Journal articles: 'Rhodopsin proteins'

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Meng, Chao Luo, Gang Dai, and Tatsuo Iwasa. "Identification of Microbial Rhodopsin Genes from Salt Lake in Inner Mongolia." Advanced Materials Research 518-523 (May 2012): 380–83. http://dx.doi.org/10.4028/www.scientific.net/amr.518-523.380.

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Microbial rhodopsins are photoactive proteins that use retinal molecule as the photoactive center. Because of the structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In the past decades microbial rhodopisns were identified from diverse environments including salt lakes, fresh water, sea water, human and plant tissues as fungal pathogens. In the present work, we tried to identify microbial rhodopsin genes from salt lake in Inner Mongolia, China. Two bacteriorhodopsin-like genes were identified. The retrieved sequences were analyzed by comparing with four most widely studied haloarchaeal rhodopsins.

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Shen, Libing, Chao Chen, Hongxiang Zheng, and Li Jin. "The Evolutionary Relationship between Microbial Rhodopsins and Metazoan Rhodopsins." Scientific World Journal 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/435651.

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Rhodopsins are photoreceptive proteins with seven-transmembrane alpha-helices and a covalently bound retinal. Based on their protein sequences, rhodopsins can be classified into microbial rhodopsins and metazoan rhodopsins. Because there is no clearly detectable sequence identity between these two groups, their evolutionary relationship was difficult to decide. Through ancestral state inference, we found that microbial rhodopsins and metazoan rhodopsins are divergently related in their seven-transmembrane domains. Our result proposes that they are homologous proteins and metazoan rhodopsins originated from microbial rhodopsins. Structure alignment shows that microbial rhodopsins and metazoan rhodopsins share a remarkable structural homology while the position of retinal-binding lysine is different between them. It suggests that the function of photoreception was once lost during the evolution of rhodopsin genes. This result explains why there is no clearly detectable sequence similarity between the two rhodopsin groups: after losing the photoreception function, rhodopsin gene was freed from the functional constraint and the process of divergence could quickly change its original sequence beyond recognition.

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Tam, Beatrice M., Orson L. Moritz, Lawrence B. Hurd, and David S. Papermaster. "Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis." Journal of Cell Biology 151, no. 7 (December 25, 2000): 1369–80. http://dx.doi.org/10.1083/jcb.151.7.1369.

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Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

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Fu, Hsu-Yuan, Yu-Cheng Lin, Yung-Ning Chang, Hsiaochu Tseng, Ching-Che Huang, Kang-Cheng Liu, Ching-Shin Huang, et al. "A Novel Six-Rhodopsin System in a Single Archaeon." Journal of Bacteriology 192, no. 22 (August 27, 2010): 5866–73. http://dx.doi.org/10.1128/jb.00642-10.

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ABSTRACT 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 six-rhodopsin 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.

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Poupault, Clara, Diane Choi, Khanh Lam-Kamath, Deepshe Dewett, Ansa Razzaq, Joseph Bunker, Alexis Perry, Irene Cho, and Jens Rister. "A combinatorial cis-regulatory logic restricts color-sensing Rhodopsins to specific photoreceptor subsets in Drosophila." PLOS Genetics 17, no. 6 (June 23, 2021): e1009613. http://dx.doi.org/10.1371/journal.pgen.1009613.

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Color vision in Drosophila melanogaster is based on the expression of five different color-sensing Rhodopsin proteins in distinct subtypes of photoreceptor neurons. Promoter regions of less than 300 base pairs are sufficient to reproduce the unique, photoreceptor subtype-specific rhodopsin expression patterns. The underlying cis-regulatory logic remains poorly understood, but it has been proposed that the rhodopsin promoters have a bipartite structure: the distal promoter region directs the highly restricted expression in a specific photoreceptor subtype, while the proximal core promoter region provides general activation in all photoreceptors. Here, we investigate whether the rhodopsin promoters exhibit a strict specialization of their distal (subtype specificity) and proximal (general activation) promoter regions, or if both promoter regions contribute to generating the photoreceptor subtype-specific expression pattern. To distinguish between these two models, we analyze the expression patterns of a set of hybrid promoters that combine the distal promoter region of one rhodopsin with the proximal core promoter region of another rhodopsin. We find that the function of the proximal core promoter regions extends beyond providing general activation: these regions play a previously underappreciated role in generating the non-overlapping expression patterns of the different rhodopsins. Therefore, cis-regulatory motifs in both the distal and the proximal core promoter regions recruit transcription factors that generate the unique rhodopsin patterns in a combinatorial manner. We compare this combinatorial regulatory logic to the regulatory logic of olfactory receptor genes and discuss potential implications for the evolution of rhodopsins.

