Functional proteome analysis of circadian phosphoproteins in Chlamydomonas reinhardtii

真核单细胞模型藻莱茵衣藻特别适合于研究特定的科学问题，如关于策略运动(趋化性、趋光性)的生物钟的机制。在昼夜节律系统中，磷酸化形式的翻译后修饰被证明对从蓝藻到哺乳动物的内源振荡器的关键成分起着至关重要的调节作用。到目前为止所表征的两种蛋白激酶，Doubletime(DBT)和Shaggy在莱茵哈迪尔乳杆菌中高度保守。在果蝇和哺乳动物中，DBT以昼夜节律的方式使振荡器成分周期磷酸化，从而门控昼夜节律反馈环。目前的项目旨在系统地描述由DBT以昼夜节律方式磷酸化的莱茵梭菌蛋白质。因此，我们将应用基于蛋白质组的差异显示方法。DBT磷酸蛋白在昼夜节律系统中的功能将通过RNAi沉默这些基因来表征，并随后分析昼夜趋光性和趋化性的周期和阶段。此外，作为战术动作基础的基础审查小组的意见(包括一项书面意见)：米塔格教授的一个项目(Teilprojekt 6，MI 373/6-1)对研究股非常重要，该项目已由副总干事资助。该项目在前一次审查过程中曾被强烈推荐资助，并再次被本次审查小组称赞为非常有趣和值得。随着这里将要讨论的项目，它也建立在上述Teilprojekt 6(MI 373/6-1)的基础上，Mittag教授希望识别在衣藻基因表达的昼夜控制系统中具有调节功能的蛋白质。提交给专家小组的项目提案建议寻找丰度可变并与RNA和/或DNA结合的蛋白质，或者在一天中的不同时间表现出不同的磷酸化变化。总的来说，专家小组非常喜欢在蛋白质组水平上分析昼夜节律控制的想法，并特别强调了计划中的磷酸蛋白质组研究的创新性。然而，小组成员一致认为，以目前提出的形式，该项目过于多样化，不太可能以这个年轻的研究小组可用的手段加以管理，因此有可能产生太多的开放式方案。只需要重新关注所建议的一个方面。

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.