Photosensory Functions of Channelrhodopsins in Native Algal Cells

Sineshchekov, Oleg A.; Govorunova, Elena G.; Spudich, John L.

doi:10.1111/j.1751-1097.2008.00524.x

Cited by 51 publications

(44 citation statements)

References 52 publications

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“…1C). Our working hypothesis has been that the position of the highly acetylated D4 rootlet directs localization of one or both of the major rhodopsin family photoreceptors in the plasma membrane, ChR1 and ChR2, to the position of the future eyespot, which in turn directs localization and assembly of other eyespot components, such as EYE2, in the chloroplast envelope and the pigment granule layers in the chloroplast (37,38,41). This hypothesis predicts that D4 rootlet assembly occurs before eyespot assembly.…”

Section: Resultsmentioning

confidence: 99%

“…1B) (22,35). Channelrhodopsins ChR1 and ChR2, rhodopsin family light-gated cation channels that initiate the phototactic response, are localized in the plasma membrane at the eyespot (36)(37)(38)(39)(40). EYE2, an eyespotspecific, thioredoxin superfamily protein of unknown function, is thought to reside in one of the two chloroplast envelope membranes (39,41,42).…”

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confidence: 99%

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Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

et al. 2013

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bLike many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell. Previous work has shown that the eyespot is disassembled prior to cell division, and new eyespots are assembled in daughter cells adjacent to the nascent four-membered rootlet associated with the daughter basal body (D4), but the chronology of these assembly events has not been determined. Here we use immunofluorescence microscopy to follow assembly and acetylation of the D4 rootlet, localization of individual eyespot components in the plasma membrane or chloroplast envelope, and flagellar emergence during and immediately following cell division. We find that the D4 rootlet is assembled before the initiation of eyespot assembly, which occurs within the same time frame as rootlet acetylation and flagellar outgrowth. Photoreceptors in the plasma membrane are correctly localized in eyespot mutant cells lacking pigment granule layers, and chloroplast components of the eyespot assemble in mutant cells in which photoreceptor localization is retarded. The data suggest that plasma membrane and chloroplast components of the eyespot are independently responsive to a cytoskeletal positioning cue.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

et al. 2013

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“…These receptors, called channelrhodopsins-1 and -2 differ by their absorption maxima, 460 nm versus 500 nm, respectively, as well as in their levels of light saturation, which extend the wavelength and intensity ranges for phototaxis by C. reinhardtii (30). Yet-undiscovered differences in properties of the two HmBR proteins are likely to provide an advantage to the organism, but our results rule out the possibility that they expand the wavelength range since they have identical absorption spectra.…”

Section: Discussionmentioning

confidence: 99%

A Novel Six-Rhodopsin System in a Single Archaeon

Lin

Chang

et al. 2010

J Bacteriol

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Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered sixrhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.Microbial rhodopsins comprise a large family of seven-transmembrane helical proteins that either mediate light-driven ion transport to harvest solar energy or serve as receptors to mediate phototaxis (13) and possibly photoadaptation (32). In archaea, four rhodopsins responding to different wavelengths of light with distinct functions in Halobacterium salinarum have been identified and characterized (20,32 The two ion-transporting rhodopsins perform light-driven outward proton transport to create a proton-electrochemical potential or inward chloride transport to maintain the osmotic and pH homeostasis of the cell. The photoactivated sensory rhodopsins, on the other hand, undergo light-triggered conformational changes to relay signals to their cognate transducers and consequently activate signaling cascades in a manner similar to that of the two-component system involved in eubacterial chemotaxis (1, 22) to control flagellum rotation and thus swimming direction. Our current understanding of microbial rhodopsins as both ion transporters and photosensory receptors has been based primarily on these four known rhodopsins.A recently completed genome project for Haloarcula marismortui (3) proposed the existence of six opsin-related genes, the greatest number ever found in a single archaeon. This proposal immediately raised three questions. (i) Are these six rhodopsins biologically expressed and functionally active? (ii) What is the maximum-absorbance-wavelength ( max ) distribution pattern of these six rhodopsins? (iii) Do the two extra rhodopsins, compared to the four in H. salinarum, perform new functions or have new features beyond those of t...

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“…Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2).…”

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confidence: 99%

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Volz

Krause

Balke

et al. 2016

Journal of Biological Chemistry

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A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2). In CrChR2 the chromophore retinal is bound to Lys-257 (3). Channelrhodopsin activation via lightinduced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (4 -6). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (3 10).The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (5). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11-13). Together these data suggest that in contrast to bR and sensory rhod...

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Photosensory Functions of Channelrhodopsins in Native Algal Cells

Cited by 51 publications

References 52 publications

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

Independent Localization of Plasma Membrane and Chloroplast Components during Eyespot Assembly

A Novel Six-Rhodopsin System in a Single Archaeon

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

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