Roberts D G, Lamb M R, Dieckmann C L
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Genetics. 2001 Jul;158(3):1037-49. doi: 10.1093/genetics/158.3.1037.
The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.
单细胞双鞭毛绿藻莱茵衣藻能够感知光线，并通过改变其游动行为做出反应。眼点是一种专门用于感知光线的结构，在每次细胞分裂时，它都会由位于两个不同细胞区室的成分重新组装而成。光感受器和相关的信号转导成分定位于质膜的一个离散斑块中。这个斑块紧密地贴靠在叶绿体膜和充满类胡萝卜素的脂质颗粒的下层夹层上，这有助于细胞区分光的方向。在之前对眼点缺陷突变株的筛选中，EYE2基因座由单眼2-1等位基因定义。通过光学显微镜观察，该突变株没有眼点，通过电子显微镜判断，它没有有组织的类胡萝卜素颗粒层。在这里，我们证明眼2-1突变株能够对光做出反应，尽管该菌株对低光强度的敏感性远低于野生型且定向不准确。因此，叶绿体中的色素颗粒层组装对于质膜中的光感受器定位不是必需的。一个质粒插入诱变筛选产生了眼2-2等位基因，这使得EYE2基因得以分离和表征。EYE2蛋白是硫氧还蛋白超家族的成员。对活性位点半胱氨酸进行定点诱变表明，EYE2在眼点组装中的功能不依赖于氧化还原，这与其他硫氧还蛋白家族成员在蛋白质折叠和复合物组装中的辅助功能类似。