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Chuang, Jen-Zen, and Ching-Hwa Sung. "The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells." Journal of Cell Biology 142, no. 5 (September 7, 1998): 1245–56. http://dx.doi.org/10.1083/jcb.142.5.1245.

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All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

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Tarlachkov, Sergey V., Taras V. Shevchuk, Maria del Carmen Montero-Calasanz, and Irina P. Starodumova. "Diversity of rhodopsins in cultivated bacteria of the family Geodermatophilaceae associated with non-aquatic environments." Bioinformatics 36, no. 6 (November 11, 2019): 1668–72. http://dx.doi.org/10.1093/bioinformatics/btz840.

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Abstract Motivation A small amount of research is focused on investigation of rhodopsins in cultivated bacteria isolated from non-aquatic environments. Furthermore, the abundance of these proteins in strains from hot and arid habitats was not reported previously. Since there is an insignificant amount of such isolates, the enigmatic role of the rhodopsins in dry ecological niches is still poorly understood. The members of the family Geodermatophilaceae could be used as interesting objects to search for new rhodopsin genes that will provide novel insights into versatility and importance of these proteins in non-aquatic conditions. Results This is the first report of the abundance of different rhodopsins in cultivated bacteria isolated from hot and arid ecological niches. A total of 31 rhodopsin genes were identified in 51 analyzed genomes of strains belonging to the family Geodermatophilaceae. Overall, 88% of the strains harbouring rhodopsins are isolated from non-aquatic environments. It was found that 82% of strains belonging to the genus Geodermatophilus have at least one gene as compared to 38% of strains of other genera which contain rhodopsins. Analysis of key amino acids revealed two types of the studied proteins: DTE type (putative proton pump) and NDQ type (putative sodium pump). Proton pumps were divided into two subtypes (DTEW and DTEF) according to phylogenetic analysis and the presence of highly conserved tryptophan or phenylalanine at position 182. Among all studied rhodopsins DTEF subtype is the most unique one, identified only in this family. Supplementary information Supplementary data are available at Bioinformatics online.

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Ahrendt, Steven R., Edgar Mauricio Medina, Chia-en A. Chang, and Jason E. Stajich. "Exploring the binding properties and structural stability of an opsin in the chytridSpizellomyces punctatususing comparative and molecular modeling." PeerJ 5 (April 27, 2017): e3206. http://dx.doi.org/10.7717/peerj.3206.

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BackgroundOpsin proteins are seven transmembrane receptor proteins which detect light. Opsins can be classified into two types and share little sequence identity: type 1, typically found in bacteria, and type 2, primarily characterized in metazoa. The type 2 opsins (Rhodopsins) are a subfamily of G-protein coupled receptors (GPCRs), a large and diverse class of seven transmembrane proteins and are generally restricted to metazoan lineages. Fungi use light receptors including opsins to sense the environment and transduce signals for developmental or metabolic changes. Opsins characterized in the Dikarya (Ascomycetes and Basidiomycetes) are of the type 1 bacteriorhodopsin family but the early diverging fungal lineages have not been as well surveyed. We identified by sequence similarity a rhodopsin-like GPCR in genomes of early diverging chytrids and examined the structural characteristics of this protein to assess its likelihood to be homologous to animal rhodopsins and bind similar chromophores.MethodsWe used template-based structure modeling, automated ligand docking, and molecular modeling to assess the structural and binding properties of an identified opsin-like protein found inSpizellomyces punctatus, a unicellular, flagellated species belonging to Chytridiomycota, one of the earliest diverging fungal lineages. We tested if the sequence and inferred structure were consistent with a solved crystal structure of a type 2 rhodopsin from the squidTodarodes pacificus.ResultsOur results indicate that theSpizellomycesopsin has structural characteristics consistent with functional animal type 2 rhodopsins and is capable of maintaining a stable structure when associated with the retinaldehyde chromophore, specifically the 9-cis-retinal isomer. Together, these results support further the homology ofSpizellomycesopsins to animal type 2 rhodopsins.DiscussionThis represents the first test of structure/function relationship of a type 2 rhodopsin identified in early branching fungal lineages, and provides a foundation for future work exploring pathways and components of photoreception in early fungi.

