Intraflagellar transport-20 coiled-coil domain mediates the channelrhodopsins trafficking to the cilia in Chlamydomonas reinhardtii

The primary cilium is a microtubule-based organelle essential for cellular signaling, whose assembly depends on intraflagellar transport (IFT). IFT20, a unique IFT-B component, localizes to both Golgi and cilium/flagellum, suggesting a role in membrane trafficking. Here, we analyzed CrIFT20-mediated ciliary membrane trafficking of channelrhodopsin-1 (ChR1) in Chlamydomonas reinhardtii. Spectroscopic analysis shows recombinant CrIFT20 has a predominantly helical structure and undergoes GTP-dependent shuttle conformational change. Co-immunolocalization of IFT20 in wild-type C. reinhardtii shows puncta distribution throughout cilia and co-localizes directly/indirectly with ChR1 in cilia. Further co-immunoprecipitation supports the cellular biochemical evidence that IFT20 guides the trafficking of ChR1 at the ciliary membrane. Protein interaction network analysis reveals that IFT20 serves as a central adaptor interfacing early ciliary trafficking components with IFT-B complex and BBSome subunits. These findings extend the mechanistic understanding of ciliary membrane protein delivery in C. reinhardtii and reveal a conserved role of IFT20 in photoreceptor trafficking. This study illustrates how coordinated adaptor, motor, and cargo selection maintain the ciliary distribution of ciliary membrane proteins. This study aids in delineating the underlying mechanism of protein trafficking in cilia using C. reinhardtii as a model system, crucial for studying human cilia-associated disorders (ciliopathies).