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.
