EC Accepts, published online ahead of print on 8 May 2009

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Eukaryotic Cell doi:10.1128/EC.00067-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Jens Boesger, Volker Wagner, Wolfram Weisheit, and Maria Mittag^*

Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena, Am Planetarium 1, 07743 Jena, Germany

^* To whom correspondence should be addressed. Email: M.Mittag{at}uni-jena.de.

Cilia and flagella are cell organelles that have been highly conserved throughout evolution. Since many years the green biflagellate alga Chlamydomonas reinhardtii serves as a model to examine 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 identity of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites is still missing. Here we used immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from purified flagella that were analyzed by mass spectrometry. 141 phosphorylated peptides were identified that belong 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 as well as many proteins whose function is still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites was found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.