Single Rhodopsin Research Articles

Transforming yeast into a facultative photoheterotroph via expression of vacuolar rhodopsin

Phototrophic metabolism, the capture of light for energy, was a pivotal biological innovation that greatly increased the total energy available to the biosphere. Chlorophyll-based photosynthesis is the most familiar phototrophic metabolism, but retinal-based microbial rhodopsins transduce nearly as much light energy as chlorophyll does,1 via a simpler mechanism, and are found in far more taxonomic groups. Although this system has apparently spread widely via horizontal gene transfer,2,3,4 little is known about how rhodopsin genes (with phylogenetic origins within prokaryotes5,6) are horizontally acquired by eukaryotic cells with complex internal membrane architectures or the conditions under which they provide a fitness advantage. To address this knowledge gap, we sought to determine whether Saccharomyces cerevisiae, a heterotrophic yeast with no known evolutionary history of phototrophy, can function as a facultative photoheterotroph after acquiring a single rhodopsin gene. We inserted a rhodopsin gene from Ustilago maydis,7 which encodes a proton pump localized to the vacuole, an organelle normally acidified via a V-type rotary ATPase, allowing the rhodopsin to supplement heterotrophic metabolism. Probes of the physiology of modified cells show that they can deacidify the cytoplasm using light energy, demonstrating the ability of rhodopsins to ameliorate the effects of starvation and quiescence. Further, we show that yeast-bearing rhodopsins gain a selective advantage when illuminated, proliferating more rapidly than their non-phototrophic ancestor or rhodopsin-bearing yeast cultured in the dark. These results underscore the ease with which rhodopsins may be horizontally transferred even in eukaryotes, providing novel biological function without first requiring evolutionary optimization.

Photoreceptor physiology and evolution: cellular and molecular basis of rod and cone phototransduction.

The detection of light in the vertebrate retina utilizes a duplex system of closely related rod and cone photoreceptors: cones respond extremely rapidly, and operate at 'photopic' levels of illumination, from moonlight upwards; rods respond much more slowly, thereby obtaining greater sensitivity, and function effectively only at 'scotopic' levels of moonlight and lower. Rods and cones employ distinct isoforms of many of the proteins in the phototransduction cascade, and they thereby represent a unique evolutionary system, whereby the same process (the detection of light) uses a distinct set of genes in two classes of cell. The molecular mechanisms of phototransduction activation are described, and the classical quantitative predictions for the onset phase of the electrical response to light are developed. Recent work predicting the recovery phase of the rod's response to intense flashes is then presented, that provides an accurate account of the time that the response spends in saturation. Importantly, this also provides a new estimate for the rate at which a single rhodopsin activates molecules of the G-protein, transducin, that is substantially higher than other estimates in the literature. Finally, the evolutionary origin of the phototransduction proteins in rods and cones is examined, and it is shown that most of the rod/cone differences were established at the first of the two rounds of whole-genome duplication more than 500 million years ago.

Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

De novo transcriptome analyses provide insights into opsin-based photoreception in the lanternshark Etmopterus spinax.

The velvet belly lanternshark (Etmopterus spinax) is a small deep-sea shark commonly found in the Eastern Atlantic and the Mediterranean Sea. This bioluminescent species is able to emit a blue-green ventral glow used in counter-illumination camouflage, mainly. In this study, paired-end Illumina HiSeqTM technology has been employed to generate transcriptome data from eye and ventral skin tissues of the lanternshark. About 64 and 49 million Illumina reads were generated from skin and eye tissues respectively. The assembly allowed us to predict 119,749 total unigenes including 94,569 for the skin transcriptome and 94,365 for the eye transcriptome while 74,753 were commonly found in both transcriptomes. A taxonomy filtering was applied to extract a reference transcriptome containing 104,390 unigenes among which 38,836 showed significant similarities to known sequences in NCBI non-redundant protein sequences database. Around 58% of the annotated unigenes match with predicted genes from the Elephant shark (Callorhinchus milii) genome. The transcriptome completeness has been evaluated by successfully capturing around 98% of orthologous genes of the « Core eukaryotic gene dataset » within the E. spinax reference transcriptome. We identified potential “light-interacting toolkit” genes including multiple genes related to ocular and extraocular light perception processes such as opsins, phototransduction actors or crystallins. Comparative gene expression analysis reveals eye-specific expression of opsins, ciliary phototransduction actors, crystallins and vertebrate retinoid pathway actors. In particular, mRNAs from a single rhodopsin gene and its potentially associated peropsin were detected in the eye transcriptome, only, confirming a monochromatic vision of the lanternshark. Encephalopsin mRNAs were mainly detected in the ventral skin transcriptome. In parallel, immunolocalization of the encephalopsin within the ventral skin of the shark suggests a functional relation with the photophores, i.e. epidermal light-producing organs. We hypothesize that extraocular photoreception might be involved in the bioluminescence control possibly acting on the shutter opening and/or the photocyte activity itself. The newly generated reference transcriptome provides a valuable resource for further understanding of the shark biology.

