Probing circadian rhythms in Chlamydomonas rheinhardtii by functional proteomics

In the unicellular flagellated green alga Chlamydomonas reinhardtii several processes are regulated by the circadian clock. To study circadian controlled processes, the cell's clock is synchronized in a 12 h light-12 h dark cycle (LD12:12) before the cells are released into constant conditions of dim light and temperature. Under these free-running conditions circadian rhythms will continue with a period of about 24 h and cells can be harvested during specific time-points of subjective day and night. These cells were then used for isolating basic proteins by heparin-affinity chromatography, separating them on two-dimensional PAGE and comparing the amount of their expression at four different time-points of subjective day and night. Among 230 proteins, we could find two proteins whose expression level changed more than fourfold throughout the circadian cycle. These proteins were identified as a protein disulfide isomerase (PDI)-like protein and a tetratricopeptide repeat (TPR) protein by liquid-chromatography-electrospray ionization mass spectrometry (LC-ESI-MS).