Photoreceptor Disk Membranes Research Articles

The cGMP-gated Channel and Related Glutamic Acid-rich Proteins Interact with Peripherin-2 at the Rim Region of Rod Photoreceptor Disc Membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.

Membrane Topology of the ATP Binding Cassette Transporter ABCR and Its Relationship to ABC1 and Related ABCA Transporters: IDENTIFICATION OF N-LINKED GLYCOSYLATION SITES

ABCR is a member of the ABCA subclass of ATP binding cassette transporters that is responsible for Stargardt macular disease and implicated in retinal transport across photoreceptor disc membranes. It consists of a single polypeptide chain arranged in two tandem halves, each having a multi-spanning membrane domain followed by a nucleotide binding domain. To delineate between several proposed membrane topological models, we have identified the exocytoplasmic (extracellular/lumen) N-linked glycosylation sites on ABCR. Using trypsin digestion, site-directed mutagenesis, concanavalin A binding, and endoglycosidase digestion, we show that ABCR contains eight glycosylation sites. Four sites reside in a 600-amino acid exocytoplasmic domain of the N-terminal half between the first transmembrane segment H1 and the first multi-spanning membrane domain, and four sites are in a 275-amino acid domain of the C half between transmembrane segment H7 and the second multi-spanning membrane domain. This leads to a model in which each half has a transmembrane segment followed by a large exocytoplasmic domain, a multi-spanning membrane domain, and a nucleotide binding domain. Other ABCA transporters, including ABC1 linked to Tangier disease, are proposed to have a similar membrane topology based on sequence similarity to ABCR. Studies also suggest that the N and C halves of ABCR are linked through disulfide bonds.

11] Functional study of rhodopsin phosphorylation in vivo

Publisher Summary Phosphorylation of rhodopsin is a key reaction in the termination of the light response. It is the first step in rhodopsin inactivation, beginning 100 msec after the light stimulus. A subsequent step, the binding of arrestin to phosphorylated rhodopsin, is necessary to complete inactivation. Extensive work has been done to address basic aspects of rhodopsin Phosphorylation. Its importance in rhodopsin shutoff was first identified by in vitro reconstitution studies. The sites of phosphorylation on rhodopsin were localized at several serine and threonine residues at the cytoplasmic carboxyl terminus of rhodopsin, and the most favored sites in vitro were reported as Ser-338 and Ser-343 by three independent studies. The carboxyl terminus of rhodopsin was isolated from photoreceptor disk membranes and analyzed by chromatographic separation and mass spectrometry. This chapter describes how to generate transgenic mice expressing rhodopsin mutants at the phosphorylation sites and how to characterize the lines in terms of transgene expression and retinal morphology. It also describes the criteria used in the selection of mice for electrophysiological recordings so that light responses elicited by mutant rhodopsins can be unequivocally assigned.