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Shtyrov, Andrey A., Dmitrii M. Nikolaev, Vladimir N. Mironov, Andrey V. Vasin, Maxim S. Panov, Yuri S. Tveryanovich, and Mikhail N. Ryazantsev. "Simple Models to Study Spectral Properties of Microbial and Animal Rhodopsins: Evaluation of the Electrostatic Effect of Charged and Polar Residues on the First Absorption Band Maxima." International Journal of Molecular Sciences 22, no. 6 (March 16, 2021): 3029. http://dx.doi.org/10.3390/ijms22063029.

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A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.

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Konno, Masae, Yumeka Yamauchi, Keiichi Inoue, and Hideki Kandori. "Expression analysis of microbial rhodopsin-like genes in Guillardia theta." PLOS ONE 15, no. 12 (December 3, 2020): e0243387. http://dx.doi.org/10.1371/journal.pone.0243387.

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The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

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Chiang, Wei-Chieh, Carissa Messah, and Jonathan H. Lin. "IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin." Molecular Biology of the Cell 23, no. 5 (March 2012): 758–70. http://dx.doi.org/10.1091/mbc.e11-08-0663.

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Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin.

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Penn, Wesley D., Andrew G. McKee, Charles P. Kuntz, Hope Woods, Veronica Nash, Timothy C. Gruenhagen, Francis J. Roushar, et al. "Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning." Science Advances 6, no. 10 (March 2020): eaay7505. http://dx.doi.org/10.1126/sciadv.aay7505.

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Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

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Asano, Y., S. Nakamura, S. Ishida, K. Azuma, and T. Shinozawa. "Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis." Journal of Experimental Biology 201, no. 9 (May 1, 1998): 1263–71. http://dx.doi.org/10.1242/jeb.201.9.1263.

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The presence of rhodopsin-like proteins in the eyes and auricles of the freshwater planarian Dugesia japonica was confirmed using anti-frog-rhodopsin rabbit IgG. The apparent relative molecular masses of these proteins were 65x10(3) and 62x10(3), and positive reactions to IgG were localized to the microvilli of the photoreceptor cells in the eyes and to the sensory cilia, rootlets and microvilli in the auricles. Eye- or head-excised planarians showed no negative phototaxis, whereas intact or auricle-excised planarians did. During regeneration in head-excised planarians, the appearance of rhodopsin-like proteins in the regenerating eyes corresponded to the recovery of negative phototaxis behaviour. Head or auricle excision enhanced asexual fission under continuous illumination. However, eye excision had no such effect. These results suggest that the rhodopsin-like proteins in the eyes work as photoreceptors for negative phototaxis behaviour and that, in the auricles, they are involved in asexual fission originating from the circadian rhythm.

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Kirpichnikov, M. P., and М. А. Оstrovsky. "Optogenetics and vision." Вестник Российской академии наук 89, no. 2 (March 20, 2019): 125–30. http://dx.doi.org/10.31857/s0869-5873892125-130.

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In this article the authors discuss electronic and optogenetic approaches for degenerative (blind) retina prosthesis as the main strategies for the restoration of vision to blind people. Primary attention is devoted to the prospects of developing retinal prostheses for the blind using modern optogenetic methods, and rhodopsins, which are photosensitive retinal-binding proteins, are examined as potential tools for such prostheses. The authors consider the question of which particular cells of the degenerative retina for which rhodopsins can be prosthetic as well as ways of delivering the rhodopsin genes to these cells. In conclusion, the authors elucidate the main provisions and tasks related to optogenetic prosthetics for degenerative retina.

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Kim, Benjamin, and Michael Z. Lin. "Optobiology: optical control of biological processes via protein engineering." Biochemical Society Transactions 41, no. 5 (September 23, 2013): 1183–88. http://dx.doi.org/10.1042/bst20130150.

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Enabling optical control over biological processes is a defining goal of the new field of optogenetics. Control of membrane voltage by natural rhodopsin family ion channels has found widespread acceptance in neuroscience, due to the fact that these natural proteins control membrane voltage without further engineering. In contrast, optical control of intracellular biological processes has been a fragmented effort, with various laboratories engineering light-responsive properties into proteins in different manners. In the present article, we review the various systems that have been developed for controlling protein functions with light based on vertebrate rhodopsins, plant photoregulatory proteins and, most recently, the photoswitchable fluorescent protein Dronpa. By allowing biology to be controlled with spatiotemporal specificity and tunable dynamics, light-controllable proteins will find applications in the understanding of cellular and organismal biology and in synthetic biology.

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Inoue, Keiichi. "Shining light on rhodopsin selectivity: How do proteins decide whether to transport H+ or Cl–?" Journal of Biological Chemistry 295, no. 44 (October 30, 2020): 14805–6. http://dx.doi.org/10.1074/jbc.h120.016032.

