The objectives of this project are to understand how rhodopsin is activated and how the signals involving rhodopsin function in the photoreceptor cell. The unicellular eukaryote, Chlamydomonas reinhardtii, was developed as a model system since it has a rhodopsin-based visual system homologous with higher animals. One of its unique advantages is that its opsin can incorporate retinal analogs in vivo. A second advantage is that the expression of opsin can be specifically regulated and the genes regulated by rhodopsin excitation can be assayed. Further, these advantages can be exploited with sensitive behavioral assays and with the microbial advantages (plate colonies, short generation time, haploid genetics, ease of growth, small genome).
The specific aims of this proposal pursue the most promising leads. Pre-eminent among these leads is the evidence from this system suggesting that cis-trans isomerization of retinal probably does not activate rhodopsin. Since several nonbleachable retinal analogs fully activate Chlamydomonas and bovine rhodopsin, the played by specific residues of opsin in activation can be explored spectroscopically. Another promising lead is the identification in Chlamydomonas of a gene homologous to mammalian rhodopsin, a corresponding eye protein on gels, and a bleachable pigment which regenerates with ll-cis-retinal. Site-specific mutagenesis and measurement of the altered behavior in vivo should be possible. Determination of rhodopsin's tertiary structure may be carried out with the semicrystalline array of rhodopsin in Chlamydomonas. The rhodopsin control of gene expression, down regulation of rhodopsin, daily reabsorption of the eye, and regulation of retinal and opsin synthesis in Chlamydomonas offers the opportunity to use a molecular genetic approach to their study. The normal pathway of rhodopsin control of gene expression will be focused on and for that purpose mutants will be isolated and their order in the pathway established. We believe this work will help to reveal how abnormal signaling may be involved in mammalian photoreceptor diseases.
