Molecular Mechanisms of in vivo Channelrhodopsin Regulation in Chlamydomonas reinhardtii
Final Report Abstract
In our project, we set out to analyze the molecular mechanisms how the green alga Chlamydomonas reinhardtii controls the abundance of the two Channelrhodopsins ChR1 and ChR2 and how their activities are integrated into its general photoprotective network(s). The analyses were focused on the dominant ChR1 and led to the following key findings: The ChR1 level decreases upon illumination in a light-intensity and quality-dependent manner. UV and blue light was most efficient, whereas it is stable in darkness. - Analysis of knockout strains of six major photoreceptors absorbing in the blue/UV range revealed major involvement of Phototropin (PHOT) in ChR1 degradation. Additionally, red light - most likely via affecting the cellular redox poise – can induce ChR1 degradation. - Illumination stimulates ChR1 internalization via endocytosis, prior or during which ChR1 is modified and forms high molecular mass complexes (HMMCs). These are the solely detectable ChR1 forms in extracellular vesicles (EVs) and their abundance therein dynamically changes upon illumination. The ChR1-containing EVs are secreted via the plasma membrane and/or the ciliary base and not via the cilia. - Beside PHOT, we identified a COP1-SPA1 E3 ubiquitin ligase, the transcription factor Hy5 and cAMP as additional components involved in ChR1 degradation. - The cysteine protease CEP1 is essential for ChR1 degradation. Its expression is stimulated by low-light and is regulated via PHOT, a COP1-SPA1 E3 ubiquitin ligase and cAMP. Additionally, a Calpain protease might be involved in ChR1 internalization. - The levels of ChR1 and ChR2 appear to be regulated in different ways, as ChR2 degradation was unaffected in ∆PHOT and ∆CEP1 strains. - Further, the small GTPase ARL11, protein SUMOylation, the intraflagellar transport machinery and the cilia are involved in targeting and regulating the ChR1 levels. We thus highlighted the presence of an adaptive network connecting ChR1 homeostasis to known general photoprotective mechanisms of this alga in this project. Further the ChR1 degradation pathway was defined for the first time and novel important elements involved in its degradation and targeting were delineated. These data thus lay the basis for a more detailed molecular view of ChR1 homeostasis and how Chlamydomonas can dynamically adapt to the highly fluctuating light environment already at the level of this photoreceptor.
Publications
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Multifactorial in vivo regulation of the photoreceptor channelrhodopsin‐1 abundance. Plant, Cell & Environment, 46(9), 2778-2793.
Wolfram, Michaela; Greif, Arne; Sizova, Irina; Baidukova, Olga; Hegemann, Peter & Kreimer, Georg
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Insights into degradation and targeting of the photoreceptor channelrhodopsin-1.
Kreimer, Georg; Wolfram, Michaela; Greif, Arne; Baidukova, Olga; Voll, Hildegard; Tauber, Sandra; Lindacher, Jana & Hegemann, Peter