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The versatile microbial rhodopsin family performs a variety of biological tasks using a highly conserved architecture, making it difficult to understand the mechanistic basis for different functions. Besaw et al. now report structures of a recently discovered cyanobacterial Cl−-pumping rhodopsin and its functionally divergent mutant that reveal how these transmembrane proteins create a gradient of activity with subtle changes. These insights are paralleled by a second recent report, which in combination answers long-standing questions about rhodopsin selectivity and will facilitate future engineering efforts.

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Church, Jonathan R., Jógvan Magnus Haugaard Olsen, and Igor Schapiro. "The Impact of Retinal Configuration on the Protein–Chromophore Interactions in Bistable Jumping Spider Rhodopsin-1." Molecules 27, no. 1 (December 23, 2021): 71. http://dx.doi.org/10.3390/molecules27010071.

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Bistable rhodopsins have two stable forms that can be interconverted by light. Due to their ability to act as photoswitches, these proteins are considered as ideal candidates for applications such as optogenetics. In this work, we analyze a recently crystalized bistable rhodopsin, namely the jumping spider rhodopsin-1 (JSR1). This rhodopsin exhibits identical absorption maxima for the parent and the photoproduct form, which impedes its broad application. We performed hybrid QM/MM simulations to study three isomers of the retinal chromophore: the 9-cis, 11-cis and all-trans configurations. The main aim was to gain insight into the specific interactions of each isomer and their impact on the absorption maximum in JSR1. The absorption spectra were computed using sampled snapshots from QM/MM molecular dynamics trajectories and compared to their experimental counterparts. The chromophore–protein interactions were analyzed by visualizing the electrostatic potential of the protein and projecting it onto the chromophore. It was found that the distance between a nearby tyrosine (Y126) residue plays a larger role in the predicted absorption maximum than the primary counterion (E194). Geometric differences between the isomers were also noted, including a structural change in the polyene chain of the chromophore, as well as changes in the nearby hydrogen bonding network.

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Huang, Huai-Wei, and Hyung Don Ryoo. "Drosophila fabp is required for light-dependent Rhodopsin-1 clearance and photoreceptor survival." PLOS Genetics 17, no. 10 (October 29, 2021): e1009551. http://dx.doi.org/10.1371/journal.pgen.1009551.

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Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

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Zernii, Evgeni Yu, Konstantin E. Komolov, Sergei E. Permyakov, Tatiana Kolpakova, Daniele Dell'orco, Annika Poetzsch, Ekaterina L. Knyazeva, et al. "Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase." Biochemical Journal 435, no. 2 (March 29, 2011): 441–50. http://dx.doi.org/10.1042/bj20110013.

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NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin–kinase complex provided insight into the protein–protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation–π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.

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Kishigami, A., T. Ogasawara, Y. Watanabe, M. Hirata, T. Maeda, F. Hayashi, and Y. Tsukahara. "Inositol-1,4,5-trisphosphate-binding proteins controlling the phototransduction cascade of invertebrate visual cells." Journal of Experimental Biology 204, no. 3 (February 1, 2001): 487–93. http://dx.doi.org/10.1242/jeb.204.3.487.

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The main phototransduction cascade in invertebrate visual cells involves the turnover of phosphatidylinositol, an important biochemical mechanism common to many signal-transduction systems. Light-activated rhodopsin stimulates guanine nucleotide exchange on the Gq class of G-protein, which activates phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol-1,4,5-trisphosphate and diacylglycerol. Subsequently, inositol-1,4,5-trisphosphate-binding proteins continue the signal cascade. Here, we report on the first inositol-1,4,5-trisphosphate-binding proteins demonstrated in an invertebrate visual system with our investigation of the photosensitive rhabdoms of squid. We screened the ability of proteins to interact with inositol-1,4,5-trisphosphate by affinity column chromatography with an inositol-1,4,5-trisphosphate analogue. We detected an inositol-1,4,5-trisphosphate-binding affinity in phospholipase C, receptor kinase and five other proteins in the cytosolic fraction and, surprisingly, rhodopsin in the membrane fraction. A binding assay with (3)H-labelled inositol-1,4,5-trisphosphate demonstrated the inositol-1,4,5-trisphosphate affinity of each of the purified proteins. Since rhodopsin, receptor kinase and phospholipase C are involved upstream of phosphatidylinositol turnover in the signal cascade, our result suggests that phosphatidylinositol turnover is important in feedback pathways in the signalling system.

