands are shown as loading controls. (D, E) Quantification of expression on the basis of spot volume for the CrPDI2-immunoblotts of crude extracts from wild-type (WT) and from the overexpressing strains. CrPDI2-ox11 shows an increased expression rate of 2.8-fold (D), whereas CrPDI2-ox14 shows a 2.6-fold stronger expression of CrPDI2 (E). Spot volumes were determined using the ImageMaster-2D software (GE Healthcare). Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Measurement of Circadian Phototaxis of C. reinhardtii Wild-Type Cells and Cells from CrPDI2-Overexpressing Strains. Cells were entrained to a 12:12 h light/dark cycle and then released into constant darkness. The peak time of circadian phototaxis of cells maintained under constant conditions is significantly shifted towards the night phase in CrPDI2-ox11 and CrPDI2-ox14 compared to the wild-type strain SAG73.72 (WT). Ordinate, cell density documented as extinction E in mV; abscissa, time in days under constant darkness. The free-running period of the strains is indicated. Phototaxis curves of wild-type (A), CrPDI2-ox11 (B), and CrPDI2-ox14 (C) strain are shown. Average periods and the standard error of the mean (S.E.M.) from six phototaxis measurements were τ = 24.2±0.21 (WT), τ = 25.2±0.19 (CrPDI2-ox11), and τ = 25.1±0.09 (CrPDI2-ox14). Amplification and comparison of phototaxis curves of CrPDI2-ox11 and WT (D) and CrPDI2-ox14 and WT (E) over the first 3 d under constant conditions are also shown. Molecular Plant 2013 6, 1503-1517DOI: (10.1093/mp/sst048) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions