Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.     

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.     

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.     

Sphingomyelin chain length influences the distribution of GPI-anchored proteins in rafts in supported lipid bilayers

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts within the membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factors controlling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins in supported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The transmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains. Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted the association of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with the rafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchored proteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchor contains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used but associated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on the distribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supported lipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the association of a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchored proteins in rafts.     

Evidence for segregation of heterologous GPI-anchored proteins into separate lipid rafts within the plasma membrane

Cholesterol and glycosphingolipid-rich membrane rafts, which are rich in GPI-anchored proteins and are distinct from caveolae, are believed to serve as platforms for signal transduction events and protein recycling. GPI-anchored proteins with diverse functions as well as caveolin may be recovered in a membrane fraction insoluble in cold non-ionic detergent. This study tests for possible heterogeneity in the protein composition of the lipid rafts and detergent-insoluble membrane complexes by examining the two GPI-anchored homologous human folate receptors (FR)-alpha and -beta, the GPI-anchored human placental alkaline phosphatase (PLAP), and caveolin (control) in transfected CHO cells. Both FR and PLAP showed the equal distribution of cell-surface vs. sequestered (recycling) protein typical of GPI-proteins. Quantitative affinity purification of detergent-insoluble complexes using biotinylated folate or specific antibodies demonstrated a strong association of the homologous FR-alpha and FR-beta in the same detergent-insoluble complex and separate complexes containing either PLAP or caveolin. Immunogold localization experiments using antibody crosslinking to produce larger aggregates of GPI-anchored proteins for visualization by electron microscopy also showed a clear separation between FR- and PLAP-rich membrane microdomains. Thus, even though functionally diverse and heterologous GPI-anchored proteins are known to share endocytic and recycling vesicles, they may be segregated in distinct lipid rafts on the basis of their ecto(protein) domains facilitating clustering, compartmentalization and homotypic protein interactions.     

Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology

Glycosylphosphatidylinositol (GPI)-anchored proteins have been regarded as typical cell surface proteins found in most eukaryotic cells from yeast to man. They are embedded in the outer plasma membrane leaflet via a carboxy-terminally linked complex glycolipid GPI structure. The amphiphilic nature of the GPI anchor, its compatibility with the function of the attached protein moiety and the capability of GPI-anchored proteins for spontaneous insertion into and transfer between artificial and cellular membranes initially suggested their potential for biotechnological applications. However, these expectations have been hardly fulfilled so far. Recent developments fuel novel hopes with regard to: (i) Automated online expression, extraction and purification of therapeutic proteins as GPI-anchored proteins based on their preferred accumulation in plasma membrane lipid rafts, (ii) multiplex custom-made protein chips based on GPI-anchored cell wall proteins in yeast, (iii) biomaterials and biosensors with films consisting of sets of distinct GPI-anchored binding-proteins or enzymes for sequential or combinatorial catalysis, and (iv) transport of therapeutic proteins across or into relevant tissue cells, e.g., enterocytes or adipocytes. Latter expectations are based on the demonstrated translocation of GPI-anchored proteins from plasma membrane lipid rafts to cytoplasmic lipid droplets and eventually further into microvesicles which upon release from donor cells transfer their GPI-anchored proteins to acceptor cells. The value of these technologies, which are all based on the interaction of GPI-anchored proteins with membranes and surfaces, for the engineering, production and targeted delivery of biomolecules for a huge variety of therapeutic and biotechnological purposes should become apparent in the near future.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

GPI-anchored proteins and lipid rafts

Several proteins are anchored to membranes via a post-translational lipid modification, the glycosylphosphatidylinositol (GPI) anchor. In mammals and other vertebrates, GPI-anchored proteins have been found in almost all tissues and cells examined. Several studies have provided significant insight into the functions of this ubiquitous modification. An intriguing relevant feature of GPI-anchored proteins is their association with lipid rafts, specialized regions of elevated cholesterol and sphingolipid content, that are present within most cell membranes. In addition to the structure and biosynthesis of the GPI-anchor, recent researches have focused on its molecular interaction with lipid rafts and the biological meaning of such interaction. The aim of this review is to examine the emerging evidences of association between lipid rafts and GPI-anchored proteins, and their relationship with the modulation of important cellular functions such as protein/lipid sorting, signaling mechanisms and with human disease.     

