Transglutaminase modification of rhodopsin in retinal rod outer segment disk membranes

Rhodopsin in rod outer segment disk membranes was enzymatically modified by erythrocyte transglutaminase, which linked small primary amines to glutamine residues. In order to avoid formation of protein crosslinks, rhodopsin was first reductively methylated to modify its lysines. From 1.9 to 2.5 mol of putrescine, ethanolamine, or dinitrophenylcadaverine were incorporated into rhodopsin by transglutaminase during 16 h reaction time. A maximum of 3.5 mol of [14C]putrescine was incorporated per mole of rhodopsin during 48 h. Essentially all of the rhodopsin sequence containing the putrescine could be removed by limited proteolysis of the membranes by thermolysin. Glutamine residues in positions 236, 237, 238, and 344 were modified to approximately equal extents, as determined by isolation of the cyanogen bromide peptides of modified rhodopsin followed by further subdigestion of the peptides. The modified glutamine residues are located in the helix V-VI (or F1-F2) connecting loop and in the carboxyl-terminal region of rhodopsin.     

The bilayer enhances rhodopsin kinetic stability in bovine rod outer segment disk membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (T_m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T_m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (E_act) was calculated from the scan rate dependence of the T_m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E_act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E_act to rhodopsin denaturation.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.     

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.     

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.     

The role of cholesterol in rod outer segment membranes

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.     

Calorimetric studies of bovine rod outer segment disk membranes support a monomeric unit for both rhodopsin and opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (T_m) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T_m of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T_ms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T_m remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T_m of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.     

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.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

Photobleaching and cyclic GMP dependences of rhodopsin phosphorylation in rod outer segment

The experimental data on the cGMP decrease under continuous illumination of rod outer segment have been theoretically analysed to study the bleaching and hence the cGMP dependence of the rhodopsin phosphorylation. From the agreement of the theoretical results with the experimental observations it has been found that the rate of phosphorylation depends on the rate of cGMP hydrolysis. If the rate of cGMP hydrolysis increases the rate of phosphorylation also increases. The results of the theoretical treatment predict that (i) the presence of cGMP in rod outer segment inhibits the rhodopsin phosphorylation and (ii) rhodopsin phosphorylation process is much faster than what has been reported in the literature.     

Monensin stimulates glycerolipid incorporation into rod outer segment membranes

Monensin is an ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells. In this study, we examined the effects of monensin on the incorporation of newly synthesized glycerolipids into retinal rod outer segment (ROS) membranes. Frog retinas were incubated in the presence or absence of monensin (50 nM) with either [1,2,3-3H]glycerol or [9,10-3H]palmitic acid as radiolabeled substrate. Total lipids were extracted from retinas and ROS membranes and resolved into individual phospholipid classes and neutral lipids by thin-layer chromatography. In the presence of monensin, the specific activity of ROS phospholipids was increased about 2-fold with [3H]glycerol and nearly 3-fold with [3H]palmitate as substrates relative to controls. In contrast, the specific activity of total retinal lipids, the relative incorporation of label into ROS and retinal phospholipids, and the total lipid phosphorous content of ROS membranes and retinas were not significantly different from control values. These data suggest that the enhanced labeling of ROS phospholipids in the presence of monensin was due to altered intracellular routing of lipids rather than increased glycerolipid synthesis. Under the same conditions, total retinal protein synthesis was about 90% of control, but light microscopic autoradiography indicated that newly synthesized proteins were not transported to the ROS for assembly into disc membranes. Thus, newly synthesized glycerolipids can be delivered to the ROS by a mechanism which is independent of protein transport to that cellular compartment.     