In Vitro Demonstration of Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven H+/Na+ pumps, ejecting Na+ from cells in the presence of Na+ and H+ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Na+ and H+ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely Na+-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing H+ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system.

In Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H+/Na+ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na+ and H+ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na+ transport and electrogenic H+ transport, respectively, in the presence and absence of Na+. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na+ transport by IAR requires Na+ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.

Exploring Light and Life

Exploring Light and Life

Quantitative Comparison of GPCR Interactions with the Lipid Bilayer of the Cubic and Lamellar Mesophases

Recent successes in the crystallographic determination of structures of transmembrane (TM) proteins in the G protein-coupled receptor (GPCR) family have established the lipidic cubic phase (LCP or “in meso”) environment as useful for growing diffraction quality crystals. The mechanism underlying in meso crystallogenesis is currently at a descriptive level. To begin developing a quantitative, energy-based nucleation and crystallization mechanism we are conducting molecular dynamics studies of the GPCR, rhodopsin, reconstituted into the LCP using the coarse-grained representations of the Martini force-field. The first aim is to quantify differences in the hydrophobic/hydrophilic exposure of the GPCR to lipids in the cubic and lamellar phases. Simulations of a single rhodopsin molecule in these monoolein-based mesophases showed more energetically unfavorable hydrophobic-hydrophilic interactions between the protein and lipid in planar bilayers of the lamellar phase. The reduced level of hydrophobic mismatch in the LCP, by contrast, is attributable to the highly curved geometry of the cubic phase that provides for more efficient shielding of the protein from unfavorable hydrophobic exposure. Since hydrophobic mismatch can drive oligomerization (Mondal et al., BJ 2011 - in press), these differences suggest that compared to the LCP, lamellar structures provide a more favorable setting in which GPCRs can oligomerize as a prelude to nucleation and crystal growth. These new findings lay the foundation for future studies of in meso crystallization mechanisms and for a rational approach to the generation of structure-quality crystals of membrane proteins.

Arrestin can Bind to a Single G-Protein Coupled Receptor

Termination of G protein-coupled receptor (GPCR) signaling typically involves phosphorylation of the receptor, followed by binding of a protein called arrestin. Here we tested the minimal stoichiometry required for this interaction, by determining if a single rhodopsin molecule can bind arrestin. To do this, we prepared nanoscale phospholipids particles, so-called nanodiscs, which contain only monomeric rhodopsin and measured their ability to bind visual arrestin. Our data clearly show that visual arrestin can bind to monomeric phosphorylated rhodopsin to stabilize its active form, called metarhodopsin II.

Arrestin Competition Influences the Kinetics and Variability of the Single-Photon Responses of Mammalian Rod Photoreceptors

Reliable signal transduction via G-protein-coupled receptors requires proper receptor inactivation. For example, signals originating from single rhodopsin molecules vary little from one to the next, requiring reproducible inactivation of rhodopsin by phosphorylation and arrestin binding. We determined how reduced concentrations of rhodopsin kinase (GRK1) and/or arrestin1 influenced the kinetics and variability of the single-photon responses of mouse rod photoreceptors. These experiments revealed that arrestin, in addition to its role in quenching the activity of rhodopsin, can tune the kinetics of rhodopsin phosphorylation by competing with GRK1. This competition influenced the variability of the active lifetime of rhodopsin. Biasing the competition in favor of GRK1 revealed that rhodopsin remained active through much of the single-photon response under the conditions of our experiments. This long-lasting rhodopsin activity can explain the characteristic time course of single-photon response variability. Indeed, explaining the late time-to-peak of the variance required an active lifetime of rhodopsin approximately twice that of the G-protein transducin. Competition between arrestins and kinases may be a general means of influencing signals mediated by G-protein-coupled receptors, particularly when activation of a few receptors produces signals of functional importance.