30] Isoprenylation/methylation and transducin function

Freshly prepared proteolyzed (deprenylated) T beta gamma and material isolated from retina are inert with respect to activating T alpha in the presence of R* in detergent and in disk membranes. In addition, proteolyzed T beta gamma is also incapable of supporting the pertussis toxin-catalyzed ADP ribosylation of T alpha-GDP. These experiments show that isoprenylation/methylation is essential for the fruitful interactions between T alpha and T beta gamma at the membrane. When tested for its ability to support GTP-for-GDP exchange catalyzed by R*, demethylated T beta gamma proved to be approximately 50% as active as methylated T beta gamma in photoreceptor disk membranes (Fig. 3) and in reconstituted liposomes containing rhodopsin. In detergent, no difference was observed between methylated and demethylated T beta gamma, suggesting no role at all for the methyl group in functional interactions between T alpha, T beta gamma, and R*. The twofold activity difference observed in membranes can be accounted for by the twofold lessened affinity of the demethylated T beta gamma, compared with its methylated counterpart, for membranes in the presence of R* and T alpha. It is interesting to note that a substantially larger difference (> 10-fold) in the relative binding of methylated versus demethylated T beta gamma to membranes is observed in the absence of R* and T alpha. However, R* has a substantial affinity for T alpha beta gamma, and the influence of R* and T alpha greatly reduces any differences resulting from the presence or absence of a methyl group on T beta gamma. The results from studies of demethylated T beta gamma demonstrate that specific lipid-receptor interactions are unlikely to play a critical role in the rhodopsin-transducin system, and further show that the effect of methylation is probably due to the increased hydrophobicity of methylated T beta gamma versus its unmethylated counterpart. These studies are, of course, relevant to heterotrimeric G proteins, and specifically to the interactions of receptor (R*) with T alpha and T beta gamma. If a hydrophobic lipid-lipid mechanism is operative, the state of methylation would be expected to have a more profound effect on the membrane-associative properties of farnesylated proteins, but not on those of geranylgeranylated proteins. The increased hydrophobicity of the C20 geranylgeranyl group relative to the C15 farnesyl group will compensate for the loss of the methyl substituent. The results obtained in the transducin-rhodopsin system can be contrasted with the effect of gamma-subunit methylation on effector enzyme activation. In the case of the geranylgeranylated beta 1 gamma 2, methylation proved to have only a small effect on PIPLC beta activation (Fig. 4B). An approximately 25% diminution in efficacy, but not potency, was observed for the demethylated geranylgeranylated beta 1 gamma 2 versus its methylated counterpart. This again shows that specific lipid-protein interactions are unimportant. The effect of methylation on membrane binding would be expected to be small, given that beta 1 gamma 2 is geranylgeranylated. It is of interest to compare these results with those found with methylated and unmethylated T beta gamma as activators of PIPLC beta. In this instance there was a large effect noted, with methylated T beta gamma being at least 10-fold more potent than its unmethylated counterpart with respect to activating either enzyme (Fig. 4A). This result is readily understandable in light of the role of methylation in selectively enhancing hydrophobicity of farnesylated proteins as opposed to geranyl-geranylated proteins. Similar results were obtained for the activation of PI3K, further strengthening the conclusion that it is lipid-lipid interactions that direct beta gamma subunit membrane association. (ABSTRACT TRUNCATED)

High-resolution freeze-etching replica images of the disk and the plasma membrane surfaces in purified bovine rod outer segments.

Molecular organization of the photoreceptor disk membrane was revealed by the freeze-deep etching replica method using purified and successively rinsed bovine rod outer segment (ROS). Various membrane particles with different shape and sizes were found on cytoplasmic surface (PS face) as well as on both P and E fracture faces, which are presumed to be peripheral membrane proteins such as transducin, phosphodiesterase, guanylate cyclase and so on. Membrane particles seen on PS face were catalogued in size. The histogram on their number and size showed that they were classified at least into two major groups, the group of particles about 50 nm2 in size and the group of particles about 115 nm2 in size. The distribution density of the 115 nm2 particle was 1200 microm(-2) in native state, but it decreased to 125 microm(-2) after washing with hypotonic buffer solution. Namely, the group of the 115 nm2-particle seems to be mainly composed of peripheral membrane proteins. Rinsing with the sucrose free buffer at the final step of the purification procedure enabled us to observe three types of filaments localized in ROS (filaments connecting disk to disk at the margin, filaments connecting disk to the plasma membrane, filaments associated with PS face of disk membrane); and also to find characteristic domains with crystal arrangement of particles on the external surface (ES face) of ROS plasma membrane.

