Contamination of nuclear fractions with plasma membrane lipid rafts

Subcellular fractionation is central to a range of cell biological, biochemical and proteomic studies. Purification of nuclear-enriched fractions is critical for studies on nuclear structure and function. Here we show that detergent-based nuclear isolation methods cause the redistribution of proteins associated with plasma membrane lipid rafts into nuclear fractions. The glycosyl-phosphatidylinositol (GPI)-anchored prion protein (PrP(C)) and a GPI-anchored construct of angiotensin converting enzyme (GPI-ACE), as well as the lipid raft markers flotillin-1 and -2, were present in the nuclear fractions derived using three different subcellular fractionation protocols. Incubation of intact cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves GPI-anchored proteins from the cell surface, significantly reduced the amount of PrP(C) and GPI-ACE in the nuclear fraction. Buoyant sucrose density gradient centrifugation in the presence of Triton X-100 of the nuclear fraction resulted in a significant proportion of the GPI-anchored proteins being recovered in the low density lipid raft fractions. These data indicate that the nuclear fraction isolated using such subcellular fractionation protocols is contaminated with components of plasma membrane lipid rafts and raises questions as to the integrity of the nuclear fraction isolated by such protocols for use in detailed cell biological studies and proteomics analysis.     

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.     

Interaction of phosphatidylinositolglycan(-peptides) with plasma membrane lipid rafts triggers insulin-mimetic signaling in rat adipocytes

The phosphoinositolglycan(-peptide) (PIG-P) portion of glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins or synthetic PIG(-P) molecules interact with proteinaceous binding sites which are located in high-cholesterol-containing detergent/carbonate-insoluble glycolipid-enriched raft domains (hcDIGs) of the plasma membrane. In isolated rat adipocytes, PIG(-P) induce the redistribution of GPI proteins from hcDIGs to low-cholesterol-containing DIGs (lcDIGs) and concomitantly provoke insulin-mimetic signaling and metabolic action. Using a set of synthetic PIG(-P) derivatives we demonstrate here that their specific binding to hcDIGs and their insulin-mimetic signaling/metabolic activity strictly correlate with respect to (i) translocation of the GPI proteins, Gce1 and 5(')-nucleotidase, from hcDIGs to lcDIGs, (ii) dissociation of the nonreceptor tyrosine kinase, pp59(Lyn), from caveolin residing at hcDIGs, (iii) translocation of pp59(Lyn) from hcDIGs to lcDIGs, (iv) activation of pp59(Lyn), (v) tyrosine phosphorylation of insulin receptor substrate proteins-1/2, and finally (vi) stimulation of glucose transport. The natural PIG(-P) derived from the carboxy-terminal tripeptide of Gce1, YCN-PIG, exhibits the highest potency followed by a combination of the separate peptidylethanolamidyl and PIG constituents. We conclude that efficient positive cross-talk of PIG(-P) to the insulin signaling cascade requires their interaction with hcDIGs. We suggest that PIG(-P) thereby displace GPI proteins from binding to hcDIGs leading to their release from hcDIGs for lateral movement to lcDIGs which initiates signal transduction from DIGs via caveolin and pp59(Lyn) to the insulin receptor substrate proteins of the insulin signaling pathway.     

Distinct localization of lipid rafts and externalized phosphatidylserine at the surface of apoptotic cells

Externalization of phosphatidylserine (PS) takes place in apoptotic cells as well as in viable cells under certain circumstances. Recent studies showed that externalized PS is localized at the lipid raft in viable activated immune cells. We found that lipid rafts and PS existed in a mutually exclusive manner in apoptotic cells. The number of PS-exposing apoptotic cells decreased when lipid rafts were disrupted. BCtheta;, which binds selectively to cholesterol in a cholesterol-rich region, did not effectively recognize lipid rafts of apoptotic cells. Lipid rafts rich in GM1 were successfully prepared from apoptotic cells, but the lipid raft protein LAT was not enriched in the preparation. Furthermore, the amount of PS and phosphatidylethanolamine but not of cholesterol in lipid rafts appeared to change after induction of apoptosis. These results suggest that lipid rafts are structurally modified during apoptosis and, despite being localized differently from PS, are involved in the externalization of PS.     

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.     

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.     

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.     

Detachment-associated changes in lipid rafts of senescent human fibroblasts

We characterized the effects of in vitro cellular aging on constituents of lipid rafts in human diploid fibroblasts, TIG-1. Cholesterol recovery from lipid rafts of senescent cells was decreased by the detaching treatment, while the decrease was far less obvious in young cells. A probe that binds selectively to cholesterol in lipid rafts revealed that the amount of lipid rafts on the cell surface decreased in senescent cells upon cell detachment. Accompanying this change was the release of the raft-associated molecules caveolin and Fyn from lipid rafts upon cell detachment, suggesting a detachment-associated disorganization of lipid rafts in senescent cells. In addition, our observations showing differential sensitivities of lipid rafts from young and senescent cells to detaching treatment indicate a caution in how to detach cells. Particular attention needs to be paid to interpreting the results when lipid rafts are prepared from mechanically detached cells under detergent-free conditions.     

