Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Abstract

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.Cilia and flagella, which are essentially identical, are among the most ancient cellular organelles, providing motility for primitive eukaryotic cells living in aqueous environments. The assembly and motility of flagella have been studied extensively with the unicellular biflagellate green alga Chlamydomonas reinhardtii. This alga uses flagella for motility and for cell-cell recognition during mating. In basal land plants, such as bryophytes and pteridophytes, the only flagellated cells are motile sperm cells, which require water to swim to the egg. With the evolution of pollen tubes in higher gymnosperms and angiosperms, these plant species lost the ability to assemble flagella (24, 42). Flagella of animals have acquired new functions in multicellular organizations during evolution (6). In mammals, cilia and flagella can be motile or immotile. Motile cilia can be found, for example, in airways (respiratory cilia), in the brain (ependymal cilia), or in the male reproductive system (sperm flagella). Defects in cilia in humans can cause severe diseases, such as polycystic kidney disease, retinal degeneration, hydrocephalus, or changes in the left-right symmetry of organs, collectively known as ciliopathies (20, 32).Although C. reinhardtii and mammals are separated by more than 10 9 years of evolution, C. reinhardtii flagella are amazingly similar in structure and function to the 9ϩ2-type axonemes of most motile mammalian flagella and cilia (42). They are composed of nine microtubular doublets surrounding two central microtubular singlets. The axoneme of motile flagella includes substructures such as dynein arms and radial spokes that generate and...

Subunit Interactions and Organization of the Chlamydomonas reinhardtii Intraflagellar Transport Complex A Proteins

“…This is in contrast to the IFT A proteins isolated from the parental strain (CC-503) that also exhibited a fairly broad sedimentation, which clearly peaked at ϳ16 S with approximately half of the IFT43 present in the 16 S fractions and the other half present near the top of the gradient. It is interesting to note that the 2 S and 16 S IFT43 ran on the SDS-PAGE as a doublet, which is consistent with a previous phosphoproteomic analysis that showed that a fraction of the flagellar form of Chlamydomonas IFT43 is phosphorylated (77). The presence of the IFT43 doublet in the ift121 mutant suggests that this phosphorylation is not dependent on the association of IFT43 with complex A.…”

“…A complete loss of an IFT A subunit, such as that observed with a murine IFT122 knock-out strain shows that the loss of the IFT A complex is likely to be embryonic lethal in humans (49,87). Based on our results and the recent mammalian IFT A studies (77), it is likely that complex A is severely disrupted in the IFT122 knock-out strain; such a disruption would explain why the cilia in these mice are considerably shorter than normal (incomplete assembly) and engorged (defective in retrograde transport). Thus, mutations resulting in more subtle phenotypes are likely required to generate disease states that allow full gestation and live birth.…”

CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor

“…CK1 is involved in biological processes including circadian rhythm establishment, vesicular trafficking, DNA repair, the cell cycle, and morphogenesis (Gross et al, 1995;Akashi et al, 2002;Cheong and Virshup, 2011). A number of cytoskeleton-related proteins have been identified as targets of CK1 kinases; these targets include cofilin, twinfilin, myosin, tropin, spectrin3, dynein, a-/b-tubulin, microtubuleassociated protein, and kinesin-like protein (Boesger et al, 2014;Knippschild et al, 2014;Peng et al, 2015). In addition to the other cytoskeleton-related proteins, CK1 directly phosphorylates actin protein in vitro (Shibayama et al, 1986;Knippschild et al, 2005).…”

Multiple Roles and Interaction Factors of an E-Box Element in Chlamydomonas reinhardtii

“…Data analysis was done using the Proteome Discoverer software (version 1.0) from Thermo Electron including the SEQUEST algorithm (Link et al, 1999). The parameters for all database searches were set to achieve a false discovery rate of not more than 1% for each individual analysis according to Boesger et al (2009). Data were searched against the Joint Genome Institute C. reinhardtii database (versions 2.0 and 4.0).…”