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.     

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.     

Probing lipid rafts with proximity imaging: actions of proatherogenic stimuli

Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to cluster in microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years, several attempts have been made to use fluorescent probes to nondisruptively label these domains in living cells. Here, we have transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show colocalization of this fluoroprobe with another marker of lipid rafts, urokinase-type plasminogen activator receptor-1. ttGFP was used to quantify the cell surface area occupied by lipid rafts and to examine the effect of various proatherogenic signals on lipid rafts. Exposure of endothelial cells to asymmetric dimethylarginine and oxidized LDL (oxLDL), as well as oxidant stress, reduced the cell surface area occupied by lipid rafts. Next, the property of ttGFP to undergo a shift in absorbance depending on the clustering of these molecules was utilized to perform proximity imaging (PRIM). PRIM showed that nitric oxide (NO) increased the distance between GPI-anchored ttGFP molecules clustered in lipid-rich microdomains. This "unclustering" of GPI-anchored ttGFP was not reproduced by prooxidant signals and was due to reduction in membrane-cytoskeletal constraints on the lipid rafts. These findings suggested that two fundamentally different mechanisms modulate lipid rafts: 1) substance regulation of lipid rafts involving modification of cholesterol and sphingolipids and 2) structural regulation of lipid rafts through disruption of membrane-cytoskeletal interactions, switching off the spatial confinement of lipid rafts.     

Identification of a subgroup of glycosylphosphatidylinositol-anchored tryptases

The tryptase locus on mouse chromosome 17A3.3 contains 13 genes that encode enzymatically active serine proteases with different tissue expression profiles and substrate specificities. Mouse mast cell protease (mMCP) 6, mMCP-7, mMCP-11/protease serine member S (Prss) 34, tryptase 6/Prss33, tryptase ε/Prss22, implantation serine protease (Isp) 1/Prss28, and Isp-2 are constitutively exocytosed enzymes. We now demonstrate that tryptase 5/Prss32, pancreasin/Prss27, and testis serine protease-1 are inserted into plasma membranes via glycosylphosphatidylinositol (GPI) anchors analogous to Prss21, and that these serine proteases can be released from the cell’s surface by a phosphatidylinositol-specific phospholipase C. These data suggest that the C-terminal residues play key roles in determining where tryptases compartmentalize in cells. GPI-anchored proteins are targeted to lipid rafts. Thus, our identification of a number of GPI-anchored tryptases whose genes reside at mouse chromosome 17A3.3 also implicates important biological functions for this new family of serine proteases on the surfaces of cells.     

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.     

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.     

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.     

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

An analysis of the Caenorhabditis elegans lipid raft proteome using geLC-MS/MS

Lipid rafts are microdomains of the phospholipid bilayer, proposed to form semi-stable "islands" that act as a platform for several important cellular processes; major classes of raft-resident proteins include signalling proteins and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteomic studies into lipid rafts have been mainly carried out in mammalian cell lines and single cell organisms. The nematode Caenorhabditis elegans, the model organism with a well-defined developmental profile, is ideally suited for the study of this subcellular locale in a complex developmental context. A study of the lipid raft proteome of C. elegans is presented here. A total of 44 proteins were identified from the lipid raft fraction using geLC-MS/MS, of which 40 have been determined to be likely raft proteins after analysis of predicted functions. Prediction of GPI-anchoring of the proteins found 21 to be potentially modified in this way, two of which were experimentally confirmed to be GPI-anchored. This work is the first reported study of the lipid raft proteome in C. elegans. The results show that raft proteins, including numerous GPI-anchored proteins, may have a variety of potentially important roles within the nematode, and will hopefully lead to C. elegans becoming a useful model for the study of lipid rafts.