Proteomic analysis of the eyespot of Chlamydomonas reinhardtii and functional characterization of components involved in tactic movements

模型绿藻莱茵衣藻表现出照相和趋化运动。这两条信号通路可能在膜去极化水平上汇聚，而钙在不同水平上错综复杂地参与了这两种反应。我们已经开发了一种程序来纯化这种藻类的趋光性原始视觉系统，即眼斑。在下面的蛋白质组学方法中，至少用两个不同的多肽(总共984个)鉴定了202个眼斑部分的蛋白质。已识别的蛋白质包括结构成分、光感受器以及用于战术运动的代谢和假定信号通路的成员。现在我们的目标是从这个蛋白质组中获得两个蛋白质的功能信息：(I)一个钙感应受体和(Ii)一个与蓝藻趋化受体相关的蛋白质。在已鉴定的蛋白质中还包括激酶和磷酸酶，这表明磷酸化/去磷酸化在与眼斑相关的信号传递中起着重要作用。酪蛋白激酶1(CK1)的沉默影响趋光性的昼夜节律控制。然而，目前还不知道眼斑中的激酶的靶标。因此，我们还将对CK1沉默细胞和野生型细胞中的眼斑蛋白进行分析，并进行比较定量的蛋白组学分析，以确定CK1和其他激酶在这个原始视觉系统中的潜在靶点。因此，我们希望对进一步了解莱茵梭子蟹战术运动中所涉及的信号通路做出贡献。

The model green alga Chlamydomonas reinhardtii exhibits photo- and chemotactic movements. Both signaling pathways converge possibly at the level of membrane depolarization and Ca2+ is intricately involved in both responses at different levels. We have developed a procedure to purify the primitive visual system for phototaxis, the eyespot, of this alga. In the following proteomic approach, 202 proteins of the eyespot fraction were identified with at least two different peptides (984 in total). The identified proteins include e.g. structural components, photoreceptors, as well as members of metabolic and putative signaling pathways for tactic movements. Now our aim is to get functional information about two selected proteins from this proteome: (i) a Ca2+ sensing receptor and (ii) a protein related to a cyanobacterial chemotaxis receptor. Among the identified proteins were also kinases and phosphatases, indicating that phosphorylation/ dephosphorylation is important for signaling related to the eyespot. Silencing of one of the kinases, casein kinase 1(CK1), affects the circadian control of phototaxis. However, nothing is yet known about the targets of kinases in the eyespot. Therefore we will also analyze the eyespot phosphoproteome and do comparative quantitative phosphoproteomics with eyespots isolated from CK1-silenced and wild-type cells to identify potential targets of CK1 and other kinases in this primitive visual system. Thereby we hope to contribute to the further understanding of signaling pathways involved in tactic movements of C. reinhardtii.