Highlights From The Literature

Highlights From The Literature

Seeing Is Believing

In order for vertebrate photoreceptors to exhibit their exquisite sensitivity that allows them to distinguish stimulation by a single photon, the sensor, rhodopsin, must have a very reproducible response. Rhodopsin propagates the signal from absorbed photons by activating transducin, but it is then inactivated by phosphorylation. Doan et al. measured the response of single rhodopsin molecules to single-photon absorption events in preparations of mouse photoreceptors. Multiple phosphorylation events provide independent inhibitory signals that together may provide the remarkable reproducibility of the amplitude and duration of rhodopsin activation observed in the vertebrate eye. T. Doan, A. Mendez, P. B. Detwiler, J. Chen, F. Rieke, Multiple phosphorylation sites confer reproducibility of the rod's single-photon responses. Science 313 , 530-533 (2006). [Abstract] [Full Text]

Highlights from the Literature

Highlights from the Literature

Smell's different

G protein amplification occurs automatically in an activated rod cell, but not so simply in olfactory cells, according to Vikas Bhandawat, Johannes Reisert, and King-Wai Yau (Johns Hopkins University, Baltimore, MD). Photon activation of a single rhodopsin molecule activates many G proteins, thereby amplifying the signal until rhodopsin is inactivated by phosphorylation. “Based on this one well-studied system, it has generally been assumed that other G protein pathways behave similarly,” says Yau. Now, his group's analyses of single olfactory receptor neurons reveal a low amplification system. An individual odorant-bound receptor exhibited a very low probability of activating even one downstream G protein molecule, as odorant receptor binding was transient—lasting 1 ms or less. “We expect many other ligand-triggered G protein pathways to behave similarly,” says Yau. Olfaction amplification therefore requires increasing the probability of G protein activation. This could be achieved either via many odorant molecules that continuously bind to receptors, or via a large number of receptors, so that odorants at low concentrations will still be able to find a receptor. “When these events are summated across all receptor molecules on the cell, and all cells express the same receptor protein,” says Bhandawat, “this produces substantial signal amplification and therefore high sensitivity in the brain.” Reference: Bhandawat, V., et al. 2005. Science. 308:1931–1934. [PubMed]

Mathematical and computational modelling of spatio-temporal signalling in rod phototransduction

Rod photoreceptors are activated by light through activation of a cascade that includes the G protein-coupled receptor rhodopsin, the G protein transducin, its effector cyclic guanosine monophosphate (cGMP) phosphodiesterase and the second messengers cGMP and Ca2+. Signalling is localised to the particular rod outer segment disc, which is activated by absorption of a single photon. Modelling of this cascade has previously been performed mostly by assumption of a well-stirred cytoplasm. We recently published the first fully spatially resolved model that captures the local nature of light activation. The model reduces the complex geometry of the cell to a simpler one using the mathematical theories of homogenisation and concentrated capacity. The model shows that, upon activation of a single rhodopsin, changes of the second messengers cGMP and Ca2+ are local about the particular activated disc. In the current work, the homogenised model is computationally compared with the full, non-homogenised one, set in the original geometry of the rod outer segment. It is found to have an accuracy of 0.03% compared with the full model in computing the integral response and a 5200-fold reduction in computation time. The model can reconstruct the radial time-profiles of cGMP and Ca2+ in the interdiscal spaces adjacent to the activated discs. Cellular electrical responses are localised near the activation sites, and multiple photons sufficiently far apart produce essentially independent responses. This leads to a computational analysis of the notion and estimate of 'spread' and the optimum distribution of activated sites that maximises the response. Biological insights arising from the spatio-temporal model include a quantification of how variability in the response to dim light is affected by the distance between the outer segment discs capturing photons. The model is thus a simulation tool for biologists to predict the effect of various factors influencing the timing, spread and control mechanisms of this G protein-coupled, receptor-mediated cascade. It permits ease of simulation experiments across a range of conditions, for example, clamping the concentration of calcium, with results matching analogous experimental results. In addition, the model accommodates differing geometries of rod outer segments from different vertebrate species. Thus it represents a building block towards a predictive model of visual transduction.

