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Four-transmembrane-domain proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organization of TERM are poorly understood. In the present study, we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase [NB-DGJ (N-butyldeoxygalactonojirimycin) and PPMP (D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol·HCl)] resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasma-membrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased, but the association with EGFR [EGF (epidermal growth factor) receptor] was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together, our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these results demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

Throughout the biological world, a 30 Å hydrophobic film typically delimits the environments that serve as the margin between life and death for individual cells. Biochemical and biophysical findings have provided a detailed model of the composition and structure of membranes, which includes levels of dynamic organization both across the lipid bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic physical phase behaviour of lipids and their interactions with membrane proteins, to create the unique compositions and multiple functionalities of their individual membranes?

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.

The EuroConference on Microdomains, Lipid Rafts and Caveolae was held in Tomar, Portugal, from 17 to 22 May 2003. This joint EURESCO Conference/EMBO workshop was organized by G. van Meer and K. Simons and was the second meeting in a series that was initiated by K. Fiedler in 2001. ![][1] The plasma membrane was described as a fluid mosaic in the early 1970s by Singer & Nicolson (1972). Since then, several studies that were designed to elucidate the temporal and spatial architecture of the plasma membrane have provided a more complicated picture (reviewed in Edidin, 2003a). Many of these studies indicate that the plasma membrane is at the very least a mosaic of compartments that is maintained by an active cytoskeleton mesh. The membrane raft hypothesis proposes another type of compartmentalization (Simons & Ikonen, 1997; Simons & van Meer, 1988), in which specific lipids may dynamically associate with each other to form platforms that are important for membrane protein sorting and the formation of signalling complexes. This conference focused on the lateral domains that occur in biomembranes. In this report, we discuss new developments in the understanding of the lateral segregation of lipids that have been obtained from studies in artificial membranes and the parallel efforts to visualize lipidic assemblies in living cell membranes. We also report on new information about the roles of rafts in several cellular processes, such as in the sorting of membrane constituents during vesicular trafficking and in signal transduction, especially in immunological processes and caveolae formation and function. ### Raft structure In artificial membranes. To address the role of lipids in the formation of membrane rafts, studies have been performed with artificial membranes, and these suggest that homogeneous membrane bilayers might be an exception rather than a rule (Edidin, 2003b; McConnell & Vrljic, 2003). In general, … [1]: /embed/graphic-1.gif

Biological activities ranging from signaling, membrane transport to pathogenic processes take place in segregated subdomains of cell membranes. A fundamental step towards understanding the organization of membrane domains arose from the raft hypothesis, which proposes that these processes occur in raft membrane domains formed by lateral lipid assemblies enriched in sphingolipid and cholesterol (1).

The plant toxin ricin binds to both glycosphingolipids and glycoproteins with terminal galactose and is transported to the Golgi apparatus in a cholesterol-dependent manner. To explore the question of whether glycosphingolipid binding of ricin or glycosphingolipid synthesis is essential for transport of ricin from the plasma membrane to the Golgi apparatus, retrogradely to the endoplasmic reticulum or for translocation of the toxin to the cytosol, we have investigated the effect of ricin and the intracellular transport of this toxin in a glycosphingolipid-deficient mouse melanoma cell line (GM95), in the same cell line transfected with ceramide glucosyltransferase to restore glycosphingolipid synthesis (GM95-CGlcT-KKVK) and in the parental cell line (MEB4). Ricin transport to the Golgi apparatus was monitored by quantifying sulfation of a modified ricin molecule, and toxicity was studied by measuring protein synthesis. The data reveal that ricin is transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum and translocated to the cytosol equally well and apparently at the same rate in cells with and without glycosphingolipids. Importantly cholesterol depletion reduced endosome to Golgi transport of ricin even in cells without glycosphingolipids, demonstrating that cholesterol is required for Golgi transport of ricin bound to glycoproteins. The rate of retrograde transport of ricin was increased strongly by monensin and the lag time for intoxication was reduced both in cells with and in those without glycosphingolipids. In conclusion, neither glycosphingolipid synthesis nor binding of ricin to glycosphingolipids is essential for cholesterol-dependent retrograde transport of ricin. Binding of ricin to glycoproteins is sufficient for all transport steps required for ricin intoxication.

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

New insights in the distribution of lipids over the various intracellular organelles have been gained by a newly developed method for the immuno-localization of lipids. The method involves the labeling of an antigenic lipid with an antibody followed by a protein A-gold probe. Labeling is performed on ultrathin sections after freeze-substitution and low temperature embedding of aldehyde-fixed and cryo-protected cells. In a first study, Forssman glycolipid was localized in epithelial MDCK cells. Abundant labeling of the plasma membrane, the Golgi complex, and the membranes of the endocytic pathway was in contrast with the complete absence of labeling from mitochondrial and peroxisomal membranes. This demonstrated the specificity of the method. Recently, the method has also been applied to the(glycophospho)lipid phosphatidylinositol 4, 5-bisphosphate(PIP2), the central component of the phosphoinositide signal transduction pathway. In MDCK cells, two monoclonal antibodies labeled the plasma membrane, whereas membranes of the Golgi complex were virtually negative. One monoclonal in addition yielded abundant labeling over the heterochromatin in the nuclear matrix, whereas the other monoclonal antibody yielded only minor labeling dispersed over the nuclear matrix. This labeling in the nuclear matrix suggests the occurrence of a non membrane-associated form of PIP2, most likely complexed to a nuclear protein. The general applicability of the method is discussed.