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Kakakhel, Mashal, Lars Tebbe, Mustafa S. Makia, Shannon M. Conley, David M. Sherry, Muayyad R. Al-Ubaidi, and Muna I. Naash. "Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival." Proceedings of the National Academy of Sciences 117, no. 34 (August 10, 2020): 20615–24. http://dx.doi.org/10.1073/pnas.2010751117.

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Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specificStx3knockouts [Stx3f/f(iCre75)andStx3f/f(CRX-Cre)] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

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Yonamine, Ikuko, Takeshi Bamba, Niraj K. Nirala, Nahid Jesmin, Teresa Kosakowska-Cholody, Kunio Nagashima, Eiichiro Fukusaki, Jairaj K. Acharya, and Usha Acharya. "Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors." Journal of Cell Biology 192, no. 4 (February 14, 2011): 557–67. http://dx.doi.org/10.1083/jcb.201004098.

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Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein–coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.

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Islam, Md Sirajul, James P. Gaston, and Matthew A. B. Baker. "Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers." Membranes 11, no. 11 (November 4, 2021): 857. http://dx.doi.org/10.3390/membranes11110857.

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Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

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Sakurai, Keisuke, Akishi Onishi, Hiroo Imai, Osamu Chisaka, Yoshiki Ueda, Jiro Usukura, Kei Nakatani, and Yoshinori Shichida. "Physiological Properties of Rod Photoreceptor Cells in Green-sensitive Cone Pigment Knock-in Mice." Journal of General Physiology 130, no. 1 (June 25, 2007): 21–40. http://dx.doi.org/10.1085/jgp.200609729.

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Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was ∼11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 × 10−7 s−1, about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.

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Geneva, Ivayla I., Han Yen Tan, and Peter D. Calvert. "Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways." Molecular Biology of the Cell 28, no. 4 (February 15, 2017): 554–66. http://dx.doi.org/10.1091/mbc.e16-07-0549.

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Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells.

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Belousov, Anatolii, Ivan Maslov, Polina Khorn, Alexander Mishin, Mikhail Baranov, Thomas Gensch, and Valentin Borshchevskiy. "Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S15. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p10.

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Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.

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Pojer, Jonathan M., Abdul Jabbar Saiful Hilmi, Shu Kondo, and Kieran F. Harvey. "Crumbs and the apical spectrin cytoskeleton regulate R8 cell fate in the Drosophila eye." PLOS Genetics 17, no. 6 (June 7, 2021): e1009146. http://dx.doi.org/10.1371/journal.pgen.1009146.

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The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.

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Wieland, Thomas, Isabel Ulibarri, Klaus Aktories, Peter Gierschik, and Karl H. Jakobs. "Interaction of small G proteins with photoexcited rhodopsin." FEBS Letters 263, no. 2 (April 24, 1990): 195–98. http://dx.doi.org/10.1016/0014-5793(90)81372-u.

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Xu, Xian-Zhong Shawn, Atish Choudhury, Xiaoling Li, and Craig Montell. "Coordination of an Array of Signaling Proteins through Homo- and Heteromeric Interactions Between PDZ Domains and Target Proteins." Journal of Cell Biology 142, no. 2 (July 27, 1998): 545–55. http://dx.doi.org/10.1083/jcb.142.2.545.

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The rapid activation and feedback regulation of many G protein signaling cascades raises the possibility that the critical signaling proteins may be tightly coupled. Previous studies show that the PDZ domain containing protein INAD, which functions in Drosophila vision, coordinates a signaling complex by binding directly to the light-sensitive ion channel, TRP, and to phospholipase C (PLC). The INAD signaling complex also includes rhodopsin, protein kinase C (PKC), and calmodulin, though it is not known whether these proteins bind to INAD. In the current work, we show that rhodopsin, calmodulin, and PKC associate with the signaling complex by direct binding to INAD. We also found that a second ion channel, TRPL, bound to INAD. Thus, most of the proteins involved directly in phototransduction appear to bind to INAD. Furthermore, we found that INAD formed homopolymers and the homomultimerization occurred through two PDZ domains. Thus, we propose that the INAD supramolecular complex is a higher order signaling web consisting of an extended network of INAD molecules through which a G protein–coupled cascade is tethered.

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Shirzad-Wasei, Nazhat, Jenny van Oostrum, Petra H. M. Bovee-Geurts, Lisanne J. A. Kusters, Giel J. C. G. M. Bosman, and Willem J. DeGrip. "Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification." Biological Chemistry 396, no. 8 (August 1, 2015): 903–15. http://dx.doi.org/10.1515/hsz-2015-0100.

