Retinylidene Phototransducers in a Eukaryotic Model System

已有令人信服的证据表明，在单细胞真核生物中存在视紫红质样光感受器。这些光感受器在自然界中构成了一类新的含有视网膜的感觉蛋白，在进化上介于高等动物的视觉色素和古细菌的感觉视紫红质之间。在莱茵衣藻中，两种不同的运动行为被证明依赖于视网膜的光检测：趋光性迁移和恐光反应。间接证据表明，视网膜生色团的厌光反应在化学上类似于其视网膜/载脂蛋白与古细菌视紫红质的相互作用。另一方面，有数据表明，趋光性受体在生色团性质上更类似于视觉色素。本研究的目的是确定导致莱茵梭子虫光行为的视紫红质样色素的数量，确定光感受器(S)的光谱和生化性质，并利用这些信息克隆相应的结构基因(S)。将通过定量运动分析来分析视网膜模拟重组细胞的趋光性和厌光性，以建立视黄亚甲基光受体(S)的光谱和生色团结合特性。光行为突变体将被分离和鉴定。这项工作将为光感受器色素的光谱鉴定以及蛋白质浓缩和放射性标记的视网膜结合片段的分离提供标准。将获得蛋白质初级序列信息，并用于设计寡核苷酸探针。%对于大多数生物来说，探测和响应光刺激的能力是它们生存的关键基本属性。在动物中，光的主要探测器是所谓的视觉色素，一种名为视紫红质的蛋白质，其功能与视网膜(维生素A)有关。在古细菌的光反应装置中发现了一种相关但不同形式的视紫红质。该实验室以前对考古细菌视紫红质所做的工作非常重要，这不仅是因为有可能扩大关于视紫红质感觉功能的一般知识，而且还为开发具有广泛应用前景的潜在的新一代基于视紫红质的微型光电探测器奠定了基础。在目前的研究中，本实验室重点研究了单细胞运动型绿藻衣藻的光响应。这种有机体表现出两种不同类型的光行为：趋光性反应和恐光性反应，前者是有机体游向光线，后者是有机体游过突然的闪光。初步数据表明，这两种光反应是由不同的视紫红质介导的，一种类似于动物的视觉色素，另一种类似于考古细菌光传感器。这些光响应将以多学科的方式进行研究，结合遗传学、分子生物学、生物化学和生物物理测量，以扩大我们对这类重要的光响应生物大分子的理解。

Compelling evidence has accumulated for the existence of retinylidene (rhodopsin-like) photosensory receptors in unicellular eukaryotes. These photoreceptors constitute a new class of retinal-containing sensory proteins in nature, evolutionarily intermediate between visual pigments in higher animals and sensory rhodopsins in archaebacteria. In Chlamydomonas reinhardtii, two distinct motility behaviors have been shown to depend on retinal for light detection: phototaxis migration and photophobic responses. Indirect evidence indicates the retinal chromophore for the photophobic response is chemically similar in its retinal /apoprotein interactions to the archaebacterial rhodopsins. On the other hand, there is data indicating the phototaxis receptor is more similar in chromophore properties to visual pigments. The aims of this research are to establish the number of rhodopsin-like pigments responsible for C. reinhardtii photobehavior, to determine spectroscopic and biochemical properties of the photoreceptor(s), and to use this information to clone the corresponding structural gene(s). Phototactic and photophobic behavior of retinal analog- reconstituted cells will be analyzed by quantitative motion analysis to establish spectroscopic and chromophore binding properties of the retinylidene photoreceptors(s). Photobehavioral mutants will be isolated and characterized. This work will provide criteria for spectroscopic identification of the photoreceptor pigments and for protein enrichment and isolation of radio-labeled retinal binding fragments. Protein primary sequence information will be obtained and used to design oligonucleotide probes. %%% For most organisms, the ability to detect and respond to light stimuli is a fundamental property critical to their survival. In animals, the primary detector of light is the so-called visual pigment, a protein called rhodopsin which functions in association with retinal (vitamin A). A related but different form of rhodopsin is found in the photoresponsive apparatus of archaebacteria. Prior work with archaebacterial rhodopsin by this laboratory has been important not only because of the potential for expanding knowledge about rhodopsin sensory function in general, but also for laying the groundwork for development of a potential new generation of rhodopsin-based miniature photodetectors with broad applications. In the present study, this laboratory is focusing on the photoresponses of a unicellular motile green alga, Chlamydomonas. This organism exhibits two different types of photobehaviors: a phototactic response, in which the organism swims toward the light, and a photophobic response, in which the organism swims away from a sudden flash of light. Preliminary data suggest that the two photoresponses are mediated by different rhodopsins, one resembling the visual pigment of animals and the other resembling the archaebacterial photosensor. These photoresponses will be studied in a multidisciplinary fashion, combining genetics, molecular biology, biochemistry, and biophysical measurements, to expand our understanding of this important class of photoresponsive biomacromolecules.