S100B and S100A1 proteins in bovine retina: their calcium-dependent stimulation of a membrane-bound guanylate cyclase activity as investigated by ultracytochemistry

The Ca 2+-binding proteins of the EF-hand type, S100B and S100A1, were detected in the outer segment of bovine retina photoreceptors where they are localized to disc membranes, as investigated by immunofluorescence and immunogold cytochemistry. S100B and S100A1 stimulate a membrane-bound guanylate cyclase activity associated with photoreceptor disc membranes in dark-adapted retina in a Ca 2+-dependent manner, although with different Ca 2+ requirements, as investigated by an ultracytochemical approach. Other retinal cell types express S100B and S100A1 as well. S100B is detected in the outer limiting membrane, fine cell processes in the outer nuclear layer and the outer plexiform layer, cell bodies in the inner nuclear layer and the ganglion cell layer, and the inner limiting membrane, whereas S100A1 has a more discrete distribution. S100B and S100A1 also stimulate a membrane-bound guanylate cyclase activity in photoreceptor cell bodies and Müller cells, but their effect appears independent of the light- or dark-adapted state of the retina and is observed at relatively high Ca 2+ concentrations. These data represent the ultrastructural counterpart of recent biochemical observations implicating S100B and, possibly, S100A1 in the Ca 2+-dependent stimulation of a photoreceptor membrane-bound guanylate cyclase activity [T. Duda, R. M. Goraczniak and R. K. Sharma (1996) Molecular characterization of S100A1–S1000B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclast. Biochemistry 35, 6263–6266; A. Margulis, N. Pozdnyakov and A. Sitaramayya (1996) Activation of bovine photoreceptor guanylate cyclast by S100 proteins. Biochem. Biophys. Res. Commun. 218, 243–247]. Our data suggest that at least S100B may take part in the regulation of a membrane-bound guanylate cyclase-based signalling pathway in both photoreceptors and Müller cells.

Investigation of Luminal Surface Structures of Rod Photoreceptor Outer Segments by Lectin Cytochemistry Combined with Freeze-Etching

The luminal surface of the rod photoreceptor disk membrane was exposed by means of osmotic shock and labeled with ferritin- conjugated concanavalin A. The structural changes of the luminal surface were examined by a freeze-etching procedure with cryoprotectant (methanol). On replicas from freeze-etched membranes with concanavalin A labeling, 6- to 10-nm particles were codistributed with ferritin particles on the luminal surface of the disk membrane. By contrast, there were few ferritin particles or less numerous 6- to 10-nm particles on the corresponding surface without concanavalin A labeling. If 6- to 10-nm particles corresponded to the carbohydrate moiety of rhodopsin, concanavalin A binding might tend to preserve this carbohydrate moiety. These results suggest that the two-dimensional analysis of lectin-induced structural changes of the membrane surface glycoconjugates may become available by lectin cytochemistry combined with freeze-etching.

Disk membrane initiation and insertion are not required for axial disk displacement in Xenopus laevis rod outer segments

PURPOSE. Mechanisms that maintain the close coupling between the formation of photoreceptor disk membranes and the displacement of disk membranes toward the pigment epithelium are poorly understood. This study was designed to determine whether the axial displacement of disk membranes requires the assembly and insertion of new disk lamellae. METHODS. Retinal detachment and treatment with cytochalasin D were employed to interrupt the normal formation of disk membranes in cultured Xenopus laevis retinas. The effect of disrupting disk initiation and assembly upon disk displacement was documented and quantified. RESULTS. Isolating retinas from the retinal pigment epithelium prevented the normal morphogenesis of disks, but previously formed disks moved distally at a rate that is greater than or equal to the rate in attached retinas or in vivo. Treatment of attached retinas in eyecups with cytochalasin D blocked initiation of new disks and resulted in the formation of ectopic, disk-like membranes, but it did not stop axial displacement of previously formed disks. Rod cells in retinas that were cultured while slightly elevated from the retinal pigment epithelium sometimes formed disks of a smaller diameter than normal, even though the rate of initiation and displacement of disks was the same as in vivo. CONCLUSIONS. Observations on detached retinas and or retinas treated with cytochalasin D suggest that disk displacement does not depend upon normal disk formation and that the motive mechanism does not involve filamentous actin. The formation of small diameter disks in elevated retinas suggests that disk initiation and displacement is independent of the completion of normal diameter disks.