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Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

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Needham, David M., Susumu Yoshizawa, Toshiaki Hosaka, Camille Poirier, Chang Jae Choi, Elisabeth Hehenberger, Nicholas A. T. Irwin, et al. "A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20574–83. http://dx.doi.org/10.1073/pnas.1907517116.

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Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae. Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

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Kunduri, Govind, Changqing Yuan, Velayoudame Parthibane, Katherine M. Nyswaner, Ritu Kanwar, Kunio Nagashima, Steven G. Britt, et al. "Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors." Journal of Cell Biology 206, no. 1 (July 7, 2014): 79–95. http://dx.doi.org/10.1083/jcb.201405020.

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The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

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KLAASSEN, Corné H. W., Petra H. M. BOVEE-GEURTS, Godelieve L. J. DECALUWÉ, and Willem J. DEGRIP. "Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system." Biochemical Journal 342, no. 2 (August 24, 1999): 293–300. http://dx.doi.org/10.1042/bj3420293.

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Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni2+-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

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Van Eps, Ned, Christian Altenbach, Lydia N. Caro, Naomi R. Latorraca, Scott A. Hollingsworth, Ron O. Dror, Oliver P. Ernst, and Wayne L. Hubbell. "Gi- and Gs-coupled GPCRs show different modes of G-protein binding." Proceedings of the National Academy of Sciences 115, no. 10 (February 20, 2018): 2383–88. http://dx.doi.org/10.1073/pnas.1721896115.

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More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand–receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR–G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin–G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin–Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR–Gs structures, providing insight into specificity determinants.

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Dizhoor, Alexander M., Elina R. Nekrasova, and Pavel P. Philippov. "The binding of G proteins to immobilized delipidated rhodopsin." Biochemical and Biophysical Research Communications 162, no. 1 (July 1989): 544–49. http://dx.doi.org/10.1016/0006-291x(89)92031-7.

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Fujita, Jun, Norika Sakurai, and Takao Shinozawa. "Presence of rhodopsin-like proteins in the planarian head." Hydrobiologia 227, no. 1 (December 1991): 93–94. http://dx.doi.org/10.1007/bf00027587.

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Schopf, Krystina, Thomas K. Smylla, and Armin Huber. "Immunocytochemical Labeling of Rhabdomeric Proteins inDrosophilaPhotoreceptor Cells Is Compromised by a Light-dependent Technical Artifact." Journal of Histochemistry & Cytochemistry 67, no. 10 (June 27, 2019): 745–57. http://dx.doi.org/10.1369/0022155419859870.

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Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.

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Besaw, Jessica E., Wei-Lin Ou, Takefumi Morizumi, Bryan T. Eger, Juan D. Sanchez Vasquez, Jessica H. Y. Chu, Andrew Harris, Leonid S. Brown, R. J. Dwayne Miller, and Oliver P. Ernst. "The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants." Journal of Biological Chemistry 295, no. 44 (July 23, 2020): 14793–804. http://dx.doi.org/10.1074/jbc.ra120.014118.

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Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

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Grime, Rachael L., Richard T. Logan, Stephanie A. Nestorow, Pooja Sridhar, Patricia C. Edwards, Christopher G. Tate, Bert Klumperman, et al. "Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles." Nanoscale 13, no. 31 (2021): 13519–28. http://dx.doi.org/10.1039/d1nr02419a.

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Using the GPCR rhodopsin as an exemplar, SMA SMI and DIBMA constitute a ‘tool-kit’ of structurally-related solubilising polymers, with each providing different advantages for studying membrane proteins encapsulated in lipid particles.

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Paolicchi, Fabio, Lara Lombardi, Nello Ceccarelli, and Roberto Lorenzi. "Are retinal and retinal-binding proteins involved in stomatal response to blue light?" Functional Plant Biology 32, no. 12 (2005): 1135. http://dx.doi.org/10.1071/fp05054.

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Stomata respond to blue light and it is generally believed that the photoreceptor for this response is located inside the guard cells. Only a small number of blue light photoreceptors have been identified so far, namely cryptochromes and phototropins, and they show overlapping functions in regulating many different responses to light. The possibility that plants may possess other receptors regulating blue light responses under different light conditions cannot be excluded. In this paper we show the presence of two retinal-binding proteins in Commelina communis and we report the identification of retinal, a chromophore usually bound to the photoreceptor rhodopsin and previously identified in algae and other higher plants. We show that, under our experimental conditions, stomata open promptly when exposed to blue light and we demonstrated that this response is dependent on retinal. We hypothesise that rhodopsin-like retinal-binding proteins might be involved in stomatal response to blue light.

