Phototropin localization and interactions regulate photophysiological processes in Chlamydomonas reinhardtii

Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. In Chlamydomonas reinhardtii, phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. Although prior studies have identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, its broader interaction network and regulatory mechanisms remain poorly understood. In this study, we identified novel protein partners of phototropin and their roles in modulating its regulatory functions in C. reinhardtii. Employing a range of intraflagellar transport (IFT) mutants of C. reinhardtii, we demonstrated that phototropin localization to the flagella and eyespot is IFT-mediated. Our results reveal novel interactions between phototropin and other photoreceptors, including-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and the signaling protein- 14-3-3. CRISPR-Cas9 generated knockouts of phototropin led to reduced expression of ChR1 and 14-3-3, accompanied by impaired photomotility of the mutants. Additionally, gene expression of LOV-HK1 and LOV-HK2 were found to be elevated under UV-light in C. reinhardtii and these had altered expression in phototropin knockout line. These findings provide novel insights into phototropin interactome and elucidate molecular mechanisms underlying its localization and signaling functions in C. reinhardtii. This work advances our understanding of phototropin-mediated signal transduction and lays the groundwork for future exploration of its broader physiological roles in cellular responses.