Site of attachment of 11-cis-retinal in bovine rhodopsin

A dipeptide containing the binding site for retinal in bovine rhodopsin has been isolated and its sequence determined. Rhodopsin containing [11-3H]retinal was prepared in chromatographically pure form, and the [3H]retinal was reductively linked to its binding site on opsin by using borane--dimethylamine. The [3H]retinylopsin in octyl glucoside was exhaustively digested with Pronase, and its peptides were separated on silica gel in chloroform/methanol/ammonia [Bownds, D. (1967) Nature (London) 216, 1178--1181] followed by silica gel thin-layer chromatography in two solvent systems. The major retinyl peptide was shown to be alanyl-N epsilon-retinyllysine by amino acid composition, 3H content, and amino acid sequence analysis. The retinyl binding site is located in the carboxyl-terminal region of rhodopsin: when rod cell disk membranes containing [3H]retinal rhodopsin were digested with thermolysin and then reacted with sodium borohydride or borane--dimethylamine, [3H]retinal was reduced onto the F2 (Mr congruent to 6000) fragment, which derives from rhodopsin's carboxyl-terminal region.     

Localization of rhodopsin in isolated, osmotically intact rod outer segment discs.

Localization of rhodopsin and its position in the membrane has been the subject of numerous studies. Most recently, immunocytochemical techniques have been employed to localize the opsin component of the molecule in in situ rod outer segments. Due to the problems inherent in localization procedures (penetration and mechanical interference) we have utilized isolated, osmotically intact rod outer segment discs in this study. Specific antibodies to chromatographically pure rhodopsin were prepared and enzymatically digested to their Fab components. The univalent Fab antibodies were conjugated to horseradish peroxidase and used to label the isolated rod outer segment discs. Discs treated with anti-opsin conjugate stained uniformly and heavily on their interdisc surfaces. Reaction product was also present on the intradisc surface in a thinner but still uniformly distributed layer. Controls treated with preimmune Fab - horseradish peroxidase conjugate showed no deposition of reaction product.     

An evaluation of purity criteria for bovine rod outer segment membranes

The purity criteria of bovine rod outer segments (ROS) purified by different procedures were evaluated. Bovine ROS were purified by flotation and/or sedimentation in a continuous concentration gradient of sucrose. The purity of the different fractions was then evaluated according to four purity criteria: (i) the A280/delta A500 ratio, (ii) the moles of phospholipid per mole of rhodopsin, (iii) the fatty acid composition, and (iv) the interfacial properties of ROS membranes. All the purity criteria, except the A280/delta A500 ratio, were found to be adequate. From our results, the A280/delta A500 ratio cannot be used alone to characterize ROS purity. Furthermore, the phospholipid-to-rhodopsin ratio appears as the best purity criterion because of its reliability, its higher sensitivity, and its ease of achievement. It is noteworthy that mechanical treatment of the retinas dramatically affects the purification of ROS.     

Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

In the presence of exogenous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition, GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5'-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.     

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.     

Isolation of rhodopsin by the combined action of cardiotoxin and phospholipase A2 on rod outer segment membranes

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom V₄^II cardiotoxin in rod outer segment membrane preparations. The extent of the morphological changes depended on the purity of the cardiotoxin. Pure cardiotoxin had no detectable effect upon the preparation, but, when contaminated with venom phospholipase A₂, let to a rapid disintegration of the membrane vesicles. With trace amounts (up to about 0.5% of the cardiotoxin) of phospholipase A₂, the membrane vesicles disintegrated into smooth lamellae and particles in solution. These two components were separated by centrifugation. The pellet, which showed the presence of smooth lamellae and aggregated particles, was composed of unbleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. The supernatant, which showed only the presence of dispersed particles, was composed of unbleached rhodopsin, lysolipids and cardiotoxin. With cardiotoxin containing larger amounts of phospholipase A₂ (more than 0.5% of the cardiotoxin), membrane vesicles were disintegrated into large aggregates of amorphous material, composed of bleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. These results confirm our previous observation on the release of integral membrane proteins from membrane vesicles by the action of cardiotoxin containing traces of phospholipase A₂ (Gulik-Krzywicki, T., Balerna, M., Vincent, J.P. and Lazdunski, M. (1981) Biochim. Biophys. Acta 643, 101–114) and suggest its possible use for isolation and purification of integral membrane proteins.