Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes†

ABSTRACT: Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pK a difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the onephoton excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connecte

Evolution of phototaxis

Phototaxis in the broadest sense means positive or negative displacement along a light gradient or vector. Prokaryotes most often use a biased random walk strategy, employing type I sensory rhodopsin photoreceptors and two-component signalling to regulate flagellar reversal. This strategy only allows phototaxis along steep light gradients, as found in microbial mats or sediments. Some filamentous cyanobacteria evolved the ability to steer towards a light vector. Even these cyanobacteria, however, can only navigate in two dimensions, gliding on a surface. In contrast, eukaryotes evolved the capacity to follow a light vector in three dimensions in open water. This strategy requires a polarized organism with a stable form, helical swimming with cilia and a shading or focusing body adjacent to a light sensor to allow for discrimination of light direction. Such arrangement and the ability of three-dimensional phototactic navigation evolved at least eight times independently in eukaryotes. The origin of three-dimensional phototaxis often followed a transition from a benthic to a pelagic lifestyle and the acquisition of chloroplasts either via primary or secondary endosymbiosis. Based on our understanding of the mechanism of phototaxis in single-celled eukaryotes and animal larvae, it is possible to define a series of elementary evolutionary steps, each of potential selective advantage, which can lead to pelagic phototactic navigation. We can conclude that it is relatively easy to evolve phototaxis once cell polarity, ciliary swimming and a stable cell shape are present

Photoinduced electric currents in carotenoid-deficient Chlamydomonas mutants reconstituted with retinal and its analogs.

Reconstitution of the photoelectric responses involved in photosensory transduction in "blind" cells of Chlamydomonas reinhardtii carotenoid-deficient mutants was studied by means of a recently developed population method. Both the photoreceptor current and the regenerative response can be restored by addition of all-trans-retinal, 9-demethyl-retinal, or dimethyl-octatrienal, while the retinal analogs prevented from 13-cis/trans isomerization, 13-demethyl-retinal and citral, are not effective. Fluence dependence, spectral sensitivity, and effect of hydroxylamine treatment on retinal-induced photoelectric responses are similar to those found earlier in green strains of Chlamydomonas, although an alternative mechanism of antenna directivity in white cells of reconstituted "blind" mutants (likely based on the focusing effect of the transparent cell bodies) leads to the reversed sign of phototaxis in mutant cells under the same conditions. The results obtained indicate that both photoreceptor current and regenerative response are initiated by the same or similar rhodopsins with arhaebacterial-like chromophore(s) and prove directly the earlier suggested identity of the photoreceptor pigment(s) involved in photomotile and photoelectric responses in flagellated algae

Photoreceptor current and photoorientation in chlamydomonas mediated by 9-demethylchlamyrhodopsin.

Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via generation of photocurrents across the plasma membrane. Chlamydomonas mutants blocked in retinal biosynthesis are "blind," but they can be rescued by the addition of exogenous retinoids. Photosignaling by chlamyrhodopsin regenerated with 9-demethylretinal was investigated by recording photocurrents from single cells and cell suspensions, and by measuring phototactic orientation. The addition of a saturating concentration of this analog led to reconstitution of all receptor molecules. However, sensitivity of the photoreceptor current in cells reconstituted with the analog was smaller compared with retinal-reconstituted cells, indicating a decreased signaling efficiency of the analog receptor protein. Suppression of the photoreceptor current in double-flash experiments was smaller and its recovery faster with 9-demethylretinal than with retinal, as it would be expected from a decreased PC amplitude in the analog-reconstituted cells. Cells reconstituted with either retinal or the analog displayed negative phototaxis at low light and switched to positive one upon an increase in stimulus intensity, as opposed to the wild type. The reversal of the phototaxis direction in analog-reconstituted cells was shifted to a higher fluence rate compared with cells reconstituted with retinal, which corresponded to the decreased signaling efficiency of 9-demethylchlamyrhodopsin