Rhodopsin Signaling in a Model Eucaryotic Microorganism

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Chlamydomonas reinhardtii exhibits two distinct photomotility responses to light: phototaxis migration and the photophobic avoidance response. These are mediated by retinal-containing receptors ("rhodopsins"). Both behaviors may be governed by the same receptor or by two different receptors. For both behaviors, photoactivation has been localized in the eyespot organelle of the cell and results in a cascade of transmembrane electrical currents, which ultimately alter calcium concentration in the flagella and induce alteration of their beating. The generation of a photoreceptor current is the earliest event in this cascade, which is a combination of at least two kinetically distinct electrochemical processes in the photoreceptor membrane. Using pulsed laser excitation to analyzed the electrical responses of the cells, it was demonstrated that the faster component is calcium-independent, is induced within three microseconds of photon absorption, and must be attributed therefore not to a secondary calcium flux but to an early photoreaction of a photoreceptor protein. The objectives of this project are to identify the rhodopsin protein(s) and elucidate their signaling mechanism. The approach is to identify and characterize Chlamydomonas gene(s) encoding the receptors and components of the photo-signaling pathway by insertional mutagenesis, selection for loss of each of the two responses by capillary selection techniques, and subsequent testing of the mutants for the presence of rhodopsin-mediated photocurrents. In parallel, the two photoreceptor currents will be analyze to elucidate their ion dependencies and distinguish whether (1) they represent consecutive steps in the signal transduction chain mediated by a single photoreceptor; (2) there is parallel activation of the two currents by a single bi-functional photoreceptor; or (3) the two currents derive from multiple photoreceptors with different effects on transmembrane ion fluxes. Two classes of rhodopsins are known: (1) visual pigments in animals and (2) archaeal rhodopsins, ion transport, and photosensory proteins studied primarily in halobacteria, and recently demonstrated as well in eukaryotes (fungi). PCR and reduced-stringency Southern hybridization, and the Chlamydomonas genome project will augment the search for genes in Chlamydomonas encoding either of the two types. The results will significantly expand our knowledge of mechanisms of photosensory transduction mediated by rhodopsin proteins, and also may clarify their evolution.