Sushmita, K. | bioRxiv Science

bioRxiv2025

The protein turnover and trafficking of Chlamyopsin6 is regulated by IFT88 and IFT52 in the Chlamydomonas reinhardtii

Microbial rhodopsin-based optogenetics has been widely applied to diverse mammalian and plant cell types for controlling membrane potential mediated responses. However, trafficking of optogenetically active protein to the desired subcellular organelle is still a major concern in optogenetic field. This could be resolved by studying the trafficking mechanism of optogenetically active protein in the native system. Current study is focused on the trafficking of two of the microbial rhodopsins named Chalmyopsin5 and Chlamyopsin6 in a green alga, Chlamydomonas reinhardtii. Chlamyopsin5 and Chlamyopsin6 are modular in nature and possess rhodopsin, histidine kinase, response regulator and cyclase domain in tandem. Immunolocalization of Chlamyopsin5 and Chlamyopsin6 in wild strain suggests their different subcellular localization; Chlamyopsin5 in eyespot and Chlamyopsin6 in flagella. Extensive immunocytochemistry of Chlamyopsin5 and Chlamyopsin6 was performed in different intraflagellar transport (IFT) components-defective strains of Chlamydomonas to dissect their trafficking mode to the destined subcellular compartment. Our results indicated the trafficking of Chlamyopsin5 to the eyespot to be independent of IFT machinery while Chlamyopsin6 to the flagella to be IFT dependent. Further, we demonstrate that IFT88 and IFT52 stabilizes Chlamyopsin6 and IFT20 interacts with Chlamyopsin6 in Chlamydomonas. Protein interactome of Chlamyopsin5 and Chlamyopsin6 indicate their role in nitrogen assimilation, gametogenesis and photoprotection in co-ordination with other photoreceptors. Collectively, our study enabled us to understand the targeting of Chlamyopsins to the subcellular compartment (eyespot and flagella). This study is important to expand optogenetic application of microbial type modular rhodopsin with histidine kinase and response regular. Further research in this direction is required to resolve the current challenge of targeting of optogenetic protein to desired subcellular compartment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/693822v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@2691edorg.highwire.dtl.DTLVardef@367a61org.highwire.dtl.DTLVardef@9c85c6org.highwire.dtl.DTLVardef@128f9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

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. While previous research has identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, many aspects of its interaction network and regulatory mechanisms remain unresolved. In this study, we explored novel interacting protein partners of phototropin and their roles in modulating its regulatory functions in Chlamydomonas reinhardtii. Employing a suite of intraflagellar transport (IFT) mutants of C. reinhardtii such as IFT172, IFT52, IFT88, IFT139, kinesin/dynein, CEP290 etc., we elucidate that phototropin localization within the flagella and eyespot is IFT-mediated. Our study highlights interaction of phototropin with other photoreceptors-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling protein-14-3-3. CRISPR-Cas9 knockouts of phototropin showed reduced ChR1, 14- 3-3 levels and exhibited impaired photomotility. Moreover, two LOV-domain containing histidine kinases, LOV-HK1 and LOV-HK2, were identified in C. reinhardtii. Gene expression of LOV-HK1 and LOV-HK2 were found to be elevated in UV-light in C. reinhardtii and their genes expression was found to be altered in phototropin CRISPR-Cas9 knockouts. This study provides new insights into phototropin signalosome and highlights molecular mechanisms governing its function. The research outcomes advances our understanding of phototropin trafficking and signal modulation in Chlamydomonas reinhardtii, and sets the stage for further exploration into the broader physiological roles of phototropin in cellular responses. Graphical abstractPhototropin, a blue-light receptor in Chlamydomonas reinhardtii, localizes to the flagella and eyespot, mediates phototaxis and photosynthesis. Its trafficking is mediated by intraflagellar transport (IFT) machinery, with mutations in IFT components (kinesin, dynein, IFT172, IFT52, IFT88, IFT139, CEP290) disrupting phototropin localization. Phototropin interacts with other photoreceptors like channelrhodospins (ChR1/2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling proteins (14-3-3), coordinating light-driven responses. These findings underscore the details of phototropin trafficking and phototropin signaling impacting light-induced physiological processes in C. reinhardtii. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/630506v1_ufig1.gif" ALT="Figure 1000"> View larger version (51K): org.highwire.dtl.DTLVardef@29f6b3org.highwire.dtl.DTLVardef@10382aeorg.highwire.dtl.DTLVardef@1786f0dorg.highwire.dtl.DTLVardef@1c2ed65_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights* Phototropin localizes in eyepot and flagella in Chlamydomonas reinhardtii. * Intraflagellar transport (IFT) mutants of C. reinhardtii suggest role of different IFT proteins in phototropin trafficking and localization. * Phototropin interacts with other photoreceptors (ChR1 & ChR2, COP6, LOV-HK1 & LOV-HK2) and signaling proteins (14-3-3), contributing to various physiological processes. * CRISPR-Cas9 knockouts of phototropin showed reduced 14-3-3 protein content and photomotility response in C. reinhardtii.

plant biology↗