Rod Outer Segment Disk Research Articles

Regulation of Membrane Proteins by Dietary Lipids: Effects of Cholesterol and Docosahexaenoic Acid Acyl Chain-Containing Phospholipids on Rhodopsin Stability and Function

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature ( T m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process ( E a) was determined from DSC thermograms by four separate methods. Both T m and E a varied with bilayer composition. Cholesterol increased the T m both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E a in the absence of DHA, but raised E a in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T m for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability ( E a) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

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.

Mesoscopic Monte Carlo Simulations of Stochastic Encounters between Photoactivated Rhodopsin and Transducin in Disc Membranes

The issue of how the molecular organization of rod outer segments (ROS) discs affects the initial timing of the photoresponse in vertebrates has been recently raised by novel structural findings that raise doubts about the classical scenario of monomeric rhodopsin (R) and heterotrimeric transducin (G) freely diffusing in the membrane milieu. In this study, we investigate this issue by means of mesoscopic Monte Carlo (MMC) simulations of the stochastic encounters between one photoactivated R and one G, explicitly taking into account the molecular size and the diffusion coefficient of each species as well as crowding effects. Three different scenarios were compared with respect to their effects on timing, namely, (a) the classical framework, where both G and monomeric R are allowed to freely diffuse in the ROS disc membrane, (b) the ideal paracrystalline organization of R dimers considered as a structural unit, where ordered rows completely cover the disc membrane patch, and (c) the scenario suggested by recent AFM data, where R dimers organize in differently sized rafts with varying local concentrations. Our simulations suggest that a similar kinetic response could arise from very different microscopic scenarios, thus opening new interpretations to the controversial recent findings. Moreover, we show that if high-density R packing on ROS discs is characterized by a highly ordered structural organization rather than unspecific aggregation, an unexpected favorable effect on the temporal response of early phototransduction reactions can occur.

Differential distribution of proteins and lipids in detergent‐resistant and detergent‐soluble domains in rod outer segment plasma membranes and disks

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

Rhodopsin palmitoylation influences the redistribution of phospholipids in bovine rod outer segment disc membranes

Rhodopsin palmitoylation influences the redistribution of phospholipids in bovine rod outer segment disc membranes

Calcium-Dependent Association of Calmodulin with the C-Terminal Domain of the Tetraspanin Protein Peripherin/rds

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.

Peripherin-2: An Intracellular Analogy to Viral Fusion Proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.

Confocal laser scanning microscopy of retinal rod outer segment intact disks: new labeling technique

Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis.

Acceleration of Age-Related Changes in the Retina in α-Tocopherol Transfer Protein Null Mice Fed a Vitamin E–Deficient Diet

To assess the role of vitamin E (VE) in age-related changes in the retinal tissues by using a mouse model of severe VE deficiency. Pups of alpha-tocopherol transfer protein null (a-TTP(-)(/)(-)) mice were fed a VE-deficient diet for 4 or 18 months (VE (-) group). Wild-type C57BL/6 mice were fed a 0.002% alpha-tocopherol-supplemented diet (VE (+) group). In various ocular tissues, the VE levels were measured by high-performance liquid chromatography; the fatty acid composition by gas chromatography (GC); and the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels, which are biomarkers for lipid peroxidation, by GC-mass spectrometry. The retinal structure was assessed by light, electron, and fluorescence microscopy. The alpha-tocopherol level in the retinas obtained from 4-month-old VE (-) animals was 71-fold lower than that in the retinas obtained from the VE (+) group. In addition, gamma-tocopherol was not detected; thus, the VE (-) group demonstrated a more severe VE deficiency than ever reported. In this group, the concentration of n-3 polyunsaturated fatty acids decreased (0.3- to 0.9-fold), whereas that of other classes of fatty acids was unchanged or increased. At 18 months of age, the number of the outer nuclear layer (ONL) nuclei was observed to be 17% lower in the VE (-) than in the VE (+) group (P < 0.05). Electron microscopy revealed larger amounts of matrix between the ONL nuclei indicating the Müller cell hypertrophy, greatly expanded rod outer segment discs, and a larger number of inclusion bodies in the retinal pigment epithelium (RPE; P < 0.05) in the VE (-) group. Fluorescence microscopy revealed that the autofluorescence signal was increased in the RPE layer in this group. When the observations of the 18-month-old animals were compared to those of the 4-month-old animals, the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels were found to be increased in the retina and RPE obtained from both the VE (-) and VE (+) groups; however, the age-related increases were more remarkable in the VE (-) group (2.6- to 43.5-fold) than in the VE (+) group (0.8- to 8.7-fold). The combined use of a-TTP(-)(/)(-) mice and a VE-deficient diet leads to a severe deficiency of VE, enhances lipid peroxidation in the retina, and accelerates degenerative damage of the retina with age.

Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.

Modeling the Role of Incisures in Vertebrate Phototransduction

Phototransduction is mediated by a G-protein-coupled receptor-mediated cascade, activated by light and localized to rod outer segment (ROS) disk membranes, which, in turn, drives a diffusion process of the second messengers cGMP and Ca 2+ in the ROS cytosol. This process is hindered by disks—which, however, bear physical cracks, known as incisures, believed to favor the longitudinal diffusion of cGMP and Ca 2+. This article is aimed at highlighting the biophysical functional role and significance of incisures, and their effect on the local and global response of the photocurrent. Previous work on this topic regarded the ROS as well stirred in the radial variables, lumped the diffusion mechanism on the longitudinal axis of the ROS, and replaced the cytosolic diffusion coefficients by effective ones, accounting for incisures through their total patent area only. The fully spatially resolved model recently published by our group is a natural tool to take into account other significant details of incisures, including their geometry and distribution. Using mathematical theories of homogenization and concentrated capacity, it is shown here that the complex diffusion process undergone by the second messengers cGMP and Ca 2+ in the ROS bearing incisures can be modeled by a family of two-dimensional diffusion processes on the ROS cross sections, glued together by other two-dimensional diffusion processes, accounting for diffusion in the ROS outer shell and in the bladelike regions comprised by the stack of incisures. Based on this mathematical model, a code has been written, capable of incorporating an arbitrary number of incisures and activation sites, with any given arbitrary distribution within the ROS. The code is aimed at being an operational tool to perform numerical experiments of phototransduction, in rods with incisures of different geometry and structure, under a wide spectrum of operating conditions. The simulation results show that incisures have a dual biophysical function. On the one hand, since incisures line up from disk to disk, they create vertical cytoplasmic channels crossing the disks, thus facilitating diffusion of second messengers; on the other hand, at least in those species bearing multiple incisures, they divide the disks into lobes like the petals of a flower, thus confining the diffusion of activated phosphodiesterase and localizing the photon response. Accordingly, not only the total area of incisures, but their geometrical shape and distribution as well, significantly influence the global photoresponse.

High reolution structural studies on peripherin, an intergal membrane protein

Peripherin is a tetraspannin transmembrane protein localized in retinal rod outer segment disk membranes whose complete 3-dimensional structure is the subject of this nuclear magnetic resonance (NMR) analysis. Peripherin promotes membrane fusion and may be involved in fusion events with rod cell membranes during the rod outer segment disk renewal processes. The 63-residue C-terminal extramembraneous region of peripherin (PERCTER) is crucial to peripherin-mediated fusion and contains both an incisure affinity region and an OS targeting signal. Several mutations in this C-terminal region have been linked to various forms of retinal degenerative disease. The first target of our structural analysis has been the PERCTER, expressed and labeled with 15N, and subjected to structural determination by solution NMR. For this analysis, 3-dimensional HSQC-NOESY and HSQC-TOCSY experiments were utilized, along with 2-dimensional 1H NOESY and TOCSY experiments at various mixing times. This analysis is followed by a similar study of the PERCTER in the presence of a membrane-mimetic, DPC micelles. The next stage of our examination is to obtain NMR data from the isotope-labeled full length peripherin. Detailed understanding of the peripherin structure provides greater insight into retinal degenerative diseases and the mechanisms of membrane fusion. NIH EY10420-14, E. Matilda Ziegler Vision Award.

Effect of Packing Density on Rhodopsin Stability and Function in Polyunsaturated Membranes

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.

Mathematical and computational modelling of spatio-temporal signalling in rod phototransduction

