Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte

In blood-stage infection by the human malaria parasite Plasmodium falciparum, export of proteins from the intracellular parasite to the erythrocyte is key to virulence. This export is mediated by a host-targeting (HT) signal present on a "secretome" of hundreds of parasite proteins engaged in remodeling the erythrocyte. However, the route of HT-mediated export is poorly understood. Here we show that minimal soluble and membrane protein reporters that contain the HT motif and mimic export of endogenous P falciparum proteins are detected in the lumen of "cleft" structures synthesized by the pathogen. Clefts are efficiently targeted by the HT signal. Furthermore, the HT signal does not directly translocate across the parasitophorous vacuolar membrane (PVM) surrounding the parasite to deliver protein to the erythrocyte cytoplasm, as suggested by current models of parasite protein trafficking to the erythrocyte. Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM. These data suggest that Maurer's clefts, which are unique to the virulent P falciparum species, are pathogen-induced secretory organelles that concentrate HT-containing soluble and membrane parasite proteins in their lumen for delivery to the host erythrocyte.     

Ca(++)-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin

Cytosolic Ca(++) induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glycosyl phosphatidylinositol-linked proteins, free of cytoskeletal components, and depleted of the major transmembrane proteins. Both types of vesicles contain 2 as-yet-unrecognized red cell proteins, synexin and sorcin, which translocate from the cytosol to the membrane upon Ca(++) binding. In nanovesicles, synexin and sorcin are the most abundant proteins after hemoglobin. In contrast, the microvesicles are highly enriched in stomatin. The membranes of both microvesicles and nanovesicles contain lipid rafts. Stomatin is the major protein of the microvesicular lipid rafts, whereas synexin and sorcin represent the major proteins of the nanovesicular rafts in the presence of Ca(++). Interestingly, the raft proteins flotillin-1 and flotillin-2 are not found in the vesicles but remain in the red cell membrane. These data indicate the presence of different types of lipid rafts in the erythrocyte membrane with distinct fates after Ca(++) entry. Synexin, which is known to be vital to the process of membrane fusion, is suggested to be a key component in the process of vesicle release from erythrocytes.     

Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors

Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.     

Localization of a 230-kD parasitophorous vacuole membrane antigen of Plasmodium berghei exoerythrocytic schizonts (LSA-2) by immunoelectron and confocal laser scanning microscopy.

Using antiserum to a 230-kD parasitophorous vacuole membrane (PVM) antigen of Plasmodium berghei exoerythrocytic schizonts as a specific probe for the PVM, we studied the three-dimensional structure of this membrane within infected host cells by immunoelectron microscopy and confocal laser scanning microscopy at 3, 4, and 50 hr after sporozoite invasion. Fluorescent label was not detected at 3 hr, but was associated with the cytoplasm of 24-hr-old exoerythrocytic parasites. Specific labeling of the PVM was not observed by immunoelectron microscopy until 50 hr, when numerous vesicles and finger-like projections of the PVM were found in the cytoplasm of infected host cells. Labeled vesicles were often isolated and located at the periphery of the infected hepatocyte. Confocal microscopy demonstrated that these vesicles formed discontinuous chains that extended from 3-10 microns away from the parasite. These structures appear to be similar to the membranous clefts of Plasmodium-infected erythrocytes, and may be important in the movement of host or parasite proteins within infected hepatocytes.     

The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca(2+)-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium- induced phospholipid scrambling

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca(2+)- induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca(2+)-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca(2+)-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca(2+)-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD- phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca(2+)-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.     

From the Cover: Membrane raft association is a determinant of plasma membrane localization

