Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway

Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI-anchored protein, and the [H⁺] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin-dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1–100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent-resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent-soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild-type form of the v-SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent-resistant and detergent-soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent-resistant fractions .     

PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation

The pathological conversion of cellular prion protein (PrP(C)) into the scrapie prion protein (PrP(Sc)) isoform appears to have a central role in the pathogenesis of transmissible spongiform encephalopathies. However, the identity of the intracellular compartment where this conversion occurs is unknown. Several lines of evidence indicate that detergent-resistant membrane domains (DRMs or rafts) could be involved in this process. We have characterized the association of PrP(C) to rafts during its biosynthesis. We found that PrP(C) associates with rafts already as an immature precursor in the endoplasmic reticulum. Interestingly, compared with the mature protein, the immature diglycosylated form has a different susceptibility to cholesterol depletion vs. sphingolipid depletion, suggesting that the two forms associate with different lipid domains. We also found that cholesterol depletion, which affects raft-association of the immature protein, slows down protein maturation and leads to protein misfolding. On the contrary, sphingolipid depletion does not have any effect on the kinetics of protein maturation or on the conformation of the protein. These data indicate that the early association of PrP(C) with cholesterol-enriched rafts facilitates its correct folding and reinforce the hypothesis that cholesterol and sphingolipids have different roles in PrP metabolism.     

Hsp90 interactions and acylation target the G protein Galpha 12 but not Galpha 13 to lipid rafts

The heterotrimeric G proteins, G(12) and G(13), are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G(12) and G(13) can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G(12) and G(13) at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Galpha(12) was in the lipid raft fractions, whereas Galpha(13) was not associated with lipid rafts. Mutation of Cys-11 on Galpha(12), which prevents its palmitoylation, partially shifted Galpha(12) from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Galpha(12) and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Galpha(12) in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Galpha(12) to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Galpha(12) only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G(12) and G(13) and control of these potent cell regulators.     

Lipid rafts: signaling and sorting platforms of cells and their roles in cancer

Lipid rafts are defined as microdomains within the lipid bilayer of cellular membranes that assemble subsets of transmembrane or glycosylphosphatidylinisotol-anchored proteins and lipids (cholesterol and sphingolipids) and experimentally resist extraction in cold detergent (detergent-resistant membrane). These highly dynamic raft domains are essential in signaling processes and also form sorting platforms for targeted protein traffic. Lipid rafts are involved in protein endocytosis that occurs via caveolae or flotillin-dependent pathways. Non-constitutive protein components of rafts fluctuate dramatically in cancer with impacts on cell proliferation, signaling, protein trafficking, adhesion and apoptosis. This article focuses on the identification of candidate cancer-associated biomarkers in carcinoma cells using state-of-the-art proteomics.     

A simplified method for the preparation of detergent-free lipid rafts

Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. Traditional methods for the biochemical isolation of lipid rafts involve the extraction of cells with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, it is important to develop procedures for the isolation of lipid rafts that do not involve detergent extraction. We report here a simplified method for the purification of detergent-free lipid rafts that requires only one short density gradient centrifugation, but yields a membrane fraction that is highly enriched in cholesterol and protein markers of lipid rafts, with no contamination from nonraft plasma membrane or intracellular membranes.     

Cholesterol-sensitive modulation of transcytosis

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-beta-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

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.     

Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.     

Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl--cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane. lipid rafts; caveolae; extracellular signal-regulated kinase     

N-Terminal Protein Acylation Confers Localization to Cholesterol, Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.     

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.     

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.     

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.     

Cholesterol and prostate cancer

Cholesterol is a neutral lipid that accumulates in liquid-ordered, detergent-resistant membrane domains called lipid rafts. Lipid rafts serve as membrane platforms for signal transduction mechanisms that mediate cell growth, survival, and a variety of other processes relevant to cancer. A number of studies, going back many years, demonstrate that cholesterol accumulates in solid tumors and that cholesterol homeostasis breaks down in the prostate with aging and with the transition to the malignant state. This review summarizes the established links between cholesterol and prostate cancer (PCa), with a focus on how accumulation of cholesterol within the lipid raft component of the plasma membrane may stimulate signaling pathways that promote progression to hormone refractory disease. We propose that increases in cholesterol in prostate tumor cell membranes, resulting from increases in circulating levels or from dysregulation of endogenous synthesis, results in the coalescence of raft domains. This would have the effect of sequestering positive regulators of oncogenic signaling within rafts, while maintaining negative regulators in the liquid-disordered membrane fraction. This approach toward examining the function of lipid rafts in prostate cancer cells may provide insight into the role of circulating cholesterol in malignant growth and on the potential relationship between diet and aggressive disease. Large-scale characterization of proteins that localize to cholesterol-rich domains may help unveil signaling networks and pathways that will lead to identification of new biomarkers for disease progression and potentially to novel targets for therapeutic intervention.     

