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, 99 (13), 8689-94

Two Rhodopsins Mediate Phototaxis to Low- And High-Intensity Light in Chlamydomonas Reinhardtii

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Two Rhodopsins Mediate Phototaxis to Low- And High-Intensity Light in Chlamydomonas Reinhardtii

Oleg A Sineshchekov et al. Proc Natl Acad Sci U S A.

Abstract

We demonstrate that two rhodopsins, identified from cDNA sequences, function as low- and high-light-intensity phototaxis receptors in the eukaryotic alga Chlamydomonas reinhardtii. Each of the receptors consists of an approximately 300-residue seven-transmembrane helix domain with a retinal-binding pocket homologous to that of archaeal rhodopsins, followed by approximately 400 residues of additional membrane-associated portion. The function of the two rhodopsins, Chlamydomonas sensory rhodopsins A and B (CSRA and CSRB), as phototaxis receptors is demonstrated by in vivo analysis of photoreceptor electrical currents and motility responses in transformants with RNA interference (RNAi) directed against each of the rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor currents differ greatly in transformants in accord with the relative amounts of photoreceptor pigments expressed. The data show that CSRA has an absorption maximum near 510 nm and mediates a fast photoreceptor current that saturates at high light intensity. In contrast, CSRB absorbs maximally at 470 nm and generates a slow photoreceptor current saturating at low light intensity. The relative wavelength dependence of CSRA and CSRB activity in producing phototaxis responses matches precisely the wavelength dependence of the CSRA- and CSRB-generated currents, demonstrating that each receptor mediates phototaxis. The saturation of the two photoreceptor currents at different light fluence levels extends the range of light intensity to which the organism can respond. Further, at intensities where both operate, their light signals are integrated at the level of membrane depolarization caused by the two photoreceptor currents.

Figures

Figure 1
Figure 1
Constructs of hairpin double-stranded (ds)RNAi. SacI/KpnI fragments of pSP124S (A), RNAi against CSOA (B), and RNAi against CSOB (C). Numbers within squares correspond to exons in the CSOA and CSOB genes. The rbcS2 sequence is a promoter for ribulose bisphosphate carboxylase/oxygenase in C. reinhardtii (27) and ble is the phleomycin-resistance gene (29).
Figure 2
Figure 2
Primary sequences of the rhodopsin domains of sensory opsins from C. reinhardtii (CSOA and CSOB) aligned with sensory rhodopsin II (NpsopII) from Natronobacterium pharaonis, and bacteriorhodopsin (BR) from Halobacterium salinarum. Conserved residues are marked with black boxes and residues predicted to be in the retinal-binding pocket are marked with a red asterisk. The drawing of CSRA and CSRB is based on known microbial rhodopsin structures and secondary structure predictions.
Figure 3
Figure 3
(A) Comparison of CSOA expression in C. reinhardtii strain 495 transformant with pSP124S (lane 1), 495 with A-RNAi (lane 2), 495 with B-RNAi (lane 3), and CSOA expressed in Pichia pastoris (lane 4). Antibody against CSOA C-terminal peptide was used in this immunoblot. The molecular mass of CSOA is 76–80 kDa on SDS/PAGE. (B) Photoreceptor currents in control cells, and A-RNAi and B-RNAi transformants. Currents were recorded under identical conditions in suspensions of nonoriented cells by unilateral excitation with a broad-band (350–550 nm) flash. (C and D) Sets of photoelectric currents excited by flashes with incrementally decreasing energy (approximately 60% steps) in A-RNAi (CSRB-enriched) and B-RNAi (CSRA-enriched) transformants, respectively.
Figure 4
Figure 4
(A) Fluence-response curves for the amplitude of the photoreceptor currents in CSRA-enriched and CSRB-enriched cells. (B) Ratio of the amplitudes of the high-light-saturating to low-light-saturating functions derived from fits of fluence-response curves for controls, CSRA-enriched, and CSRB-enriched cells. Experimental data were fit by the sum of two hyperbolae.
Figure 5
Figure 5
(A) Action spectra for photoreceptor currents in CSRA-enriched and CSRB-enriched cells. Sensitivity is the relative effectiveness of monochromatic light determined from fluence-response curves as the reciprocal of the fluence that gives a photoreceptor-current amplitude equal to that at a reference wavelength (500 nm for CSRA and 470 nm for CSRB), expressed in percent of the reference value. Spectra were measured at fluences of ≈1 μE/m2 (1 μE = 1 μmol of photons). Reproducibility was better than 90%. (B) Relative effectiveness of a spectral band absorbed predominantly by CSRA (480–520 nm) compared with that absorbed predominantly by CSRB (430–470 nm) in the generation of photoreceptor currents measured in the nonoriented mode (red columns), preoriented mode (green columns), and in the phototaxis-motility response (blue columns) in CSRA- and CSRB-enriched cells. Relative effectiveness was determined from fluence-response curves as a reciprocal of fluence (for photocurrents) and fluence rate (for phototaxis) giving equal responses. (See text for details.)
Figure 6
Figure 6
Scheme of light signal transduction for photomotility responses initiated by the two sensory rhodopsin pigments in Chlamydomonas.

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