Functional proteome analysis of circadian phosphoproteins in Chlamydomonas reinhardtii

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The eukaryotic unicellular model alga Chlamydomonas reinhardtii is especially suited to investigate specific scientific questions such as the mechanism of the circadian clock with regard to tactic movements (chemotaxis, phototaxis). Within the circadian system posttranslational modifications in form of phosphorylation have been shown to play a crucial regulatory role for key components of the endogenous oscillator from cyanobacteria to mammals. Two of the so far characterized kinases, DOUBLETIME (DBT) and SHAGGY are highly conserved in C. reinhardtii. DBT phosphorylates the oscillator component PERIOD in a circadian manner in flies and mammals, thereby gating the circadian feedback loop. The current project aims to depict systematically proteins from C. reinhardtii that are phosphorylated in a circadian manner by DBT. Thereby, we will apply a proteome based differential display approach. The function of the DBT phosphoproteins within the circadian system will then be characterized by silencing of these genes by RNAi and consequent analysis of the period and phase of circadian photo- and chemotaxis. In addition, circadian regulatory components of the basal appara- tus, the basis for the tactic movements, will be analyzed together with M. Melkonian. Opinion of the review panel (including one written comment): One project of Prof. Mittag (Teilprojekt 6, MI 373/6-1), which will be of high importance for the Research Unit, is already being funded by the DFG. This project had been highly recommended for funding in the previous reviewing process and was praised again by this review panel as very interesting and worthwhile. With the project to be discussed here, which also builds upon the above-mentioned Teilprojekt 6 ( MI 373/6-1), Prof. Mittag wants to identify proteins that have regulatory function within the system of circadian control of gene expression in Chlamydomonas. The project proposal submitted to the panel suggested to search for proteins that either show variable abundance and bind to RNA and/or DNA, or exhibit variation in phosphorylation at different times of the day. In general, the panel really liked the idea of analysing circadian control on the level of the proteome, and especially emphasized the innovative character of th e planned study of the phospho-proteome. However, the panel members agreed that in the form presented, the project is too diversified and unlikely to be manageable by the means available to this young research group and, therefore, would be at risk of generating too many open ends. Re-focussing on just one of the suggested aspects was demanded.