Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.     

Structure and cholesterol dynamics of caveolae/raft and nonraft plasma membrane domains

Despite recognition that the plasma membrane (PM) is comprised of lipid raft domains that are key organizing sites of multiple signaling pathways and other cell functions, limited information is available regarding the structure and function in sterol dynamics of these microdomains. To begin to resolve these issues, MDCK membranes were subfractionated by three different techniques to produce (i) detergent-resistant membranes (DRM) and detergent-soluble membranes (DSM), (ii) nondetergent caveolae/rafts (NDCR), and (iii) nondetergent, affinity-purified caveolae/rafts (ACR) and noncaveolae/nonrafts (NR). ACR exhibited the least cross contamination with other PM domains or intracellular membranes, in marked contrast to DRM that contained the highest level of cross contaminants. Spectral properties of dehydroergosterol (DHE), a naturally occurring fluorescent sterol, showed that ACR, NDCR, and NR did not contain crystalline sterol, consistent with the lack of crystalline sterol in PM of intact cells. In contrast, DRM contained significant levels of crystalline sterol. Fluorescence polarization of membrane probes showed that ACR were the least fluid and had the highest transbilayer fluidity gradient, the most liquid ordered phase, and the sterol dynamics most responsive to sterol carrier protein-2 (SCP-2). In contrast, DRM had structural properties similar to those of NR, anomalous (very fast) spontaneous sterol dynamics, and sterol dynamics that were unresponsive to SCP-2. Differences between the structural and functional properties of DRM and those of the nondetergent preparations (ACR and NDCR) were not due to the presence of detergent. A nondetergent, affinity-purified (ACR) lipid domain fraction isolated from MDCK cells for the first time revealed unique structural (noncrystalline sterol, liquid-ordered, high transbilayer fluidity gradient) and functional (cholesterol dynamics) properties of lipid rafts as compared to nonrafts (NR). In summary, this study showed membrane microdomains (rafts/caveolae) isolated by three different methodologies have unique structural, functional, and organizational characteristics.     

Active transducin alpha subunit carries PDE6 to detergent-resistant membranes in rod photoreceptor outer segments

cGMP-Phosphodiesterase 6 (PDE6) is the central effector enzyme in the phototransduction system of vertebrate photoreceptors. We have recently found that PDE6 accumulates in a detergent-resistant membrane (DRM) fraction in response to excitation of bovine rod phototransduction system. Here, we studied the molecular mechanism of the PDE6 translocation to DRM. Pertussis toxin inhibited the translocation of PDE6. Upon addition of AlF(4)(-) to dark-adapted ROS, PDE6 translocated to DRM along with a minor fraction of the alpha subunit of transducin (T alpha). The addition of an excess of the inhibitory subunit of PDE6 blocked its accumulation in the DRM, but did not block the translocation of the minor fraction of T alpha. These data suggested that the formation of a complex between activated T alpha and PDE6 imparted upon T alpha a high affinity for the DRM. The translocation of PDE6 to the DRM may be involved in the spatiotemporal regulation of its activity on disk membranes.     

Hypoxia-induced modifications in plasma membranes and lipid microdomains in A549 cells and primary human alveolar cells

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O(2) for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment.     

Relationship between localization on cellular membranes and cytotoxicity of Vibrio vulnificus hemolysin

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MβCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MβCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MβCD treatment. In addition, the amount of VVH oligomer did not decrease in MβCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.     

Enhancement of phototransduction protein interactions by lipid surfaces

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

Cholesterol-dependent association of caveolin-1 with the transducin alpha subunit in bovine photoreceptor rod outer segments: disruption by cyclodextrin and guanosine 5'-O-(3-thiotriphosphate)