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Gerrard, Elliot, Eshita Mutt, Takashi Nagata, Mitsumasa Koyanagi, Tilman Flock, Elena Lesca, Gebhard F. X. Schertler, Akihisa Terakita, Xavier Deupi, and Robert J. Lucas. "Convergent evolution of tertiary structure in rhodopsin visual proteins from vertebrates and box jellyfish." Proceedings of the National Academy of Sciences 115, no. 24 (May 23, 2018): 6201–6. http://dx.doi.org/10.1073/pnas.1721333115.

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Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue—the counterion—to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position—from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)—is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle—E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge—R186—that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket—neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113—we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems.

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Deretic, D., and D. S. Papermaster. "Polarized sorting of rhodopsin on post-Golgi membranes in frog retinal photoreceptor cells." Journal of Cell Biology 113, no. 6 (June 15, 1991): 1281–93. http://dx.doi.org/10.1083/jcb.113.6.1281.

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We have isolated a subcellular fraction of small vesicles (mean diameter, 300 nm) from frog photoreceptors, that accumulate newly synthesized rhodopsin with kinetics paralleling its appearance in post-Golgi membranes in vivo. This fraction is separated from other subcellular organelles including Golgi and plasma membranes and synaptic vesicles that are sorted to the opposite end of the photoreceptor cell. The vesicles have very low buoyant density in sucrose gradients (rho = 1.09 g/ml), a relatively simple protein content and an orientation of rhodopsin expected of transport membranes. Reversible inhibition of transport by brefeldin A provides evidence that these vesicles are exocytic carriers. Specific immunoadsorption bound vesicles whose protein composition was indistinguishable from the membranes sedimented from the subcellular fraction. Some of these proteins may be cotransported with rhodopsin to the rod outer segment; others may be involved in vectorial transport.

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Yun, Ji-Hye, Mio Ohki, Jae-Hyun Park, Naito Ishimoto, Ayana Sato-Tomita, Wonbin Lee, Zeyu Jin, et al. "Pumping mechanism of NM-R3, a light-driven bacterial chloride importer in the rhodopsin family." Science Advances 6, no. 6 (February 2020): eaay2042. http://dx.doi.org/10.1126/sciadv.aay2042.

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A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium Nonlabens marinus, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues—Cys105, Ser60, Gln224, and Phe90—were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.

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Nakao, Yutaka, Kazumi Shimono, Takashi Kikukawa, Kunio Ihara, and Naoki Kamo. "1P090 Photochemistry of Sensory Rhodopsin III from Haloarcula marismortui(HmSRIII)(Membrane proteins,Poster Presentations)." Seibutsu Butsuri 47, supplement (2007): S46. http://dx.doi.org/10.2142/biophys.47.s46_1.

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Ye, Shixin, Caroline Köhrer, Thomas Huber, Manija Kazmi, Pallavi Sachdev, Elsa C. Y. Yan, Aditi Bhagat, Uttam L. RajBhandary, and Thomas P. Sakmar. "Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis." Journal of Biological Chemistry 283, no. 3 (November 8, 2007): 1525–33. http://dx.doi.org/10.1074/jbc.m707355200.

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G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

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Ulshafer, R. J., W. W. Hauswirth, and A. van der Langerijt. "EM immunocytochemical localization of rhodopsin and IRBP during retinal development." Proceedings, annual meeting, Electron Microscopy Society of America 47 (August 6, 1989): 800–801. http://dx.doi.org/10.1017/s0424820100155979.

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Two rod photoreceptor cell-specific proteins, rhodopsin and interphotoreceptor retinoid binding protein (IRBP), were localized during fetal development of the bovine retina using immuncicrytcichemistry. Rhodopsin is the light sensitive protein that, when activated, begins the process of transducing light energy to an electrical response. IRBP is a carrier protein that shuttles light-isomerized vitamin A (retinol) between the rod outer segment and the overlying retinal pigment epithelium where it is recycled to its light-sensitive form. Rhodopsin has been previously imimmocytochemically localized to rod (but not cone) photoreceptor outer segment membranes. IRBP has been localized to the subretinal space using immunocytochemistry.Retinas were obtained from fetuses at approximately 4, 5, 6, 7, and 8 months of gestation, fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded at 40°C in epoxy resin. Thin sections were mounted on Ni grids and incubated with antibodies raised against the purified antigens: a mouse monoclonal anti-rhodopsin or a rabbit polyclonal anti-IRBP. A second antibody (Goat-anti-mouse or Goat-anti-rabbit) was conjugated with 15 nm Au particles and reacted with the sections. Control incubations were made using pre-immune rabbit serum and mouse monoclonals made against other tissue or bacterial sources.

