The role of a temporal expressed protein disulfide isomerase in the circadian clock of the green alga Chlamydomonas reinhardtii

• Applicant: Dr. Volker Wagner
• Subject Area: Plant Physiology
• Term: from 2007 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 46567767

The presence of the entire genome sequence of Chlamydomonas reinhardtii and its easy growth and biochemical fractionation have opened an avenue for functional proteomics. With a combination of heparin affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry, we could identify a protein disulfide isomerase (CrPDI2) that is specifically enriched by heparin in samples taken during subjective night. Protein disulfide isomerases are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. To study the role of CrPDI2 in the circadian system, we have overexpressed its gene. In two transgenic strains, we have observed a clear change in the acrophase using the automated rhythm of phototaxis as indicator. Its maximum occurred at the end of night-phase instead of the middle of subjective day. We could show that the recombinant protein is a redox active protein. CrPDI2 could be reduced by thioredoxin reductase and catalyzed itself the reduction of insulin chains. In immunoblots using anti-peptide antibodies, we revealed a five-fold circadian change in abundance of the heparin bound CrPDI2 in contrast to crude extracts, indicating that it may have some specific interaction partners during night that cause this high amplitude change. Indeed, we find that the CrPDI2 is present in protein complexes of different sizes at both day and night and could identify an interaction partner (a 2-cys peroxiredoxin; CrPRX2) that is present at night. In the forthcoming experiments, we will address further aspects of the regulatory mechanism of posttranslational control of CrPDI2 and CrPRX2 via protein-protein interaction and redox signaling in the circadian clock of C. reinhardtii.

• DFG Programme: Research Grants