Evidence suggests that caveolins, 21-24 kDa cholesterol-binding proteins that generally reside in specialized detergent-resistant membrane microdomains, act as signaling scaffolds. Detergent-resistant membranes isolated from rod outer segments (ROS) have been previously shown to contain the photoreceptor G-protein, transducin. In this report we show, by subcellular fractionation, that caveolin-1 is an authentic component of purified ROS. We demonstrate that caveolin-1 in ROS almost exclusively resides in low-buoyant-density, cholesterol-rich, detergent-resistant membranes that can be disrupted by cholesterol depletion using methyl-beta-cyclodextrin (MCD). Cholesterol depletion was also observed to extract a pool of transducin alpha (Talpha) from ROS membranes. Immunoprecipitation with anti-caveolin-1 revealed the association of Talpha in the absence of Tbetagamma. Treatment of ROS with MCD resulted in a 2-fold decrease in recovery of Talpha in anti-caveolin-1 immunoprecipitates. This interaction was also completely disrupted when ROS were exposed to light in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), a nonhydrolyzable GTP analogue. In addition, caveolin-1/Talpha association in the immune complex was disrupted by a peptide based on the primary sequence of the caveolin-1 scaffolding domain. Finally, we confirm the colocalization of caveolin-1 and Talpha in photoreceptors by immunofluorescence microscopy. These results strongly suggest that the association between Talpha and caveolin-1 occurs in cholesterol-rich, detergent-resistant membranes and is likely to be dependent upon the activation state of Talpha.     

Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.     

The lipid raft proteome of Borrelia burgdorferi

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft‐associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC‐MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 ( http://proteomecentral.proteomexchange.org/dataset/PXD002365 ).     

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL₃ additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL₃ reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL₃-mediated lipid efflux.     

Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D(2)R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors and delta opioid receptors are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of mu opioid receptor. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D(2)R and that DRM is involved in the formation of a D(2)R signaling complex. D(2)R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.     

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR?) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR? is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR? was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

Lipid composition of microdomains is altered in a cell model of Gaucher disease

The formation of cholesterol and sphingolipids into specialized liquid-ordered membrane microdomains (rafts) has been proposed to function in the intracellular sorting and transport of proteins and lipids. Defined by biochemical criteria, rafts resist solubilization in nonionic detergents, enabling them to be isolated as detergent-resistant membranes (DRM). In this study, we characterized the lipid composition of DRM from a cell model of the sphingolipid storage disorder, Gaucher disease, in which the catabolism of the sphingolipid glucosylceramide (GC) is impaired. In this cell model, we showed that GC accumulated primarily in the DRM, with smaller secondary increases in ceramide, dihexosylceramide, trihexosylceramide, and phosphatidylglycerol. This suggested that not only was lipid metabolism altered as a consequence of the cells' inability to degrade GC, but this affected the DRM rather than other regions of the membrane. This increase in lipids in the DRM may be responsible for the altered lipid and protein sorting seen in Gaucher disease. Analysis of individual lipid species revealed preservation of the shorter and fully saturated fatty acid species in the DRM, suggesting that the highly ordered and tightly packed nature of the DRM is maintained.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

Accumulation of macular xanthophylls in unsaturated membrane domains

The distribution of macular xanthophylls, lutein and zeaxanthin, between domains formed in membranes made from an equimolar ternary mixture of dioleoylphosphatidylcholine/sphingomyelin/cholesterol, called a raft-forming mixture, was investigated. In these membranes, two domains are formed: the raft domain enriched in saturated lipids and cholesterol (detergent-resistant membranes, DRM), and the bulk domain enriched in unsaturated lipids (detergent-soluble membranes, DSM). These membrane domains have been separated using cold Triton X-100 extraction from membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that xanthophylls are substantially excluded from DRM and remain concentrated in DSM. Concentrations of xanthophylls in DRM and DSM calculated as the mole ratio of either xanthophyll to phospholipid were 0.005 and 0.03, respectively, and calculated as the mole ratio of either xanthophyll to total lipid (phospholipid + cholesterol) were 0.003 and 0.025, respectively. Thus, xanthophylls are over eight times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. It was also demonstrated using saturation-recovery EPR that at 1 mol%, neither lutein nor zeaxanthin affect the formation of membrane domains. The location of xanthophylls in domains formed from unsaturated lipids is ideal if they are to act as a lipid antioxidant, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular diseases.     

Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis

Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.     

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.