The various membranes in eukaryotic cells have unique lipid compositions. Despite important discoveries in lipid research over recent decades, the basic principles by which cells define their membrane compositions are essentially unknown. Cells must sense the concentration of each lipid, integrate such signals and regulate the activity of their metabolic enzymes and transport routes to dynamically meet their needs in terms of membrane composition. Sphingolipids constitute a lipid category that is essential for eukaryotic life and appears to be key to differences in lipid composition. Here we discuss recent findings that assign an important role to lipid transfer proteins in the regulation of sphingolipid metabolism, organization and function.

Mammalian cells synthesize ceramide in the endoplasmic reticulum (ER) and convert this to sphingomyelin and complex glycosphingolipids on the inner, non-cytosolic surface of Golgi cisternae. From there, these lipids travel towards the outer, non-cytosolic surface of the plasma membrane and all membranes of the endocytic system, where they are eventually degraded. At the basis of the selective, anterograde traffic out of the Golgi lies the propensity of the sphingolipids to self-aggregate with cholesterol into microdomains termed 'lipid rafts'. At the plasma membrane surface these rafts are thought to function as the scaffold for various types of (glyco) signaling domains of different protein and lipid composition that can co-exist on one and the same cell. In the past decade, various unexpected findings on the sites where sphingolipid-mediated events occur have thrown a new light on the localization and transport mechanisms of sphingolipids. These findings are largely based on biochemical experiments. Further progress in the field is hampered by a lack of morphological techniques to localize lipids with nanometer resolution. In the present paper, we critically evaluate the published data and discuss techniques and potential improvements.

International audience; Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Until some 15 years ago, sphingolipids were generally believed to protect the cell surface against harmful factors in the environment by forming a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. Furthermore, complex glycosphingolipids were found to be involved in specific functions like recognition and signaling (1). Whereas the first feature would depend on physical properties of the sphingolipids, the signaling functions involve specific interactions of the complex glycan structures on the glycosphingolipids with similar lipids on neighboring cells or with proteins. Since then, two findings have revolutionized the field. (i) Simple sphingolipid metabolites, like ceramide and sphingosine 1-phosphate, have been found to be important mediators in signaling cascades of apoptosis, proliferation, and stress responses (reviews by Hannun and Obeid (66) and Spiegel and Milstien (67)). (ii) It has been realized that ceramidebased lipids self-aggregate in cellular membranes to form a separate phase that is less fluid (liquid-ordered) than the bulk liquid-disordered phospholipids based on diacylglycerol. Sphingolipid-based microdomains or “rafts” were originally proposed to sort membrane proteins along the cellular pathways of membrane transport (2). Presently, most excitement focuses on their organizing functions in signal transduction (3). Sphingolipids are synthesized in the ER 1 and the Golgi but are enriched in plasma membrane and endosomes where they perform many of their functions. Thus, sphingolipids travel between organelles. Transport occurs via transport vesicles and via monomeric transport through the cytosol. Furthermore, some sphingolipids efficiently translocate across cellular membranes. That transport is not random is clear from the heterogeneous distribution of sphingolipids over the cell; sphingolipids are virtually absent from mitochondria and the ER but constitute 20 –35 mol % of the plasma membrane lipids (Table I). Furthermore, signaling pools of sphingolipids do not freely mix with pools of biosynthesis and degradation (reviews by Hannun and Obeid (66), Merrill (68), and Spiegel and Milstien (67)). The specificity in sphingolipid transport is the topic of the present review.

The year 2000 marks the 75th anniversary of the existence of the bilayer model of biological membranes as originally proposed by Gorter and Grendel (1925). It also marks a period of excitement in understanding membrane lipids. Many lipids appear to lead double lives, serving both as membrane building blocks and as modulators of important cellular processes. Membrane lipid behavior is becoming understood at molecular and even atomic levels, through the application of sophisticated biophysical and computational approaches. The molecular causes of membrane lipid-based diseases are being established, and new technologies whereby membranes and their components can be manipulated are now being used in medical and industrial applications. These topics were discussed at ‘Anno 2000. A lipid milestone’, and the highlights of this discussion are summarized below.

Intracellular organelles have specific lipid and protein compositions. Although great progress has been made concerning the mechanisms that govern the transport of proteins between organelles, little information is available about the mechanisms of inter-organellar lipid transport. New work identifies a protein that specifically transports ceramide, a precursor of sphingolipid synthesis, between intracellular organelles.

Each cellular membrane is a unique mixture of some 50 out of about 500 different membrane lipids, but the function of these complex lipid compositions is still far from clear. While the major lipid classes are the phospho(glycero)lipids and cholesterol, another important class of membrane lipids in mammalian cells are the glycosphingolipids. Whereas usually they make up some 10% of the lipids on the cell surface, their headgroups appear to completely cover the apical surface of epithelial cells (Simons and van Meer, 1988).

Many mammalian ABC transporters move membrane lipids to acceptor lipid assemblies in the extracellular aqueous milieu. Because the desorption from the membrane costs more energy than provided by two ATPs, the transporter probably only translocates the lipid to a partially hydrophilic site on its extracellular face. From this high-energy site, the lipid may efficiently move to the acceptor, which ideally is bound to the transporter, or, in the absence of an acceptor, fall back into the membrane. If the lipid originated from the cytosolic membrane surface, this represents lipid flop and is probably a side activity of the transporters.