An intermolecular disulfide-based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii
Version of Record online: 13 SEP 2012
© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd
The Plant Journal
Volume 72, Issue 3, pages 378–389, November 2012
How to Cite
Schwarz, C., Bohne, A.-V., Wang, F., Cejudo, F. J. and Nickelsen, J. (2012), An intermolecular disulfide-based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii. The Plant Journal, 72: 378–389. doi: 10.1111/j.1365-313X.2012.05083.x
- Issue online: 29 OCT 2012
- Version of Record online: 13 SEP 2012
- Accepted manuscript online: 22 JUN 2012 01:27PM EST
- Received 12 September 2011; revised 30 May 2012; accepted 15 June 2012; published online 13 September 2012.
Figure S1. Purification of recombinant RBP40(rRBP40). Purification steps were analyzed by SDS–PAGE andCoomassie Blue staining. The recombinant protein is marked by anarrowhead. M, molecular weight markers; P, insoluble fraction ofoverproducing extracts; S, soluble fraction of overproducingextracts; FT, flow through; W1–W4, wash fractions;E1–E3, eluates from Ni2+-NTA agarose.
Figure S2. Schematic representation of the Nac2amino acid sequence. The putative transit peptide predicted by theTargetP 1.1 algorithm (30 amino acids,http://www.cbs.dtu.dk/services/TargetP) is indicated by scissors.The C-terminal TPR repeat region and cysteine residues (C) aredepicted. Possible disulfide bonds between the Cystein residueswere predicted by the DIpro algorithm with the disulfide bridge inthe TPR region being the most probable one(http://www.ics.uci.edu/~baldig/scratch; Cheng et al. 2006). A possible Rossman fold for FAD/NADP recognition (aa 402-413) detected by the FAD or NADP binding Rossmann fold detector of the NIH MBI laboratory servers (nihserver.mbi.ucla.edu//cgi-bin/Rossmann/Rossmann.cgi; Kleiger and Eisenberg 2002) is illustrated.
Figure S3. Disulfide bridge formation of Nac2and RBP40 in mbb1. (a) Stromal proteins (100 μg)from light-grown cells of the PSII mutant mbb1 werefractionated by 2D redox SDS-PAGE, and Nac2 and RBP40 werelocalized by immunoblot analysis. The diagonal along whichpolypeptides that form no S–S bonds lie is visualized by CBBstaining of gels. For further explanation, see text. (b) Controlshowing the distribution of Nac2 and RBP40 after incubation ofsamples with 5 m<SMALLCAPS>M</SMALLCAPS>glutathione prior to electrophoresis. The arrow marks theredox-dependent Nac2 signal at 170 kDa.
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