Analysis of the Tβγ-binding domain of MEKA/phosducin

MEKA/phosducin, a 33 kDa phosphoprotein in the photoreceptor cell, associates with transducin βγ(Tβγ) with its N-terminal domain (N-terminal 105 amino acids of MEKA), and translocates Tβγ from the photoreceptor disc membrane to the soluble fraction. The present study further localized the Tβγ-binding domain to aa 17–105 of MEKA, and showed that the activity of MEKA to translocate Tβγ depends on the domain. A series of deletion mutant MEKA proteins were prepared to investigate the domain of MEKA which binds to and translocates Tβγ. Both binding and translocation activities were not impaired by the deletion of the N-terminal 16 amino acids of MEKA, but completely abolished by further deletion to 42Val. Although anti-MEKA serum inhibited the Tβγ-MEKA association, the antiserum absorbed with a recombinant peptide corresponding to aa 17–105 of MEKA did not, confirming that aa 17–105 of MEKA directly interacts with Tβγ.

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 microns 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 x 10(7) photoreceptors (packing density 374,000 mm-2) with an average disk membrane surface area of 2600 microns2. Mean nuclear volume in photoreceptor cells was 59 microns3 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 microns2 per disk and a convergence ratio of approximately 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.

SHORT COMMUNICATION: Photoreceptor rim protein: partial sequences of cDNA show a high degree of similarity to ABC transporters

PURPOSE. To isolate and sequence cDNA for bovine rim protein, a large membrane-bound glycoprotein found in photoreceptor outer segments. METHODS. Bovine rim protein was N-terminally sequenced (22 residues) and fragments were prepared by partial proteolysis. Two internal sequences of 21 and 18 amino acid residues were obtained from 35 kDa and 32 kDa fragments, respectively. Sense and anti-sense oligonucleotide primers were constructed, based on the peptide sequences derived from the 35 kDa and 32 kDa fragments, respectively, and the polymerase chain reaction (PCR) was used to amplify a 150 bp sequence from bovine retinal cDNA. RESULTS. The amplified sequence coded for the remainder of the peptide sequence determined from the 35 kDa fragment, which was not present in the primer, confirming that it was derived from the rim protein. The 150 bp sequence was translated to give a 50 amino acid peptide. Part of this peptide was compared with Dna sequence databases using the TFastA program, which found 94.6% identity with an EST derived from human retina and 86.1% identity to the mouse abc1 transporter. CONCLUSIONS. It is proposed that rim protein is a member of the ATP transporter family of proteins. It may be involved in transport of molecules involved in visual transduction across the photoreceptor disk membrane.

MEKA/Phosducin Attenuates Hydrophobicity of Transducin βγ Subunits without Binding to Farnesyl Moiety

Hydrophobic modifications of transducin (T)γ,such as farnesyl-and carboxyl-methylation, are essential for the association of Tβγ with the photoreceptor disc membrane, and MEKA/phosducin is known to inhibit the association. In this study, we examined the effect of MEKA on the hydrophobicity of Tβγ.MEKA could bind to Tβγ without farnesyl/carboxyl-methyl moieties as well as native Tβγ.In the Triton X-114 phase separation assay, Tβγ-MEKA complex was recovered in the aqueous phase, whereas Tβγ was recover in the detergent phase. N-terminal portion of MEKA which includes Tβγ-binding domain was not sufficient to reduce the hydrophobicity of Tβγ or to dissociate Tβγ from the membrane. The data suggest that MEKA attenuates the hydrophobicity of Tβγ to result in the dissociation of Tβγ from the membrane without directly binding to farnesyl/carboxyl-methyl moieties.

