Intraflagellar transport-20 mediates the ciliary membrane trafficking of channelrhodopsin 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, and plays a role in ciliary membrane protein trafficking. Here, we analyzed IFT20-mediated ciliary membrane trafficking of channelrhodopsin-1 (ChR1) in Chlamydomonas reinhardtii . IFT20 and ChR1 was found to be co-localized throughout the cilia of C. reinhardtii wild-type strain. In bbs1 mutants, IFT20 exhibited distribution along the flagellar length and basal body region. In contrast, ChR1 was found to be accumulating in the distal region of the cilia. Further, protein interaction network analysis reveals that IFT20 serves as a central adaptor, interfacing ancillary ciliary trafficking components, including Arf, the IFT complex, and BBSome subunits. The Arf co-localized with IFT20 in the cilia of the wild-type strain, suggesting a potential interaction. The fluorescence spectroscopic analysis shows that upon GTP binding, IFT20 undergoes concentration-dependent fluorescence quenching. From the far-UV CD spectroscopic analysis, it was observed that recombinant IFT20 has a predominantly helical structure, which does not change upon GTP binding. These findings extend the mechanistic understanding of ciliary membrane protein delivery in C. reinhardtii and reveal a conserved role of IFT20 in photoreceptor trafficking.