Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii
Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii
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DOI：
10.1128/ec.00067-09
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发表时间：
2009-07-01
期刊：
影响因子：
--
通讯作者：
Mittag, Maria
中科院分区：
文献类型：
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作者：
Boesger, Jens;Wagner, Volker;Mittag, Maria
纤毛和鞭毛是在整个进化过程中高度保守的细胞器。多年来，绿色双鞭藻莱茵衣原体一直是研究其鞭毛结构和功能的模型，其鞭毛与某些哺乳动物的纤毛相似。蛋白质组学分析显示，在这些细胞器中存在几种激酶和蛋白磷酸酶。可逆的蛋白质磷酸化可以控制纤毛搏动、运动、信号传导、长度和组装。尽管这种翻译后修饰的重要性，许多纤毛磷蛋白的身份和知识，他们在体内磷酸化位点仍然失踪。本文采用固定化金属亲和层析法从纯化的鞭毛中富集磷酸肽，并进行质谱分析。共鉴定出141个磷酸化肽段，分别属于32个鞭毛蛋白。由此，确定了126个体内磷酸化位点。鞭毛磷酸化蛋白质组包括不同的结构和动力蛋白、激酶、具有蛋白质相互作用结构域的蛋白质以及许多功能尚不清楚的蛋白质。在几种情况下，一个动态的磷酸化模式和磷酸化位点的集群被发现，表明一个复杂的生理状态和特定的控制可逆的蛋白质磷酸化的鞭毛。
Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.