Bacterial rhodopsins monitored with fluorescent dyes in vesicles andin Vivo

Abstract

Summary Three retinal-containing pigments have been detected inHalobacterium halobium membranes: bacteriorhodopsin (bR), halorhodopsin (hR), and slow-cycling rhodopsin (sR). The first two hyperpolarize the cell membrane by electrogenic transport of H⁺ and Cl⁻, respectively. The third pigment, sR, may be a photosensory receptor since mutants lacking bR and hR retain their retinal-dependent phototaxis responses. We monitored light-induced changes in fluorescence of several voltage-sensitive dyes in cells and membrane vesicles. Red light-induced potential changes generated by bR and hR were similar to signals described previously. Signals generated by hR could be identified using four criteria: wavelength dependence, Cl⁻ dependence, shunting by valinomycin and K⁺, and the absence of these signals in hR-deficient mutants. The absence (detection limit ∼0.5 mV) of hyperpolarization signals in bR⁻hR⁻sR⁺ vesicles and cells shows that sR photochemical reactions are nonelectrogenic. Two signals independent of bR and hR were measured: blue light caused a decrease and red light an increase in dye fluorescence. Both signals appear to derive from sR on the basis of their retinal-dependence and action spectra. In a retinal-deficient mutant strain (Flx3R), both sR signals appeared after addition of all-trans retinal. In this strain retinal also restores phototaxis sensitivity within the same time scale. The retinal concentration dependence for all four parameters monitored—the attractant (red) and repellent (blue) phototaxis, and the red light and blue light-induced fluorescence signals—is the same. This correlation is consistent with the hypothesis that both attractant and repellent responses are mediated by sR, as suggested by Bogomolni and Spudich (Proc. Natl. Acad. Sci. USA.79:6250–6254 (1982)).