Thermal stability of rhodopsins and opsins in warm- and cold-blooded vertebrates

Thermal stability of rhodopsins and opsins has been studied in endothermic (sheep, cattle, pig, rat) and ectothermic (frog) animals under two different conditions -- in the intact photoreceptor membranes (PM) and after substitution of the lipid surrounding of rhodopsins by molecules of a detergent Triton X-100. Lipid composition of PM in these animals was also studied, as well as the effect of proteases (pronase and papaine) upon thermal stability of rhodopsins in PM and in 1% Triton X-100 solutions. The thermal resistance of rhodopsins in PM was found to vary in the animals used to a great extent. The maximal differences in thermal stability of rhodopsins in ecto- and endothermic animals were due to the properties of photoreceptor protein itself, whereas in ectothermic animals they resulted mainly from differences in the lipid composition of PM. PM of endothermic animals differ from those of ectothermic ones by a lower content of polyenoic fatty acids and by a higher amount of phosphatidyl ethanolamine. The thermal stability of rhodopsins is not due to rhodopsin molecule as a whole, and depends mainly on its part which is directly bound to 11-cis retinal, located in hydrophobic region of PM and inaccessible to protease attack.     

Rhodopsin Receptors of Phototaxis in Green Flagellate Algae

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.     

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

Phototaxis of the green algae: the new class of rhodopsin receptors

Photomotility behavior in green flagellate algae is mediated by rhodopsin-like receptors, which was initially suggested on the basis of physiological evidence. The cascade of rapid Ca(2+)-dependent electrical responses in the plasma membrane plays a key role in the signal transduction chain during both phototaxis and the photophobic response. The photoreceptor current through the plasma membrane is the earliest detectable event upon photoexcitation of the photoreceptors. Analysis of this current revealed that it consists of at least two components with different characteristics. Genes encoding two archaeal-type rhodopsins (type I rhodopsins) were recently identified in the genome of Chlamydomonas reinhardtii and named (Chlamydomonas Sensory Rhodopsins A and B CSRA and CSRB). The measurements of photoelectric and motor responses in genetic transformants of C. reinhardtii enriched in each of these receptor proteins showed that the two components of the photoreceptor current are mediated by the two rhodopsins, and that both CSRA and CSRB are involved in phototaxis and the photophobic response. The CSRA-mediated current dominates at high light intensities and contributes primarily to the photophobic response. The CSRB-initiated transduction involves an efficient amplification cascade and mediates the highly sensitive phototaxis at low light intensities. CSRA and CSRB expressed heterologously in oocytes of Xenopus laevis act as light-gated proton channels, although it is unclear whether this channel activity plays a functional role in the initiation of motor responses and/or occurs in the native system.     

Demonstration of rod and cone photoreceptors in the lamprey retina by freeze-replication and immunofluorescence

Summary In common with other cyclostomata, the Japanese river lamprey (Lampetra japonica) has a retina consisting of distinct types of photoreceptor cells called long and short photoreceptor cells. After freeze-fracture, disc membranes of these photoreceptor cells were characterized in common by a homogeneous distribution of intramembrane particles on the protoplasmic fracture faces, in contrast to those of the myeloid bodies bearing scattering particles.Immunofluorescent examination was applied to the retina with monoclonal antibodies raised against bovine and chicken rhodopsins. Positive immunoreactivity was found to be limited to outer segments of the short cell, leaving the entire body of the long cell and all other components of the retina negative. The results suggest that the short cell is more closely related to a rod-type photoreceptor cell characterized by rhodopsin as its visual pigment.     

Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane

Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.     

The light‐driven sodium ion pump: A new player in rhodopsin research

Rhodopsins are one of the most studied photoreceptor protein families, and ion‐translocating rhodopsins, both pumps and channels, have recently attracted broad attention because of the development of optogenetics. Recently, a new functional class of ion‐pumping rhodopsins, an outward Na⁺ pump, was discovered, and following structural and functional studies enable us to compare three functionally different ion‐pumping rhodopsins: outward proton pump, inward Cl^? pump, and outward Na⁺ pump. Here, we review the current knowledge on structure‐function relationships in these three light‐driven pumps, mainly focusing on Na⁺ pumps. A structural and functional comparison reveals both unique and conserved features of these ion pumps, and enhances our understanding about how the structurally similar microbial rhodopsins acquired such diverse functions. We also discuss some unresolved questions and future perspectives in research of ion‐pumping rhodopsins, including optogenetics application and engineering of novel rhodopsins.

     

Rhodopsin-mediated photoreception in cryptophyte flagellates

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.     

An expanded set of photoreceptors in the Eastern Pale Clouded Yellow butterfly, Colias erate

We studied the spectral and polarisation sensitivities of photoreceptors of the butterfly Colias erate by using intracellular electrophysiological recordings and stimulation with light pulses. We developed a method of response waveform comparison (RWC) for evaluating the effective intensity of the light pulses. We identified one UV, four violet-blue, two green and two red photoreceptor classes. We estimated the peak wavelengths of four rhodopsins to be at about 360, 420, 460 and 560 nm. The four violet-blue classes are presumably based on combinations of two rhodopsins and a violet-absorbing screening pigment. The green classes have reduced sensitivity in the ultraviolet range. The two red classes have primary peaks at about 650 and 665 nm, respectively, and secondary peaks at about 480 nm. The shift of the main peak, so far the largest amongst insects, is presumably achieved by tuning the effective thickness of the red perirhabdomal screening pigment. Polarisation sensitivity of green and red photoreceptors is higher at the secondary than at the main peak. We found a 20-fold variation of sensitivity within the cells of one green class, implying possible photoreceptor subfunctionalisation. We propose an allocation scheme of the receptor classes into the three ventral ommatidial types.     