Rod photoreceptors are activated by light through activation of a cascade that includes the G protein-coupled receptor rhodopsin, the G protein transducin, its effector cyclic guanosine monophosphate (cGMP) phosphodiesterase and the second messengers cGMP and Ca2+. Signalling is localised to the particular rod outer segment disc, which is activated by absorption of a single photon. Modelling of this cascade has previously been performed mostly by assumption of a well-stirred cytoplasm. We recently published the first fully spatially resolved model that captures the local nature of light activation. The model reduces the complex geometry of the cell to a simpler one using the mathematical theories of homogenisation and concentrated capacity. The model shows that, upon activation of a single rhodopsin, changes of the second messengers cGMP and Ca2+ are local about the particular activated disc. In the current work, the homogenised model is computationally compared with the full, non-homogenised one, set in the original geometry of the rod outer segment. It is found to have an accuracy of 0.03% compared with the full model in computing the integral response and a 5200-fold reduction in computation time. The model can reconstruct the radial time-profiles of cGMP and Ca2+ in the interdiscal spaces adjacent to the activated discs. Cellular electrical responses are localised near the activation sites, and multiple photons sufficiently far apart produce essentially independent responses. This leads to a computational analysis of the notion and estimate of 'spread' and the optimum distribution of activated sites that maximises the response. Biological insights arising from the spatio-temporal model include a quantification of how variability in the response to dim light is affected by the distance between the outer segment discs capturing photons. The model is thus a simulation tool for biologists to predict the effect of various factors influencing the timing, spread and control mechanisms of this G protein-coupled, receptor-mediated cascade. It permits ease of simulation experiments across a range of conditions, for example, clamping the concentration of calcium, with results matching analogous experimental results. In addition, the model accommodates differing geometries of rod outer segments from different vertebrate species. Thus it represents a building block towards a predictive model of visual transduction.

N-Retinylidene-phosphatidylethanolamine Is the Preferred Retinoid Substrate for the Photoreceptor-specific ABC Transporter ABCA4 (ABCR)

ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

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.

DHA-Rich phospholipids optimize G-Protein–coupled signaling

Objective To assess the effects of n–3 polyunsaturated phospholipid acyl chains on the initial steps in G-protein–coupled signaling. Study design Isolated components of the visual signal transduction system, rhodopsin, G protein (G t), and phosphodiesterase (PDE), were reconstituted in membranes containing various levels of n–3 polyunsaturated phospholipid acyl chains. In addition, rod outer segment disk membranes containing these components were purified from rats raised on n–3-deficient and n–3-adequate diets. The conformation change of rhodopsin, coupling of rhodopsin to G t, and PDE activity were each measured separately. Results The ability of rhodopsin to form the active metarhodopsin II conformation and bind G t were both compromised in membranes with reduced levels of n–3 polyunsaturated acyl chains. The activity of PDE, directly related to the integrated cellular response, was reduced in all membranes lacking or deficient in n–3 polyunsaturated acyl chains. PDE activity in membranes containing 22:5n–6 PC was 50% lower than in membranes containing either 22:6n–3 PC or 22:5n–3 PC. Conclusions The earliest events in G-protein–coupled signaling; receptor conformation change, receptor–G-protein binding, and PDE activity are reduced in membranes lacking n–3 polyunsaturated acyl chains. Efficient and rapid propagation of G-protein–coupled signaling requires polyunsaturated n–3 phospholipid acyl chains.

Atomic-force microscopy: Rhodopsin dimers in native disc membranes.

Neat rows of paired photon receptors are caught on camera in their natural state. In vertebrate retinal photoreceptors, the rod outer-segment disc membranes contain densely packed rhodopsin molecules for optimal light absorption and subsequent amplification by the visual signalling cascade1, but how these photon receptors are organized with respect to each other is not known. Here we use infrared-laser atomic-force microscopy to reveal the native arrangement of rhodopsin, which forms paracrystalline arrays of dimers in mouse disc membranes. The visualization of these closely packed rhodopsin dimers in native membranes gives experimental support to earlier inferences about their supramolecular structure2,3 and provides insight into how light signalling is controlled.

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.

Structure of rhodopsin in monolayers at the air-water interface: a PM-IRRAS and X-ray reflectivity study.

Monomolecular films of the membrane protein rhodopsin have been investigated in situ at the air-water interface by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and X-ray reflectivity in order to find conditions that retain the protein secondary structure. The spreading of rhodopsin at 0 or 5 mN m(-1) followed by a 30 min incubation time at 21 degrees C resulted in the unfolding of rhodopsin, as evidenced from the large increase of its molecular area, its small monolayer thickness, and the extensive formation of beta-sheets at the expense of the alpha-helices originally present in rhodopsin. In contrast, when spreading is performed at 5 or 10 mN m(-1) followed by an immediate compression at, respectively, 4 or 21 degrees C, the secondary structure of rhodopsin is retained, and the thickness of these films is in good agreement with the size of rhodopsin determined from its crystal structure. The amide I/amide II ratio also allowed to determine that the orientation of rhodopsin only slightly changes with surface pressure and it remains almost unchanged when the film is maintained at 20 mN m(-1) for 120 min at 4 degrees C. In addition, the PM-IRRAS spectra of rod outer segment disk membranes in monolayers suggest that rhodopsin also retained its secondary structure in these films.