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

The primary cilium is a microtubule-based structure essential for cellular signaling, whose assembly and function depend on targeted delivery of cargo via intraflagellar transport (IFT). Intraflagellar transport-20 (IFT20), a unique IFT-B component, localizes to both the Golgi and cilia, suggesting a central role in ciliary trafficking. We analyzed the IFT20-mediated trafficking (CrIFT20) of photoreceptor channelrhodopsin-1 (ChR1) in the cilia of Chlamydomonas reinhardtii using an integrative approach that combines computational and experimental methods. The sequence homology and structural modelling reveal a conserved C-terminal coiled-coil domain in IFT20, supporting its canonical function in IFT-B assembly. Furthermore, the heterologous expression and spectroscopic characterization demonstrate that recombinant CrIFT20 is structurally stable and undergoes GTP-dependent structural rearrangements. This is consistent with our docking analyses, which show that human Arf4 homolog in C. reinhardtii , small ARF-related GTPase (CrARFA1A) interacts with the conserved coiled-coil region of CrIFT20 in a GTP-binding interface. Interaction network mapping and structural docking supported a conserved mechanism in which CrIFT20 couples ciliary trafficking with IFT-B and BBSome components, such as BBS1, for precise delivery of ciliary membrane cargo. The IFT81 and BBS1 mutants of C. reinhardtii mislocalizes IFT20-ChR1 complex in cilia and disrupts photoreceptor distribution, as supported by structural models that reveal the corresponding adaptor-cargo interfaces. Our results identify CrIFT20 as an essential adaptor linking IFT-B complex, CrARFA1A-mediated membrane trafficking, and BBSome-dependent sorting to direct ChR1 localization within the cilia of Chlamydomonas . These findings expand the mechanistic understanding of photoreceptor delivery in algae, and reveal a conserved role of IFT20 in photoreceptor trafficking, illuminating how coordinated adaptor, motor, and cargo selection maintain the polarized architecture of the cilia.