Abstract：
In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.
摘要：
在衣藻中，直接光门，质膜定位的阳离子通道视紫红质ChR1和ChR2是趋光性的主要光感受器。它们的目标和丰富度对于最佳运动反应至关重要。然而,我们对衣藻如何实现这一目标的了解仍处于起步阶段。在这里，我们表明ChR1内化是通过光刺激的内吞作用发生的。在内吞之前或期间,ChR1被修饰并形成高分子量复合物。这些是细胞外囊泡中唯一可检测的ChR1形式，并且它们的丰度在照射时动态变化。含ChR1的细胞外囊泡通过质膜和/或纤毛碱基分泌。与此相符,纤毛发生突变体表现出增加的ChR1降解速率。Further,我们确定了半胱氨酸蛋白酶CEP1的参与，这是木瓜蛋白酶C1A亚家族的成员。ΔCEP1敲除菌株缺乏光诱导的ChR1降解，而ChR2降解不受影响。低光照刺激CEP1表达，它是通过促光素调节的，SPA1泛素连接酶和环AMP。Further,突变体和抑制剂分析显示，小GTP酶ARL11和SUMO化参与了靶向眼点和纤毛的ChR1。因此，我们的研究定义了衣藻的这种中央光感受器的降解途径，并确定了参与其同型平衡和靶向的新元件。
