The flagellar membrane of Chlamydomonas is a specialized domain of the plasma membrane enriched in raft lipids
A. Castillo-Flores1, J. Evans2, and G.B. Witman1
1) Department of Cell Biology and 2) Department of Biochemistry & Molecular Pharmacology
University of Massachusetts Medical School, Worcester, MA 01655, U.S.A.
Chlamydomonas has been extensively investigated as a model for the role of cilia in human health and disease. However, almost all studies of flagella have focused exclusively on proteins with no attention to lipids. Because lipids are likely to be important in sorting of flagellar proteins at the TGN, in intraflagellar transport, and in the function of cilia, our objective is to establish the lipidome of the Chlamydomonas flagellum. Wild-type and cell-wall mutant cells were grown under controlled conditions and deflagellated by treatment with dibucaine. The lipid profile of whole cells, cell bodies, flagella, and cell body plasma membrane (PM), isolated by two-phase partitioning, were compared by mass spectrometry. The analyses revealed that flagella, cell bodies, and PM have different lipid profiles. The most abundant fatty acids (FAs) in flagella, representing 95% of the total, were the short-chain saturated palmitic (37%) and stearic (22%) acids, and the unsaturate d oleic (15%) and gamma-linolenic (21%) acids. Gamma-linolenic acid is highly enriched in flagella as compared to the PM (2%) and to cell bodies (5%). The ratio of saturated to unsaturated FAs in flagella was higher (1.5) than in cell bodies (0.4), but lower than in PM (7.4). The plant membrane raft lipids phytosphingosine, ergosterol, stigmasterol, beta-sitosterol, and phosphatidylethanolamine (PE), together with diacylglycerols (DAGs) and five unidentified lipids are enriched in flagella 2-30x vs. the PM. These results show for the first time that the flagellar membrane of Chlamydomonas has a distinct lipid profile vs. the PM with which it is ultrastructurally continuous, and that flagella are enriched, by an unknown mechanism, with phytosphingosine, phytosterols, DAGs, and PE. The lipid raft-like composition of the flagellar membrane of Chlamydomonas may have a pivotal role in the assembly and function of flagella.
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