Mathematical Model of the Spatio-Temporal Dynamics of Second Messengers in Visual Transduction

A model describing the role of transversal and longitudinal diffusion of cGMP and Ca2+ in signaling in the rod outer segment of vertebrates is developed. Utilizing a novel notion of surface-volume reaction and the mathematical theories of homogenization and concentrated capacity, the diffusion of cGMP and Ca2+ in the interdiscal spaces is shown to be reducible to a one-parameter family of diffusion processes taking place on a single rod cross section; whereas the diffusion in the outer shell is shown to be reducible to a diffusion on a cylindrical surface. Moreover, the exterior flux of the former serves as a source term for the latter, alleviating the assumption of a well-stirred cytosol. A previous model of visual transduction that assumes a well-stirred rod outer segment cytosol (and thus contains no spatial information) can be recovered from this model by imposing a “bulk” assumption. The model shows that upon activation of a single rhodopsin, cGMP changes are local, and exhibit both a longitudinal and a transversal component. Consequently, membrane current is also highly localized. The spatial spread of the single photon response along the longitudinal axis of the outer segment is predicted to be 3–5μm, consistent with experimental data. This approach represents a tool to analyze pointwise signaling dynamics without requiring averaging over the entire cell by global Michaelis-Menten kinetics.

A2-rhodopsin: a new fluorophore isolated from photoreceptor outer segments.

A2E and iso-A2E are fluorescent amphiphilic pyridinium bisretinoids involved in age-related macular degeneration (AMD). It is now shown that the presence of high exogenous concentrations of all-trans-retinal in photoreceptor outer segments leads to the formation of A2-rhodopsin (A2-Rh), an unprecedented fluorescent rhodopsin adduct which consists of bisretinoids (A2) linked to each of three lysine residues in rhodopsin (Rh) and which exhibits an emission spectrum similar to A2E. The fluorophore to protein ratio was determined by MALDI-TOF-MS and UV-VIS spectroscopy. Enzymatic degradation with thermolysin and cathepsin D showed that two of the A2 moieties were located in the region of the third cytoplasmic loop and 8th helix of Rh. Examination of A2-Rh and A2-PE (the precursor of A2E) fluorescence in relation to all-trans-retinal concentration indicated that whereas A2-PE formation is favored over that of A2-Rh, for a single rhodopsin molecule only one phosphatidylethanolamine molecule is available to react with all-trans-retinal; this phosphatidylethanolamine is probably tightly associated with the protein.

Molecular Basis of Amplification in Drosophila Phototransduction : Roles for G Protein, Phospholipase C, and Diacylglycerol Kinase

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. We now find that quantum bumps in such mutants are reduced ∼3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules, identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction.

Origin of Reproducibility in the Responses of Retinal Rods to Single Photons

The single photon responses of retinal rod cells are remarkably reproducible, allowing the number and timing of photon absorptions to be encoded accurately. This reproducibility is surprising because the elementary response arises from a single rhodopsin molecule, and typically signals from single molecules display large intertrial variations. We have investigated the mechanisms that make the rod's elementary response reproducible. Our experiments indicate that reproducibility cannot be explained by saturation within the transduction cascade, by Ca 2+ feedback, or by feedback control of rhodopsin shutoff by any known element of the cascade. We suggest instead that deactivation through a series of previously unidentified transitions allows the catalytic activity of a single rhodopsin molecule to decay with low variability. Two observations are consistent with this view. First, the time course of rhodopsin's catalytic activity could not be accounted for by the time required for the known steps in rhodopsin deactivation—phosphorylation and arrestin binding. Second, the variability of the elementary response increased when phosphorylation was made rate-limiting for rhodopsin shutoff.

Photoreception in the planktonic larvae of two species of pullosquilla, a lysiosquilloid stomatopod crustacean

Optical microscopy, electron microscopy and microspectrophotometry were used to characterize pigments in the eyes of planktonic larvae of two species of the lysiosquilloid stomatopod Pullosquilla, P. litoralis and P. thomassini, which live sympatrically in French Polynesia. In contrast to the adult retina, which contains a diverse assortment of visual pigments in the main rhabdoms, the principal photoreceptors throughout the larval eyes of both species were found to contain a single rhodopsin with an absorption maximum (<IMG src="/images/symbols/&lgr ;.gif" WIDTH="8" HEIGHT= "12" ALIGN="BOTTOM" NATURALSIZEFLAG="3">max) close to 446 nm. The expression of this visual pigment may survive metamorphosis, since several adult rhodopsins occur at a similar spectral position. The retinas of these planktonic larvae also contain a novel yellow photostable pigment, which is arrayed in a regular pattern at the distal margin of the larval retina. The absorption spectrum of this pigment is well matched to the larval rhodopsin, suggesting that it acts to screen the rhabdoms from stray light. By replacing opaque, black screening pigment, the transparent yellow pigment may act together with a blue iridescent layer in the larval retina to reduce the visual contrast of the larval eye against downwelling and sidewelling light, while simultaneously acting as a retinal screen.