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Khorana, H. G. "Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin." Proceedings of the National Academy of Sciences 90, no. 4 (February 15, 1993): 1166–71. http://dx.doi.org/10.1073/pnas.90.4.1166.

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48

Castiglione, Gianni M., Frances E. Hauser, Brian S. Liao, Nathan K. Lujan, Alexander Van Nynatten, James M. Morrow, Ryan K. Schott, Nihar Bhattacharyya, Sarah Z. Dungan, and Belinda S. W. Chang. "Evolution of nonspectral rhodopsin function at high altitudes." Proceedings of the National Academy of Sciences 114, no. 28 (June 22, 2017): 7385–90. http://dx.doi.org/10.1073/pnas.1705765114.

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High-altitude environments present a range of biochemical and physiological challenges for organisms through decreases in oxygen, pressure, and temperature relative to lowland habitats. Protein-level adaptations to hypoxic high-altitude conditions have been identified in multiple terrestrial endotherms; however, comparable adaptations in aquatic ectotherms, such as fishes, have not been as extensively characterized. In enzyme proteins, cold adaptation is attained through functional trade-offs between stability and activity, often mediated by substitutions outside the active site. Little is known whether signaling proteins [e.g., G protein-coupled receptors (GPCRs)] exhibit natural variation in response to cold temperatures. Rhodopsin (RH1), the temperature-sensitive visual pigment mediating dim-light vision, offers an opportunity to enhance our understanding of thermal adaptation in a model GPCR. Here, we investigate the evolution of rhodopsin function in an Andean mountain catfish system spanning a range of elevations. Using molecular evolutionary analyses and site-directed mutagenesis experiments, we provide evidence for cold adaptation in RH1. We find that unique amino acid substitutions occur at sites under positive selection in high-altitude catfishes, located at opposite ends of the RH1 intramolecular hydrogen-bonding network. Natural high-altitude variants introduced into these sites via mutagenesis have limited effects on spectral tuning, yet decrease the stability of dark-state and light-activated rhodopsin, accelerating the decay of ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates—properties of rhodopsin that mediate rod sensitivity and visual performance. Our results support a role for natural variation in enhancing the performance of GPCRs in response to cold temperatures.

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Deretic, D., and D. S. Papermaster. "Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer segment disk formation in frog retinal photoreceptors." Journal of Cell Science 106, no. 3 (November 1, 1993): 803–13. http://dx.doi.org/10.1242/jcs.106.3.803.

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The biogenesis of light sensitive membranes in retinal rod photoreceptors involves polarized sorting and targeting of newly synthesized rhodopsin to a specialized domain, the rod outer segment (ROS). We have isolated and characterized the population of post-Golgi membranes that mediate intracellular transport of rhodopsin. In the present study we have examined the association of small (20-25 kDa) GTP-binding (G) proteins with these membranes. We found that one of the small G proteins, rab6, behaves like an integral membrane protein of the post-Golgi vesicles, although approximately 30% of rab6 is soluble. The distribution of the membrane-associated and the soluble forms is highly polarized. By confocal and EM immunocytochemistry it can be seen that most of rab6 is associated with the photoreceptor trans-Golgi cisternae, trans-Golgi network (TGN) and post-Golgi vesicles. The photoreceptor axon and synaptic terminal are unlabeled, but dendrites of deeper retinal layers are labeled. The distribution of rab6 across sucrose density gradient fractions parallels the distribution of sialyltransferase (a TGN marker) activity. About 9% of membrane-bound rab6 is associated, however, with the rhodopsin-bearing sialyltransferase-free post-Golgi vesicles, which represent a very small fraction (< 1%) of the total retinal membranes. Rab6 is absent from the mature ROS disk membranes but it is present at the sites of new ROS disk formation and in the ROS cytoplasm. This suggests that rab6 becomes soluble upon disk membrane formation. Therefore, rab6 may function not only as a component of the sorting machinery of photoreceptors that delivers rhodopsin to its appropriate subcellular domain but may also participate in some aspects of ROS disk morphogenesis.

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Deretic, D., L. A. Huber, N. Ransom, M. Mancini, K. Simons, and D. S. Papermaster. "rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis." Journal of Cell Science 108, no. 1 (January 1, 1995): 215–24. http://dx.doi.org/10.1242/jcs.108.1.215.

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Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.

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Journal articles on the topic 'Rhodopsin proteins'

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