Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Kinetic study of transfer of 11-cis-retinal between rod outer segment membranes using regeneration of rhodopsin

The present study demonstrates some important facts on the regeneration of rhodopsin in rod outer segment membranes. 11-cis-Retinal added to a rod outer segment membrane suspension did not react directly with opsin but was rapidly solubilized into membranes and then recombined with opsin in the membrane. It was also revealed that the regeneration of rhodopsin was perturbed by the formation of retinylidene Schiff base with phosphatidylethanolamine in rod outer segment membranes, which decreased with increasing temperature. The activation energy of rhodopsin regeneration in rod outer segment membranes was 18.7 kcal/mol, being smaller than the value of 22 kcal/mol in 1% digitonin solution. 11-cis-Retinal could be found to transfer relatively fast (tau-1/k(1) R 10(3) s) between rod outer segment membranes by using the regeneration of rhodopsin. It was demonstrated that the kinetic measurement for the transport of membrane-soluble molecules such as retinal between membranes could be perform ed with ease and precisely by the method described in this paper.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3'',5''-cyclic monophosphate in frog photoreceptor membranes

The concentration of guanosine 3',5'-cyclic monophosphate (cyclic GMP) has been examined in suspensions of freshly isolated frog rod outer segments using conditions which previously have been shown to maintain the ability of outer segments to perform a light-induced permeability change (presence of calf serum, anti-oxidant, and low calcium concentration). Illumination causes a rapid decrease in cyclic GMP levels which has a half-time approximately 125 ms. With light exposures that bleach less than 100 rhodopsin molecules in each rod outer segment, at least 10(4)-10(5) molecules of cyclic GMP are hydrolyzed for each rhodopsin molecule bleached. Half of the total cyclic GMP in each outer segment, approximately 2 X 10(7) molecules, is contained in the light-sensitive pool. If outer segments are exposed to continuous illumination, using intensities which bleach between 5.0 X 10(1) and 5.0 X 10(4) rhodopsin molecules/outer segment per second, cyclic GMP levels fall to a value characteristic for the intensity used. This suggests that a balance between synthesis and degradation of cyclic GMP is established. This constant level appears to be regulated by the rate of bleaching rhodopsin molecules (by the intensity of illumination), not the absolute number of rhodopsin molecules bleached...     

Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.     

Dark Ionic Flux and the Effects of Light in Isolated Rod Outer Segments

We have determined the permeability properties of freshly isolated frog rod outer segments by observing their osmotic behavior in a simple continuous flow apparatus. Outer segments obtained by gently shaking a retina are sensitive but nonideal osmometers; a small restoring force prevents them from shrinking or swelling quite as much as expected for ideal behavior. We find that Na⁺, Cl^-, No₃^-, glycerol, acetate, and ammonium rapidly enter the outer segment, but K⁺, SO₄⁼, and melezitose appear impermeable. The Na flux is rectified; for concentration gradients in the physiological range, 2 x 10⁹ Na⁺ ions/sec enter the outer segment, but we detect no efflux of Na⁺, under our conditions, when the gradient is reversed. Illumination of the outer segment produces a specific increase in the resistance to Na⁺ influx, but has no effect on the flux of other solutes. This light-dependent Na⁺ resistance increases linearly with the number of rhodopsin molecules bleached. We find that excitation of a single rhodopsin molecule produces a transient (~1 sec) "photoresistance" which reduces the Na⁺ influx by about 1%, thus preventing the entry of about 10⁷ Na⁺ ions. At considerably higher light levels, a stable afterimage resistance appears which reduces the Na influx by one-half when 10⁶ rhodopsin molecules are bleached per rod. We have incorporated these findings into a model for the electrophysiological characteristics of the receptor.     

Reduction of all-trans retinal to all-trans retinol in the outer segments of frog and mouse rod photoreceptors

The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal.     

Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments

Phosphoinositides of chick and rat retina were labelled with [3H]inositol. Exposure of retinal preparations to light for 30 s caused loss of labelled phosphatidylinositol 4,5-bisphosphate and to a smaller extent of the other phosphoinositides. Similar light-induced changes were seen when rod outer segment preparations were used and, when these were illuminated in calcium-free media, phosphatidylinositol 4,5-bisphosphate was the only lipid affected. No inositol 1,4,5-trisphosphate was seen after either 30 s or 5 s of illumination of retina or 30 s illumination of rod outer segments. It is concluded that this compound plays no direct part in vertebrate photoreceptor light transduction, though phosphoinositide metabolism might relate to adaptation mechanisms.     

Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium

Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with 32Pi. The proteins, designated component I (13,000 daltons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains approximately 10(6( molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 x 10(5) rhodopsin molecules/outer segment per second. Light which bleaches 5.0 x 10(3) rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J. Gen Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977. J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the light-induced dephosphorylation is observed only in intact retinas.     

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N₂ atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin.We have found a large seasonal variation in the endogenous level of α-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of α-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol.This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.     

Characterization of the recombination reaction of rhodopsin.

The kinetics of recombination of 11-cis-retinal with bleached rod outer segments and sodium cholate solubilized rhodopsin have been investigated. At neutral pH, it was found that bleached rod outer segments in the presence of an excess of 11-cis-retinal follow pseudo-first-order kinetics. The results suggest the second-order formation of an intermediate addition compound followed by a first-order dehydration step to form a protonated aldimine linkage. In addition, at pH values above 7.5 or below 6.5 the kinetics of recombination are complex, indicating the formation of a molecular species inactive in recombination which is in equilibrium with the active form of opsin. Based upon the observed rate constants as a function of pH, a scheme is presented to describe the recombination reaction in bleached rod outer segments. The kinetics of recombination of sodium cholate solubilized opsin were also analyzed. In terms of formation of an intermediate addition compound and subsequent dehydration, the values for the individual rate constants for both bleached rod outer segments and cholate-solubilized opsin were found to compare very favorably. These results demonstrate that the sodium cholate (2 mg/ml) maintains opsin in a conformation very similar to that in the rod outer segment membrane and suggest that the cholate-opsin complex is an excellent model system for studies on opsin-membrane interactions.     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

Bovine rod rhodopsin. 1. Bleaching by luminescence in vitro by recombination of radicals from polyunsaturated fatty acids

Rod outer segments of photoreceptors are characterized by rhodopsin, a membrane protein surrounded by phospholipids containing a very high concentration of polyunsaturated fatty acids. These fatty acids can propagate free radicals, initiated by peroxidation, whose recombination is eventually associated with light emission as chemiluminescence. The results reported here indicate that this effect produces an isomerization of the retinal (bleaching effect) of the rhodopsin, similar to that induced by light in normal vision. In vitro experiments on detergent-suspended rod outer segments (RdOS) from bovine eyes, using an enzymatic source of radicals, xanthine/xanthine oxidase, were carried out. The results indicate that the proposed mechanism is likely, because they can show the bleaching of rhodopsin in RdOS, owing to its extraordinary sensitivity. Thus this mechanism is, also, a possible explanation for anomalous visual effects such as light flashes (phosphene-like) perceived by humans. The functionality of the rhodopsin in the RdOS was first tested by visible light. Rhodopsin reactivation after bleaching was obtained by adding cis-retinal to the suspension, demonstrating the reversibility of the bleaching process. A special experimental system was developed to observe the bleaching from luminescence by radical recombination, avoiding physical contact between the rod outer segment suspension and the radicals to prevent radical-induced damage and modifications of the delicate structure of the rod outer segment.     

Spatial arrangement of rhodopsin in retinal rod outer segment membranes studied by spin-labeling and pulsed electron double resonance

We have determined the spatial arrangement of rhodopsin in the retinal rod outer segment (ROS) membrane by measuring the distances between rhodopsin molecules in which native cysteines were spin-labeled at ～1.0mol/mol rhodopsin. The echo modulation decay of pulsed electron double resonance (PELDOR) from spin-labeled ROS curved slightly with strong background decay. This indicated that the rhodopsin was densely packed in the retina and that the rhodopsin molecules were not aligned well. The curve was simulated by a model in which rhodopsin is distributed randomly as monomers in a planar membrane.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.     

Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R*-G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (less than 10% R*) in absence of GTP therefore create predominantly R*-G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP-dependent protein kinase. a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. b) The phosphorylation of R* is strongly inhibited under conditions of R*-G complex formation as compared to free R*. Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R*; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R*. We also observed a slow and nucleotide-dependent light-induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light-dependent protein previously identified in cattle rod outer segments.