The Protein Composition of the Chlamydomonas Flagellar Membrane Is Dynamically Regulated by Cilium-Generated Signaling During Fertilization



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The cellular and molecular mechanisms specifying the membrane protein composition of cilia and flagella at steady state and during cilium-generated signaling are poorly understood. Our laboratory uses the biflagellated green alga Chlamydomonas as a model system to study regulated movement of the flagellar adhesion receptor SAG1 from the cell body to the flagella. Interactions between the plus flagellar receptor (agglutinin) SAG1 and its cognate receptor SAD1 on flagella of minus gametes induces flagellar adhesion and activation of a cAMP-dependent signaling pathway ultimately leading to cell-cell fusion. Although previous work from our laboratory and others suggested that pathway activation triggers mobilization of a pool of SAG1 from the cell body to the flagella, those studies depended on indirect adhesion bioassays detecting the activity of SAG1, not the protein. Here, I report use of new tools to study directly the regulation of SAG1 localization. I show that the SAG1 gene bearing a C-terminal HA tag rescues flagellar adhesion and cell fusion in the flagellar adhesion mutant, sag1-5. Immunofluorescence studies of resting SAG1-HA/sag1-5 mt+ plus gametes show that the protein is present mostly on cell bodies. Biochemical studies show that only gametes express SAG1-HA. Detection of precursor forms indicates SAG1 undergoes cleavage soon after its synthesis to yield an HA-tagged 65 kDa, C-terminal portion (SAG1-HA-C65), and that SAG1-HA-C65 is on the cell surface. Consistent with the predicted 3 transmembrane domains at the C-terminus, release of SAG1-HA-C65 in a soluble form requires detergent and is not achieved upon mechanical disruption, high salt, or high pH treatments. Cell fractionation demonstrates that in resting gametes the majority of SAG1-HA-C65 is present on cell bodies and only a small amount is on flagella. Within minutes after signaling is triggered by flagellar adhesion, however, SAG1-HA-C65 is mobilized from the cell body to the flagella and the organelles become highly enriched in the protein, where it forms large detergent-resistant complexes. I show that Chlamydomonas can regulate the amount of SAG1 in the flagella through cilium-generated signaling, and thus provides the first system for studying regulation of the membrane protein composition of the cilium/flagellum in a biochemically tractable system. [Keywords: Chlamydomonas reinhardii, flagella, cilia, fertilization, signaling, SAG1, cell adhesion, agglutinins, ciliary membrane]