Long Huan, Huang Kaiyao
Institute of Hydrobiology, Chinese Academy of Sciences, Wuchang District, Wuhan, China.
Methods Enzymol. 2013;524:59-73. doi: 10.1016/B978-0-12-397945-2.00004-4.
Flagella/cilia are conserved organelles existing in unicellular protists and multicellular animals, where they perform essential motile and sensory functions. Their assembly and disassembly are coordinated with the cell cycle, and recent evidence shows that posttranslational modifications such as phosphorylation, methylation, and ubiquitination are involved in these two processes, perhaps through interacting with intraflagellar transport (IFT), a specialized intracellular transport that is required for the assembly and maintenance of flagella/cilia. In this chapter, we summarize the components of the ubiquitination system published in proteomic databases of flagella/cilia. Furthermore, we describe procedures to analyze the ubiquitin-conjugating system in Chlamydomonas flagella and to analyze flagellar protein ubiquitination during flagellar shortening and the mating process. These results and tools will be valuable for the characterization of substrates of ubiquitination and their roles in flagellar disassembly and in regulating signal transduction pathways in flagella/cilia.
鞭毛/纤毛是存在于单细胞原生生物和多细胞动物中的保守细胞器，它们在其中执行基本的运动和传感功能。它们的组装和拆卸与细胞周期协调，最近的证据表明，诸如磷酸化、甲基化和泛素化等翻译后修饰参与了这两个过程，可能是通过与鞭毛内运输（IFT）相互作用，IFT是鞭毛/纤毛组装和维持所需的一种特殊的细胞内运输。在本章中，我们总结了鞭毛/纤毛蛋白质组数据库中公布的泛素化系统的组成成分。此外，我们描述了分析衣藻鞭毛中泛素结合系统以及分析鞭毛缩短和交配过程中鞭毛蛋白泛素化的程序。这些结果和工具对于表征泛素化底物及其在鞭毛拆卸和调节鞭毛/纤毛信号转导途径中的作用将是有价值的。