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.Recent advances in superresolution microscopy (1), lipid analysis (2, 3), and plasma membrane (PM) isolation (4, 5) have confirmed the coexistence of lipid-driven, fluid domains in biological membranes. The relatively ordered domains, known as “membrane rafts,” have been proposed to be involved in protein sorting (6), viral/pathogen trafficking (3, 7), and PM signaling in a variety of contexts (8). However, despite the increasing evidence confirming the existence of dynamic, nanoscopic membrane rafts, the functional consequences of this phenomenon remain speculative because of the limitations of the previously used methods for defining raft association, i.e., the resistance of membrane components to solubilization by nonionic detergents (9).Lipid-mediated domains have been implicated as a mechanism for protein sorting in the latter stages of the secretory pathway (trans-Golgi network to the PM) (2, 6, 10–12), with analogous pathways mediating endosomal sorting/recycling (13, 14). Raft lipids (i.e., sterols and sphingolipids) are significantly enriched at the PM (15–17), and recent observations confirm that these lipids also are enriched in sorting vesicles destined for the PM (2, 11). For proteins, several specific cytosolic signals exist for adapter/coat-mediated sorting between cellular organelles (18); in parallel, protein–lipid interactions through hydrophobic transmembrane domains (TMDs) also have been shown to regulate trafficking. For example, a strong correlation exists between the TMD length of bitopic proteins and their organelle specificity (19, 20), with longer TMDs targeting proteins to the PM and shorter TMDs found in the endoplasmic reticulum (ER), Golgi apparatus, and endocytic organelles. These findings suggest cargo sorting in the secretory and endocytic pathways, with proteins containing longer TMDs, together with sphingolipids and cholesterol, being specifically trafficked to the PM, although the mechanism for this observation remains unresolved.One possibility for sorting of specific lipid classes along with proteins containing longer TMDs is lateral segregation and coalescence of ordered domains, followed by either domain-induced (21) or cytoskeleton-assisted (22) budding of raft-enriched transport vesicles. Proteins using this “raft pathway” would not require cytosolic sorting signals but rather would be recruited to transport vesicles by their raft affinity, i.e., their propensity to interact with specific lipids, ordered domains, or other raft-embedded proteins. Because ordered phases in lipid model systems consistently have been shown to be 0.6–1.5 nm thicker than disordered domains (23, 24), raft-associated transmembrane (TM) proteins would be predicted to have longer TMDs. TMD length-dependent protein sorting between coexisting lipid domains has been addressed experimentally only recently by measuring partitioning of an oligomeric toxin (perfringolysin O) with multiple (35–40) TM segments in synthetic, phase-separated liposomes (25). Whether these observations extend to single-pass TM proteins in biological membranes is unknown.To evaluate the role of lipid-driven raft domains as a mechanism for subcellular protein sorting, we quantitatively compared the raft association of 30 TM protein variants with their subcellular localization. To quantify raft partitioning of the constructs comprising single-pass TM proteins with varying TMD lengths and sequences, we used giant PM vesicles (GPMVs). GPMVs are cell-detached PM blebs whose protein (26) and lipid (27) diversity mirrors that of the native PM. These PM vesicles separate into coexisting liquid phases (4) with different order (28), which recruit membrane components in accordance with their predicted raft affinity, i.e., saturated lipids, glycosphingolipids (29), glycosylphosphatidyl inositol-anchored proteins (4), and palmitoylated proteins (30) partition to the ordered phase, denoted here as the “raft phase.” Most importantly, these vesicles provide a platform for repeatable, direct, and quantitative analysis of raft partitioning (30), allowing investigation of the structural determinants of raft association and its effect on protein function. We find that perturbation of raft partitioning by three independent means (decreasing TMD length, mutation of palmitoylation sites, and TMD sequence manipulation) perturbed subcellular localization, leading to missorting of PM proteins to late endosomes and lysosomes because of a failure to recycle nonraft proteins from early endosomes (EEs). These results confirm the presence of a raft-mediated recycling route in nonpolarized cells, begin to define the molecular parameters for protein association with raft domains, and suggest an explanation for the accumulation of proteins with longer TMDs at the PM.     

Functional evidence for presence of lipid rafts in erythrocyte membranes: Gsalpha in rafts is essential for signal transduction

Membrane microdomains enriched in cholesterol and sphingolipids and containing specific membrane proteins are designated as lipid rafts. Lipid rafts have been implicated in cell signaling pathways in various cell types. Heterotrimeric guanine nucleotide-binding protein (Gsalpha) has been shown to be a raft component of erythrocytes and has been implicated in cell signaling. Rafts are isolated as detergent-resistant microdomains (DRMs) for biochemical analysis. Cholesterol depletion is widely used to disrupt raft structures to study their function in biological membranes. In the present study, we developed an alternate strategy for disrupting raft structures without altering membrane cholesterol content. Lidocaine hydrochloride, an amphipathic local anesthetic, is shown to reversibly disrupt rafts in erythrocyte membranes and alter the Gsalpha dependent signal transduction pathway. These findings provide evidence for the presence of rafts while maintaining normal cholesterol content in erythrocyte membranes and confirm a role for raft-associated Gsalpha in signal transduction in erythrocytes.     

Order of lipid phases in model and plasma membranes

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165–3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.     

Local anesthetic-membrane interaction: a multiequilibrium model.

We report the detection of electrostatic interactions between local anesthetics and membrane phospholipids and proteins. A spin-labeled local anesthetic was used to study how membrane-bound tertiary amine anesthetics interact with major molecular components in the membrane. The nitroxyl reporter group of this spin label is located at the polar end of the amphiphilic local anesthetic; it is therefore a uniquely suitable probe for detecting immobilization of the anesthetic due to binding interactions at the polar regions of the bilayer. The binding properties of this spin-labeled anesthetic to human erythrocyte membranes and to vesicles made from human erythrocyte lipids were studied. Lipid vesicle-bound spin labels give rise to a composite electron spin resonance spectrum from which two subcomponent spectra were resolved. Both components are membrane-bound; the first component has a narrower linewidth, indicating a greater mobility of the nitroxyl moiety of the anesthetic probe. The second component has a broader linewidth, indicating a population of constrained spin labels. We infer from the experimental results that electrostatic binding between cationic anesthetics and anionic phosphate of phospholipids produced the constrained component. In similar studies using erythrocyte ghost membranes, both a mobile (nonelectrostatic) component and a constrained (electrostatic) component were resolved from the composite spectrum. However, the constrained component in this case is much broader than the corresponding constrained component from the vesicles. We interpret this broad component in the erythrocyte membrane as an electrostatic interaction of cationic anesthetic probes with phospholipids and with membrane proteins. We conclude that membrane-bound tertiary amine anesthetics in cationic form do interact selectively with phospholipids and proteins.     