Phospholipid Solubilization during Detergent Extraction of Rhodopsin from Photoreceptor Disk Membranes

The solubilization of rhodopsin and phospholipids from disks prepared from bovine retinal rods was studied using five different detergents. The relative amounts of rhodopsin and lipid extracted during membrane solubilization differed dramatically with the nature of the surfactant; the two nonpolar detergents, Emulphogene (polyoxyethylene-10 tridecylether) and octylglucoside, removed more protein than lipid; two bile salt-related detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) and taurocholate, released relatively more lipid than protein; and digitonin, which shares characteristics with both groups of detergents, extracted more lipid per mole of rhodopsin than the former two but less than the latter two. Solubilization was temperature-dependent with all five detergents, though particularly so with octylglucoside: concentrations adequate for the total micellation of disks at 23°C were ineffectual at 4°C. In total solubilizates of disks, the amount of lipid recovered in rhodopsin–lipid–detergent micelles showed a closer correlation with the critical micellar concentration (CMC) than with the chemical nature of the detergent (octylglucoside > taurocholate > Chaps > digitonin > Emulphogene). The higher the CMC, the larger the amount of lipid associated to the solubilized rhodopsin and the larger the amount of lipid reassociated to rhodopsin upon surfactant dilution. For all five detergents, the lipid progressively extracted from disks during solubilization was relatively richer in phosphatidylcholine (PC) than the lipid in the original membranes. The lipid which tended to be associated with rhodopsin in protein–lipid–detergent mixed micelles was also consistently richer in PC than that present in lipid–detergent micelles. Bleaching of solubilized rhodopsin decreased the amount of lipid in protein–lipid–detergent micelles. Rhodopsin photolytic transitions were faster in nonionic than in bile salt-related detergents.

Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes.

Peripherin/rds is a 39 kDa integral membrane glycoprotein essential for normal photoreceptor cell development in vertebrates. It has been implicated in several human retinal degenerative diseases including retinitis pigmentosa and macular degeneration and is thought to play a structural role at photoreceptor outer segment disk rims, where it forms a tightly-associated complex with rom-1, a nonglycosylated 37 kDa homologue. Western blot analysis of COS-1 cells transiently transfected with full-length cDNA coding for either peripherin/rds or rom-1 indicates that each protein is expressed primarily as a disulfide-linked homodimer; recombinant peripherin/rds is glycosylated while recombinant rom-1 is not--akin to their counterparts in rod photoreceptor disk membranes. Upon cotransfection of the two cDNAs, the specific assembly of a stable peripherin/rds--rom-1 complex is observed. Immunofluorescence microscopy studies demonstrate that both singly and coexpressed peripherin/rds and rom-1 complexes are localized primarily within internal membranes of transfected cells. Velocity sedimentation data indicate that the recombinant complexes (4.9 S) are assembled with a subunit stoichiometry similar to those extracted from ROS membranes (4.5 S) and are most consistent with a tetrameric arrangement of polypeptides. Sedimentation analyses of individually expressed peripherin/rds (5.1 S) and rom-1 (4.3 S) suggest that each polypeptide can also assemble into a tetrameric form in the absence of its homologue partner. Subunit assembly and interactions are discussed in terms of their potential role in hereditary retinal diseases.

Pathways for the uptake and conservation of docosahexaenoic acid in photoreceptors and synapses: biochemical and autoradiographic studies.

Docosahexaenoic acid (22:6n-3) esterified into phospholipids represents by far the most prevalent fatty acid of rod photoreceptor disc membranes and synaptic terminals. During synaptogenesis and photoreceptor biogenesis, plasma lipoproteins, secreted mainly by the liver, are the main source of plasma 22:6n-3 for the central nervous system. This systemic route (the long loop) also operates in mature animals for morphogenesis and maintenance of excitable membranes (e.g., during constant renewal of photoreceptor disc membranes). When radiolabeled 18:3n-3, the dietary precursor of 22:6n-3, is systemically supplied to 3-day-old mouse pups, it is elongated and desaturated in the liver, leading to the synthesis of 22:6n-3-lipoproteins that shuttle the fatty acid through the bloodstream to retina and brain. When radiolabeled 22:6n-3 was used, a more efficient labeling of brain and retinal lipids was achieved. The retinal pigment epithelium is involved, not only in the uptake of 22:6n-3 from circulating lipoproteins in the choriocapillaris but also in the recycling of 22:6n-3 from degraded phagosomal phospholipids back to the inner segments of photoreceptors (the short loop), following each phagocytic event. An interplay among efficient 22:6n-6 delivery from the liver, selective uptake by retinal pigment epithelium photoreceptor cells, and avid retinal retention may contribute to the enrichment of excitable membranes of the retina with 22:6n-3-phospholipids.

