Analysis of flagellar protein ubiquitination

Abstract

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.