Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.     

Lipid microdomains--plant membranes get organized

The plant plasma membrane is now known to be a more sophisticated structure than was previously thought. Sebastien Mongrand et al. and Georg Borner et al. have isolated specific plasma membrane microdomains ('lipid rafts') that are enriched in sterols and sphingolipids. These rafts contain distinct sets of proteins and might help to explain how plasma membrane proteins are positioned in certain parts of the cell to function in development and signalling.     

Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains.

Lipid rafts are liquid-ordered membrane microdomains with a unique protein and lipid composition found on the plasma membrane of most, if not all, mammalian cells. A large number of signalling molecules are concentrated within rafts, which have been proposed to function as signalling centres capable of facilitating efficient and specific signal transduction. This review summarizes current knowledge regarding the composition, structure, and dynamic nature of lipid rafts, as well as a number of different signalling pathways that are compartmentalized within these microdomains. Potential mechanisms through which lipid rafts carry out their specialized role in signalling are discussed in light of recent experimental evidence.     

Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.     

Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.

We show that a protein with a glycosylphosphatidyl inositol (GPI) anchor can be recovered from lysates of epithelial cells in a low density, detergent-insoluble form. Under these conditions, the protein is associated with detergent-resistant sheets and vesicles that contain other GPI-anchored proteins and are enriched in glycosphingolipids, but do not contain a basolateral marker protein. The protein is recovered in this complex only after it has been transported to the Golgi complex, suggesting that protein-sphingolipid microdomains form in the Golgi apparatus and plasma membrane and supporting the model proposed by Simons and colleagues for sorting of certain membrane proteins to the apical surface after intracellular association with glycosphingolipids.     

Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains.     

Proteomic analysis of membrane microdomains derived from both failing and non-failing human hearts

Eukaryotic cells plasma membranes are organized into microdomains of specialized function such as lipid rafts and caveolae, with a specific lipid composition highly enriched in cholesterol and glycosphingolipids. In addition to their role in regulating signal transduction, multiple functions have been proposed, such as anchorage of receptors, trafficking of cholesterol, and regulation of permeability. However, an extensive understanding of their protein composition in human heart, both in failing and non-failing conditions, is not yet available. Membrane microdomains were isolated from left ventricular tissue of both failing (n = 15) and non-failing (n = 15) human hearts. Protein composition and differential protein expression was explored by comparing series of 2-D maps and subsequent identification by LC-MS/MS analysis. Data indicated that heart membrane microdomains are enriched in chaperones, cytoskeletal-associated proteins, enzymes and protein involved in signal transduction pathway. In addition, differential protein expression profile revealed that 30 proteins were specifically up- or down-regulated in human heart failure membrane microdomains. This study resulted in the identification of human heart membrane microdomain protein composition, which was not previously available. Moreover, it allowed the identification of multiple proteins whose expression is altered in heart failure, thus opening new perspectives to determine which role they may play in this disease.     

Lipid rafts in T cell receptor signalling

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.     

Pseudotypes of vesicular stomatitis virus with CD4 formed by clustering of membrane microdomains during budding

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.     

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.     

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% ¹. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient ². Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K¹⁵NO₃ as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest ³. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control ⁴.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

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

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

Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation

Stomatin-like protein 2 (SLP-2) is a member of the stomatin-prohibitin-flotillin-HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane.     

Glucosylceramide Biosynthesis is Involved in Golgi Morphology and Protein Secretion in Plant Cells

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that d ,l ‐threo‐1‐phenyl‐2‐decanoyl amino‐3‐morpholino‐propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high‐pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.     

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.     

心肌肌浆网膜含有膜质微区结构

细胞膜质微区(microdomain)是细胞膜上特殊的结构域,在细胞信号转导和物质运输过程中起着非常重要的作用.绝大多数膜质微区来源于全细胞膜,即包括质膜和细胞器膜.最新研究表明细胞器膜如高尔基体膜也有膜质微区,因此分离了猪心肌浆网膜的膜质微区.首先获得了没有质膜污染的猪心肌浆网,用去污剂TritonX-100处理该肌浆网,获得了去污剂不溶的质膜微区(SR-DRM),该微区富集胆固醇和鞘磷脂.质膜微区的标记脂和蛋白质:神经节苷脂GM1和Caveolin-3也在该区富集.同时还研究了心肌浆网Ca2 -ATPase(SERCA2a)的分布,结果表明,相当数量的SERCA2a分布在膜质微区,并且有正常的生理功能.上述研究结果表明,在心肌浆网膜上有膜质微区的存在,进一步证明膜质微区不仅存在于细胞质膜,也普遍存在于细胞器膜.     

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.