Identification, expression and characterization of archaeal-type opsins of Chlamydomonas reinhardtii

Phototaxis and photophobic responses of green algae are mediated by rhodopsin-based photoreceptors that use microbial-type chromophores (all-trans retinal). Analysis of stimuli-response curves of the C. reinhardtii photoreceptor current led to the suggestion that they are based on two photosystems, one of which is more active at low flash intensities, whereas the other dominates at high flash energies. Two cDNA sequences were identified in the EST database of the C.reinhardtii that encoded microbial-type opsins, which were named Chlamyopsin-3 and 4 (Cop-3 and Cop-4) respectively, based on their homology to the known microbial-type opsins. The seven-transmembrane helices at the N-terminus of these opsins showed homology to the light-activated proton pump, bacteriorhodopsin (BR). However, after functional expression in the oocytes of X. laevis these opsins were renamed as Channelopsin-1 and 2 (Chop-1 and Chop-2) based on their ion channel activity. The amino acids that form the H+-conducting network in BR are conserved in Chop-1 and Chop-2, whereas the rest of their sequences are different. Heterologoulsy expressed Channelopsins (Chop-1 and 2) in E.coli formed inclusion bodies, and thus recombinant proteins were not functional. Expression of Chop-1 in P. pastoris led to the production of non-functional protein, since it did not bind all-trans retinal. Functional expression of Chop-1 mRNA in the oocytes of X. laevis (Chop-1 + all-trans retinal =ChR1) showed a light-gated ion channel conductance, which was studied in detail using a two-electrode voltage clamp technique (Nagel et al., 2002). The observed transport activity was purely passive and directly dependent on the membrane potential and the proton concentration gradient in bath solution. Outward photocurrents could be observed at high exracellular pH or low intracellular pH. The conductance was highly selective for protons, and other monovalent or divalent ions were not found to be permeating. It was also observed by mutational analysis that the H173 residue of Chop-1 does not function as a proton donor of a deprotonated Schiff base. Therefore, it was suggested that in ChR1 the retinal Schiff base is not de-protonated during the photocycle. It is likely that such light sensitive ion channels are widely distributed in other phototactic microalgae, as well as in gametes and zoospores of the macroalgae. This claim is corroborated by the observation that Volvoxopsin-2 (Vop-2) a partial opsin like sequence was identified in the V. carteri genome project, which showed 75% identical amino acid residues to Channelopsins in the helices 5-7 of the opsin domain. Heterologous expression of Chop-2 was also carried out in E.coli to produce functional recombinant protein. It was observed that the expression characteristic of Chop-2 were similar to that of Chop-1. Therefore, Chop-2 was directly expressed in Xenopus oocytes, in the presence of all-trans retinal to produce functional Channelrhodopsin-2 (ChR2) (Nagel et al., 2003). Photocurrents were recorded from these oocytes using two-electrode voltage clamp method. However, the cells not only became conductive for protons but also, most surprisingly, for monvalent and divalent cations like Na+ K+ and Ca++. It was concluded that ChR-2 functions as a cation-selective channel. Surprisingly, and in contrast to ChR1, the light-gated conductance of ChR-2 inactivates in continuous light to a smaller steady-state level. Western blotting analysis with membrane fractions of C. reinhardtii using anti-Chop1 and Chop2 antibodies revealed that both proteins were abundant, when cells were grown in low light conditions, both are degraded under high light conditions and that ChR2 was degraded more rapidly than ChR1. In conclusion, it is likely that both channelrhodopsins control photophobic responses and only indirectly influence phototaxis. Very recently, three more protein sequences were found in C. reinhardtii genome database, which showed homology to the sensory opsin. Surprisingly, all three sequences were coupled to a transducer like protein (HtrI and II). We have provisionally named these sequences Cop5, Cop6 and Cop7. Isolation, sequencing and bioinformatic analysis of Cop-5 protein sequence revealed that it is a unique putative opsin, which has four modular domains (Opsin, HK, RR and CYCc) in one protein. The assumption that one of these new rhodopsins could be responsible for phototaxis movement in C. reinhardtii seems to be justified. Nevertheless, other functions like control of retinal biosynthesis or developmental processes should also be taken into account