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].     

Immunological studies on vertebrate photoreceptors

Many monoclonal antibodies to the rhodopsin and other visual pigments have been reported by a number of research groups. The antibodies are available for cell classification, detecting some molecular difference(s) among various visual pigments, and also recently for protein purification and gene cloning. In this review article, we paid attention to precedingly reported 20 anti-photoreceptor antibodies in order to compare them with our own two anti-lamprey photoreceptor antibodies, H16 and B11. From the point of view of immunohistochemical reactivity, the H16 antibody was regarded as a marker for a universal component of vertebrate rhodopsins and a certain number of cone pigments; meanwhile the B11 antibody would recognize more specifically lower-vertebrate rhodopsins and, perhaps, blue-sensitive cone pigments in higher vertebrate retinas.     

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.     

Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus

Background

The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila.

Methodology/Principal Findings

Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs.

Conclusions/Significance

Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres.     

Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin are tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disk membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin, thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.     

Photoreceptor for curling behavior in Peranema trichophorum and evolution of eukaryotic rhodopsins

When it is gliding, the unicellular euglenoid Peranema trichophorum uses activation of the photoreceptor rhodopsin to control the probability of its curling behavior. From the curled state, the cell takes off in a new direction. In a similar manner, archaea such as Halobacterium use light activation of bacterio- and sensory rhodopsins to control the probability of reversal of the rotation direction of flagella. Each reversal causes the cell to change its direction. In neither case does the cell track light, as known for the rhodopsin-dependent eukaryotic phototaxis of fungi, green algae, cryptomonads, dinoflagellates, and animal larvae. Rhodopsin was identified in Peranema by its native action spectrum (peak at 2.43 eV or 510 nm) and by the shifted spectrum (peak at 3.73 eV or 332 nm) upon replacement of the native chromophore with the retinal analog n-hexenal. The in vivo physiological activity of n-hexenal incorporated to become a chromophore also demonstrates that charge redistribution of a short asymmetric chromophore is sufficient for receptor activation and that the following isomerization step is probably not required when the rest of the native chromophore is missing. This property seems universal among the Euglenozoa, Plant, and Fungus kingdom rhodopsins. The rhodopsins of animals have yet to be studied in this respect. The photoresponse appears to be mediated by Ca2+ influx.     

Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment

The R7 and R8 photoreceptor cells of the Drosophila retina are thought to mediate color discrimination and polarized light detection. This is based on the patterned expression of different visual pigments, rhodopsins, in different photoreceptor cells. In this report, we examined the developmental timing of retinal patterning. There is genetic evidence that over the majority of the eye, patterned expression of opsin genes is regulated by a signal from one subtype of R7 cells to adjacent R8 cells. We examined the onset of expression of the rhodopsin genes to determine the latest time point by which photoreceptor subtype commitment must have occurred. We found that the onset of rhodopsin expression in all photoreceptors of the compound eye occurs during a narrow window from 79% to 84% of pupal development (approximately 8 h), pupal stages P12-P14. Rhodopsin 1 has the earliest onset, followed by Rhodopsins 3, 4, and 5 at approximately the same time, and finally Rhodopsin 6. This sequence mimics the model for how R7 and R8 photoreceptor cells are specified, and defines the timing of photoreceptor cell fate decisions with respect to other events in eye development.     

Isolation and characterization of lamprey rhodopsin cDNA.

Genomic DNA fragments coding a visual pigment of the lamprey were amplified by polymerase chain reaction, using oligonucleotide mixtures as primers. The complete coding region of the cDNA was obtained by separate amplification of both cDNA ends. The deduced amino acid sequence of the coding region showed 78-82% identity with those of rhodopsins of higher vertebrates, but only 43-47% identity with those of human color pigments. The cloned DNA appears to be the cDNA of a lamprey rhodopsin, which is expressed in the "short" photoreceptor cell.     

Reconstructing the eyes of Urbilateria

The shared roles of Pax6 and Six homologues in the eye development of various bilaterians suggest that Urbilateria, the common ancestors of all Bilateria, already possessed some simple form of eyes. Here, we re-address the homology of bilaterian cerebral eyes at the level of eye anatomy, of eye-constituting cell types and of phototransductory molecules. The most widespread eye type found in Bilateria are the larval pigment-cup eyes located to the left and right of the apical organ in primary, ciliary larvae of Protostomia and Deuterostomia. They can be as simple as comprising a single pigment cell and a single photoreceptor cell in inverse orientation. Another more elaborate type of cerebral pigment-cup eyes with an everse arrangement of photoreceptor cells is found in adult Protostomia. Both inverse larval and everse adult eyes employ rhabdomeric photoreceptor cells and thus differ from the chordate cerebral eyes with ciliary photoreceptors. This is highly significant because on the molecular level we find that for phototransduction rhabdomeric versus ciliary photoreceptor cells employ divergent rhodopsins and non-orthologous G-proteins, rhodopsin kinases and arrestins. Our comparison supports homology of cerebral eyes in Protostomia; it challenges, however, homology of chordate and non-chordate cerebral eyes that employ photoreceptor cells with non-orthologous phototransductory cascades.     

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.