Plasma membrane biogenesis in eukaryotic cells: translocation of newly synthesized lipid.

We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag, in a linear manner up to 1 hr. Labeled sterol and phospholipids appeared in the plasma membrane, after a 30-min lag, at approximately the same rate. However, the ratio of newly synthesized sterol to phospholipid was significantly enriched in the plasma membrane relative to the whole cell, even at the earlier time points. Pulse-chase experiments indicated that sterols and phospholipids are turned over in the plasma membrane with similar, rather short half-lives. The results of these studies suggest that, although sterols and phospholipids are transported to the cell surface with similar kinetics, some sorting of the lipids must occur at an early stage in membrane biogenesis. The data are consistent with a model of lipid translocation by vesicular transport.     

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.     

Nonvesicular phospholipid transfer between peroxisomes and the endoplasmic reticulum

The endoplasmic reticulum (ER) plays an important role in peroxisome biogenesis; some peroxisomal membrane proteins are inserted into the ER and trafficked to peroxisomes in vesicles. These vesicles could also provide the phospholipids required for the growth of peroxisomal membranes, because peroxisomes lack phospholipid biosynthesis enzymes. To test this, we established a novel assay to monitor phospholipid transfer between the ER and peroxisomes and found that phospholipids are rapidly trafficked between these compartments. This transport is not blocked in mutants with conditional defects in Sec proteins required for vesicular trafficking from the ER or in Pex3p, a protein required for peroxisome membrane biogenesis. ER to peroxisome lipid transport was reconstituted in vitro and does not require cytosolic factors or ATP. Our findings indicate that lipids are directly transferred from the ER to peroxisomes by a nonvesicular pathway and suggest that ER to peroxisome vesicular transport is not required to provide lipids for peroxisomal growth.     

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.     

Asymmetric phospholipid distribution drives in vitro reconstituted SNARE-dependent membrane fusion

Insulin-stimulated glucose uptake requires the fusion of GLUT4 transporter-containing vesicles with the plasma membrane, a process that depends on the SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment receptor) proteins VAMP2 (vesicle-associated membrane protein 2) and syntaxin 4 (Stx4)/SNAP23 (soluble N-ethylmaleimide-sensitive fusion factor attachment protein 23). Efficient SNARE-dependent fusion has been shown in many settings in vivo to require the generation of both phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidic acid (PA). Addition of PA to Stx4/SNAP23 vesicles markedly enhanced the fusion rate, whereas its addition to VAMP2 vesicles was inhibitory. In contrast, addition of PIP2 to Stx4/SNAP23 vesicles inhibited the fusion reaction, and its addition to VAMP2 vesicles was stimulatory. The optimal distribution of phospholipids was found to trigger the progression from the hemifused state to full fusion. These findings reveal an unanticipated dependence of SNARE complex-mediated fusion on asymmetrically distributed acidic phospholipids and provide mechanistic insights into the roles of phospholipase D and PIP kinases in the late stages of regulated exocytosis.     

Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes

After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P. falciparum-infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P. falciparum-infected RBC membrane.     

Pseudo-angiomatous growth pattern in recurrent plasma cell myeloma

Erythrocytes are remarkably dynamic structures, possessing multiple and complex pathways for regulating cell membrane properties to compensate for the absence of a nucleus and internal membranes. Unlike the invasion strategies of many viruses and bacteria into their eukaryotic hosts, however, the accepted model for malaria parasite entry into human erythrocytes casts the host cell in a largely passive role. This is in contrast to mounting evidence for a suite of dynamic alterations that the erythrocyte membrane undergoes during the rapid process of invasion by the blood stage malaria parasite – the merozoite. Here we review the cellular and molecular basis for merozoite invasion of the erythrocyte and explore the idea that radical changes in the erythrocyte membrane protein and lipid architecture probably accompany this key step in the establishment of human malaria disease.     

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37 degrees C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 microm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.     

Generation of an erythrocyte vesicle transport system by Plasmodium falciparum malaria parasites

The asexual maturation of Plasmodium falciparum is accompanied by the transport of parasite-encoded proteins to the erythrocyte plasma membrane. Activation of G proteins by treatment with aluminum fluoride produced an accumulation within the erythrocyte cytosol of vesicles coated with Plasmodium homologues of COPII and N-ethylmaleimide-sensitive factor, proteins involved in intracellular transport between the Golgi apparatus and the endoplasmic reticulum. These vesicles contain malarial proteins that appear on the erythrocyte plasma membrane, as well as actin and myosin. It is proposed that the parasite adapted a process well established for intracellular transport to mediate the extracellular movement of its proteins through the erythrocyte cytosol to the surface membrane.     

Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes

Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.     

SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts ( approximately 25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.