Cholesterol-dependent separation of the beta2-adrenergic receptor from its partners determines signaling efficacy: insight into nanoscale organization of signal transduction

Determining the role of lipid raft nanodomains in G protein-coupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on beta(2) adrenergic receptor (beta(2)AR) signaling. The data revealed that whereas beta(2)AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of Galpha(s) and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted beta(2)AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on beta(2)AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of beta(2)AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G(s) and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of beta(2)AR signaling machinery within larger signaling platforms.     

A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans

In Caenorhabditis elegans, the gene unc-1 controls anesthetic sensitivity and normal locomotion. The protein UNC-1 is a close homolog of the mammalian protein stomatin and is expressed primarily in the nervous system. Genetic studies in C. elegans have shown that the UNC-1 protein interacts with a sodium channel subunit, UNC-8. In humans, absence of stomatin is associated with abnormal sodium and potassium levels in red blood cells. Stomatin also has been postulated to participate in the formation of lipid rafts, which are membrane microdomains associated with protein complexes, cholesterol, and sphingolipids. In this study, we isolated a low-density, detergent-resistant fraction from cell membranes of C. elegans. This fraction contains cholesterol, sphingolipids, and protein consistent with their identification as lipid rafts. We then probed Western blots of protein from the rafts and found that the UNC-1 protein is almost totally restricted to this fraction. The UNC-8 protein is also found in rafts and coimmunoprecipitates UNC-1. A second stomatin-like protein, UNC-24, also affects anesthetic sensitivity, is found in lipid rafts, and regulates UNC-1 distribution. Mutations in the unc-24 gene alter the distribution of UNC-1 in lipid rafts. Each of these mutations alters anesthetic sensitivity in C. elegans. Because lipid rafts contain many of the putative targets of volatile anesthetics, they may represent a novel class of targets for volatile anesthetics.     

How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells

Sphingolipid and cholesterol-rich liquid ordered lipid domains (lipid rafts) have been studied in both eukaryotic cells and model membranes. However, while the coexistence of ordered and disordered liquid phases can now be easily demonstrated in model membranes, the situation in cell membranes remains ambiguous. Unlike the usual situation in model membranes, under most conditions, cell membranes rich in sphingolipid and cholesterol may have a "granular" organization in which the size of ordered and/or disordered domains is extremely small and domains may be of borderline stability. This review attempts to explain the origin of the divergence between of our understanding of rafts in model membranes and in cells, and how the physical properties of model membranes can help explain many of the ambiguities concerning raft formation and properties in cells. How physical principles of ordered domain formation relate to limitations of detergent insolubility and cholesterol depletion methods used to infer the presence of rafts in cells is also discussed. Possible modifications of these techniques that may increase their reliability are considered. It will be necessary to study model membrane systems more closely approximating cell membranes in order gain a complete understanding of raft properties in cells. Very high concentrations of membrane cholesterol and proteins may explain key physical characteristics of domains in cellular membranes, and are the two of the most obvious factors requiring additional study.     

Effects of cholesterol manipulation on the signaling of the human oxytocin receptor

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-beta-cyclodextrin (MbetaCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MbetaCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MbetaCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.     

Depletion of sphingolipids facilitates endosome to Golgi transport of ricin

It has been previously demonstrated that depletion of cholesterol inhibits endosome to Golgi transport. Whether this inhibition is due to disruption of sphingolipid- and cholesterol-containing lipid rafts that are selected for Golgi transport or whether there is a physical requirement of cholesterol for either membrane deformations, facilitating formation of transport vesicles, or for recruitment of cytosolic constituents is not obvious. To investigate this in more detail, we have studied endosome to Golgi transport of ricin in sphingolipid-deficient cells using either a mutant cell line that does not express serine palmitoyltransferase, the first enzyme in sphingolipid biosynthesis, or a specific inhibitor, myriocin, of the same enzyme. Depletion of sphingolipids gave an increased sensitivity to ricin, and this increased sensitivity was inhibited by addition of sphingolipids. Importantly, endosome to Golgi transport of ricin, measured as sulfation of a modified ricin molecule, was increased in sphingolipid-deficient cells. No effect was seen on other pathways taken by ricin. Interestingly, cholesterol depletion inhibited endosome to Golgi transport even in cells with reduced levels of sphingolipids, suggesting that cholesterol as such is required for formation of transport vesicles. Our results indicate that the presence of sphingolipids actually limits and may function to control endosome to Golgi transport of ricin.