A null mutation in the human peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens

The murine rds (retinal degeneration slow) allele is a semidominant null allele that causes photoreceptor degeneration. The wild-type sequence at the rds locus encodes a photoreceptor disc membrane protein named peripherin/RDS. Mutations in the homologous human peripherin/RDS gene can cause autosomal dominant retinitis pigmentosa, but these are missense mutations or deletions of single codons. No obvious null alleles have been reported in humans, so that the human phenotype corresponding to rds is as yet unknown. Here we report a 2-basepair deletion in codon 25 of the human gene in a family with autosomal dominant retinitis punctata albescens, suggesting that this disease, rather than retinitis pigmentosa, is the comparable human phenotype.

Association of myosin with the connecting cilium of rod photoreceptors.

The cilium of a vertebrate photoreceptor cell connects the phototransductive outer segment of the cell to the inner segment. Previous studies have shown that, within the connecting cilium, there is a small cluster of actin filaments, which play a critical role in the formation of new disk membranes. Here, we have detected a polypeptide in rat rod outer segments that is recognized by myosin heavy chain antibodies and was found to possess other characteristics of conventional non-muscle myosin heavy chain: it comigrates in SDS-PAGE with non-muscle myosin heavy chain; it associates with the cytoskeleton of rod outer segments in an ATP-sensitive manner; and it binds to purified actin filaments in the absence of ATP. Myosin ATPase activity was also detected in isolated rod outer segments. Electron immunomicroscopy revealed that myosin is present in the small actin-containing domain within the connecting cilium at the site of disk membrane morphogenesis. These results pose the possibility that an actin-myosin contractile mechanism functions in the formation of new photoreceptor disk membranes.

Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse.

Retinal degeneration slow (rds) is a retinal disorder of an inbred strain of mice in which the outer segment of the photoreceptor cell fails to develop. A candidate gene has recently been described for the rds defect [Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. & Sutcliffe, J. G. (1989) Nature (London) 338, 70-73]. Neither the identity of the normal gene product nor its intracellular localization had been determined. We report here that the amino acid sequence of the bovine photoreceptor-cell protein peripherin, which was previously localized to the rim region of the photoreceptor disk membrane, is 92.5% identical to the sequence of the mouse protein encoded by the normal rds gene. The differences between the two sequences can be attributed to species variation. Monoclonal antibodies were used with Western blot analysis to localize the wild-type mouse peripherin/rds protein to isolated mouse rod outer segments and to show that it, like bovine peripherin, exists as two subunits linked by one or more disulfide bonds. The relative amounts of peripherin/rds protein and rhodopsin in retinal extracts of normal and rds mutant mice were also compared. Identification of peripherin as the protein encoded by the normal rds gene and its localization to membranes of rod outer segments will serve as a basis for studies directed toward defining the role of this protein in the morphogenesis and maintenance of the outer segment and toward understanding the mechanism by which the rds mutation causes retinal degeneration.

Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane

Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.

Distribution of charge on photoreceptor disc membranes and implications for charged lipid asymmetry

A novel spin labeling technique is used to determine both the inner and outer surface potentials of isolated rod outer segment disc membranes and of reconstituted membranes containing rhodopsin with defined lipid compositions. It is shown that these potentials can be accounted for in a consistent manner by the accepted model of rhodopsin, the known lipid composition, and the Gouy-Chapman theory, provided the charged lipid is asymmetric in the membrane, with approximately 75% on the external surface.