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14/CD16 circulating monocytes

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4 μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63 ± 3 vs 13 ± 1%, p < 0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63 ± 3 vs 41 ± 2%, p < 0.001), with less distribution in caveolae (13 ± 1 vs 34 ± 2%, p < 0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41 ± 2 vs 66 ± 3%, p < 0.001) and caveolae (34 ± 2 vs 18 ± 1%, p < 0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins.     

Proteomic analysis of ganglioside‐associated membrane molecules: Substantial basis for molecular clustering

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid‐enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X‐100 extracts and MS. We also examined GD3‐associated molecules using enzyme‐mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti‐GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3‐associating rafts exist. Representative examples of GD3‐associated molecules such as neogenin and MCAM were shown.     

The death-inducing signalling complex is recruited to lipid rafts in Fas-induced apoptosis

Membrane microdomains known as lipid rafts have been shown recently to be involved in Fas signalling and apoptosis in T and B cell lines. Here, we have investigated further the role of lipid rafts in Fas-induced apoptosis in non-transformed human CD4 T cells. We show that Fas-induced apoptosis in CD4 T cells was inhibited by the lipid raft disrupter methyl-beta-cyclodextrin. When lipid rafts were isolated from control and Fas ligand treated cells, we found that a small proportion of Fas was present in the raft fraction in untreated cells and that this was greatly increased upon Fas ligation. The other components of the Death Inducing Signalling Complex (DISC), FADD, and procaspase 8, were also present at higher levels in the raft fraction isolated from Fas ligand treated cells. We conclude that formation of the DISC occurs in lipid rafts and that these membrane microdomains are required for efficient Fas signalling and apoptosis.     

Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts

Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.     

Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes

Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs. Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein. A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by [(14)C]N-ethylmaleimide. The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide. Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs. The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Lipid rafts are essential for the regulation of SOCE by plasma membrane resident STIM1 in human platelets

STIM1 is a transmembrane protein essential for the activation of store-operated Ca²⁺ entry (SOCE), a major Ca²⁺ influx mechanism. STIM1 is either located in the endoplasmic reticulum, communicating the Ca²⁺ concentration in the stores to plasma membrane channels or in the plasma membrane, where it might sense the extracellular Ca²⁺ concentration. Plasma membrane-located STIM1 has been reported to mediate the SOCE sensitivity to extracellular Ca²⁺ through its interaction with Orai1. Here we show that plasma membrane lipid raft domains are essential for the regulation of SOCE by extracellular Ca²⁺. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn²⁺ entry, which was inhibited by increasing concentrations of extracellular Ca²⁺. Platelet treatment with methyl-β-cyclodextrin, which removes cholesterol and disrupts the lipid raft domains, impaired the inactivation of Ca²⁺ entry induced by extracellular Ca²⁺. Methyl-β-cyclodextrin also abolished translocation of STIM1 to the plasma membrane stimulated by treatment with TG and prevented TG-evoked co-immunoprecipitation between plasma membrane-located STIM1 and the Ca²⁺ permeable channel Orai1. These findings suggest that lipid raft domains are essential for the inactivation of SOCE by extracellular Ca²⁺ mediated by the interaction between plasma membrane-located STIM1 and Orai1.     

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation

Lipid rafts are membrane microdomains of unique lipid composition that segregate proteins with poorly understood consequences for membrane organization. Identification of raft associated proteins could therefore provide novel insight into raft-dependent functions. Monocytes process antigens for presentation to T cells by ingesting pathogens into calcium-dependent plasma membrane invaginations called "phagosomes" which develop by sequential fusion with the endoplasmic reticulum, early and late endosomes. We investigated the protein composition of Triton X-100 insoluble low density membranes of the monocyte cell-line THP-1 by matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry. The ganglioside GM1 colocalized on the plasma membrane with the raft markers flotillin 1 and 2, which were enriched in low buoyant density fractions containing 52 identifiable proteins, 28 of which have not been reported in rafts, and nine of which are associated with the endoplasmic reticulum (ER). Remarkably, 27 of the 52 proteins are components of phagosomes, including the ER protein calnexin which we demonstrate is phosphorylated on serine 562, a switch controlling calcium homeostasis. The presence of the early and late endosome trafficking proteins Rab-1, and Rab-7 together with the late endosome protein LIMPII, indicate lipid rafts are present throughout endosome maturation. Identification of vacuolar ATP synthase, and synaptosomal-associated protein-23, proteins implicated in membrane fusion, together with the cytoskeletal proteins actin, alpha-actinin, and vimentin, and Rac 1, 2, and 3, regulators of cytoskeletal assembly, indicate monocyte lipid rafts contain the machinery to direct vesicular fusion and actin based vesicular migration throughout phagosome development.