Proteomic analysis of the retinal rod outer segment disks

The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.     

Calcium adenosinetriphosphatase of bovine retinal rod outer segment disks.

A Ca(2+)-pumping ATPase activity is present in bovine retinal rod outer segment purified disks. The ATPase has a high Ca2+ affinity (KM = 25 microM). Low Ca2+ (n-microM) concentrations stimulate an ATP-dependent Ca2+ uptake and the ATP hydrolysis in the absence of exogenous Mg2+. Electrophoretic analysis of disk proteins after treatment with (gamma-32P)ATP shows the existence of the enzyme-phosphate acid-stable, hydroxylamine-sensitive intermediate complex of molecular mass of about 135 kDa. The results would indicate the presence of an inwardly directed Ca(2+)-ATPase pump acting on the disk membrane, that could be involved in the regulation of cytosolic free Ca2+ levels inside ROS.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

A barrier to lateral diffusion of porphyropsin in Necturus rod outer segment disks

Microspectrophotometry was used to study lateral diffusion of the visual pigment, porphyropsin , in the disk membrane in intact mudpuppy (Necturus maculosus) rod outer segments (ROS), isolated in frog Ringer's solution. A concentration gradient of unbleached visual pigment was produced on the disks by rapidly photobleaching 40% of the pigment in an area spanning 1/4 or 1/2 of the cell's width. The change in optical density of the cells at 580 nm was then followed with time on either the bleached or unbleached side. The temperature dependence of porphyropsin diffusion yielded a Q10 of 2.5 between 10 and 20 degrees C with an activation energy of 12 +/- 2 kcal. At completion of pigment diffusion, the center and edge of the disk had, respectively, attained only 90 and 55% of the concentration expected. Computed diffusion coefficients (5.4 X 10(-9) cm2/s) were similar at the center and periphery of the disk immediately after the flash, however, an additional slow component for diffusion was detected at the periphery. A comparison of optical density at 525 nm along the diameter of ROS before and after the flash showed a persistent (20 min) postbleach concentration gradient of unbleached porphyropsin . This suggests that 15% of the prophyropsins may be sequestered into distinct areas on a mudpuppy disk and are not free to diffuse over the whole surface. This argument is supported by the observation that mudpuppy disks are separated into petal -shaped regions by incisures, some of which penetrate nearly to the disk center.     

Preparation and characterization of monoclonal antibodies to several frog rod outer segment proteins

Monoclonal antibodies to proteins important in phototransduction in the frog rod outer segment have been obtained. These include 6 different antibodies to rhodopsin, 50 to a guanine nucleotide binding protein (G-protein; 40,000 daltons), and 2 to cytoplasmic proteins. The antigens used were Percoll-purified rod outer segments, a rod outer segment soluble protein fraction, or a soluble plus peripheral membrane protein fraction. Antibodies were assayed by solid phase assay using a fluorogenic detection system. Proteins to which antibodies bound were assayed on Western blots, and the sensitivities of three different detection systems were compared. Most antibodies bound to only one rod outer segment protein band on Western blots. Immunofluorescence microscopy demonstrated binding of both anti-rhodopsin and anti-G-protein to isolated frog rod outer segments. Antibodies were purified from either culture supernatants or ascites fluid on protein A affinity columns. Two purified anti-G-protein antibodies have binding affinities to 125I-labeled G-protein of less than 10(-6) M-1. Of 11 antibodies to frog or bovine G-protein tested in solid phase and Western blot assays, all bind to the alpha rather than the beta or gamma subunits. Procedures developed here are being used in preparing other antibodies that affect reactions in the phototransduction pathway.     

Membrane fusion in the rod outer segment disk membrane

The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

External calcium inhibits the efflux of calcium from isolated retinal rod outer segment disks

Increasing the concentration of calcium in the external buffer flowing past isolated, intact bovine retinal rod outer segment disks immobilized in a flow system reduced the rate of radioactive calcium efflux from within the disks in the dark. We interpret these results as extradiskal calcium acting at an inhibitory binding site to block the calcium efflux. A Scatchard analysis of the external calcium dependence of the efflux yields an apparent dissociation constant of 50 microM, which further suggests that the inhibition is mediated by a specific membrane binding site. The observed inhibition of calcium efflux may represent a functional role for the high-affinity calcium binding site which has been identified by others in previous physical studies of the disk membrane. This external calcium inhibited permeability may explain some of the discrepancies in the reported calcium transport properties of disks. Variations in the external calcium concentration may alter the calcium content of isolated disks, thereby indirectly affecting other transport functions including the measured light-induced release of calcium. No evidence was found for either Na/Ca or Ca/Ca exchange processes across the disk membrane. Lanthanum was even more effective than calcium in inhibiting calcium efflux in the dark. Neither lanthanum nor calcium inhibited the light-induced efflux of calcium from disks, which implies either that light and extradiskal calcium regulate separate permeability processes in the disk membrane or that light greatly reduces the affinity of the inhibitory site for calcium and lanthanum.     

Sulfhydryl group distribution along the axis of the rod outer segment in the frog

The existence of SH-group concentration axial gradient in frog's retinal rod outer segments has been shown. A diminution of SH-groups in the outer segment apical part points to a damage of the vision pigment during the life span of the rod disks.     

The C terminus of peripherin/rds participates in rod outer segment targeting and alignment of disk incisures

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.     

Light-stimulated GTP binding to a membrane protein in rod outer segments.

Bovine as well as frog rod outer segments contain a membrane-bound protein which binds the GTP analog GppNp in the light (Kd=0.3μM). The amount of GppNp bound is 2.5–3.5 mmole per mol rhodopsin. The binding protein (M.Wt. ? 54,000) can be extracted from rod membranes with detergent and purified on an Agarose column. The chromatographic profile indicates that the binding protein is distinct from rhodopsin, GTPase or cyclic nucleotide phosphodiesterase.     

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.     

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning³¹P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 Å) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.