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.     

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.     

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.     

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.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

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.     

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.     

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.     

Light-dependent association of Src with photoreceptor rod outer segment membrane proteins in vivo.

In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.     

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.     

Rhodopsin lateral diffusion as a function of rod outer segment disk membrane axial position

Rhodopsin lateral diffusion was measured at three points along the axis of frog outer segments using the method of absorbance recovery after photobleaching. Mean recovery times were slightly longer in distal disk membranes than in proximal disks. A small reduction of pigment mobility with disk age may reflect subtle changes in membrane composition.     

X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers.

Phase separation in artificially stacked multilayers of isolated bovine retinal rod outer segment (ROS) membranes has been examined via x-ray diffraction and electron microscopy. Specimens were prepared by isopotential spin drying followed with partial hydration by equilibration against moist gas streams. Upon dehydration, the multilamellar membrane phase assumes a binary phase composition consisting of concentrated protein-containing lamellae interspersed with microdomains of hexagonally packed tubes of lipid in a HII configuration. The HII lattice is geometrically coupled to the lamellar phase with one set of hexagonal crystal planes co-planar to the local membrane lamellae. The hexagonal microdomains bear a striking resemblance to the "paracrystalline inclusions" observed in fast-frozen, intact frog ROS (Corless and Costello. 1981. Exp. Eye Res. 32:217). The lamellar lattice is characterized by an unusually small degree of disorder. Sharp lamellar diffraction with a 120 A unit cell is observed (at near total dehydration) to a resolution of 6 A. A model consistent with the data is that a multilamellar array of ROS disks is stable as long as the external disk surfaces are kept sufficiently far apart. If the distance between the membranes is allowed to shrink below a certain critical value, the disk lipids spontaneously convert to a nonbilayer phase. This suggests that the structure of the ROS is stabilized by an internal framework that acts to keep the disks apart from one another and from the plasmalemma. Thus, the necessity of avoiding phase separations may provide a rationale for the peculiar morphology of the ROS.     

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.     

Differential Ca(2+) sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling

Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ～1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.     

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.     

The asymmetric transmembrane distribution of phosphatidylethanolamine,phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane

Summary The transmembrane distribution of the major aminophospholipids in the bovine retinal rod outer segment disk membrane, phosphatidylethanolamine and phosphatidylserine, was determined using a novel pair of permeable and impermeable covalent modification reagents. The values for the percentages of phosphatidylethanolamine and phosphatidylserine in the outer monolayer were calculated from a simple expression which takes into account the leakage of impermeable reagent into the disk lumen as monitored by the extent of labeling of lysine entrapped in the lumen. We infer from our results that at least 73 to 87% of the disk phosphatidylethanolamine and 77 to 88% of the disk phosphatidylserine are in the outer disk membrane monolayer. The fatty acid composition of the inner aminophospholipids is slightly more saturated than the outer aminophospholipids. Calculations using the lateral surface areas occupied by the disk membrane lipids suggest that 65 to 100% of the disk phosphatidylcholine is on the inner membrane surface. Since the disk phosphatidylcholine is also somewhat more saturated than the phosphatidylethanolamine and phosphatidylserine of the outer monolayer, the total inner membrane monolayer fatty acid composition is more saturated than that of the outer monolayer fatty acid composition.     

Association of a photoreceptor-specific tetraspanin protein,ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.     

Photoreceptor number and outer segment disk membrane surface area in the retina of the rat: stereological data for whole organ and average photoreceptor cell

A random sampling scheme is employed to obtain stereological estimates of disk membrane surface area in the entire retina and in the average photoreceptor cell. The scheme involves the use of vertical sections with combined light and electron microscopy at several magnification levels. Left and right retinas from six albino animals were analysed. There were no significant lateral differences. On average, the retina had a volume of 16 mm3, thickness of 200 μm and surface area of 80 mm2 (representing about 56% of the external surface of the eyeball). Photoreceptor disk membranes within outer segments amplified total retinal surface by almost 1000-fold (final surface 770 cm2 per retina). The retina contained 3×107 photoreceptors (packing density 374 000 mm-2) with an average disk membrane surface area of 2600 μm2. Mean nuclear volume in photoreceptor cells was 59 μm3 and the coefficient of variation for the distribution of nuclear volumes was 57%. The data are consistent with an average of 700 disks per photoreceptor cell, a membrane area of 4 μm2 per disk and a convergence ratio of ～260 photoreceptors per optic nerve fibre. The basic scheme could be modified for other species and for direct cell counts conducted on rods and cones separately.