I. INTRODUCTION

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Sphingolipids are typically found in eukaryotic cells where they comprise a small but vital fraction (10-20%) of the membrane lipids. Their lipidic part consists of a sphingoid base, a straight-chain amino alcohol of 18-20 carbon atoms, which normally carries a long saturated fatty acid amide bonded to the amino group at the C2 position (Fig. 1). Based on the type of headgroup attached to the C1, sphingolipids are classified as phosphosphingolipids or glycosphingolipids. The phosphosphingolipids sphingomyelin (SM) in animals and inositol phosphoceramide (IPC) in plants and fungi carry the polar headgroups phosphocholine and phosphoinositol, just like the major glycerolipids phosphatidylcholine (PC) and phosphatidylinositol (PI). In glycosphingolipids, the headgroup can contain a variety of monosaccharides linked by various types of glycosidic bonds. Since the discovery of the sphingolipids more than 100 years ago by Thudichum (376), their special lipid backbone and their bewildering structural heterogeneity have fascinated biologists, biochemists, and biophysicists alike.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Common lipids in fungi and various animals. a, Sphingomyelin (SM); b, glucosylceramide (GlcCer), and a derived glycosphingolipid, e.g., GM1; c, glycerolipids; d, cholesterol; e, SM; f, glycosphingolipid of the arthro series; g, see b; h, sterol; i and j, see f; k, see b; l, sterol; m, M(IP)2C; n, see c; o, ergosterol. Fat print indicates the sphingoid bases in the sphingolipids a, b, e, f, i, j, and m; glycerol in the glycerolipids c, g, k, and n; and the sterols d, h, l, and o. A, head group: choline, ethanolamine, serine, or inositol; C, choline; E, ethanolamine; G, glucose; Ino, inositol; M, mannose; P, phosphate (note that insects and nematodes are sterol auxotrophs).

The major mission of the sphingolipid field is to understand the specific functions of these lipids in eukaryotic organisms and to evaluate their significance both for the functioning of individual cells and the organism as a whole. This is an arduous task, not in the least because, unlike glycerolipids and sterols, sphingolipids display a striking structural variation between distinct organisms, and even between different tissues and cell types within one organism. Moreover, sphingolipids are no longer viewed as primarily inert structural components of cellular membranes, but also increasingly recognized as diverse and dynamic regulators of a multitude of cellular processes. An important development in this respect has been the realization that sphingoid bases, ceramides, and other intermediates of sphingolipid metabolism act as signaling molecules in mediating cell cycle control, stress responses, and apoptosis (75, 127, 356). In addition, considerable attention has been drawn to the concept that sphingolipids drive the lateral differentiation of cellular membranes into a mosaic of areas with unique molecular compositions. In essence, it has been postulated that a differential miscibility of sphingolipids, glycerolipids, and sterols triggers the formation of lateral lipid assemblies, termed microdomains or rafts, that acquire specific functions by concentrating or excluding specific membrane proteins (341).¹ Rafts are now believed to serve as platforms for various cellular events including polarized protein sorting, signal transduction, and cell adhesion (35, 118, 178, 305, 340).

Sphingolipids are essential to sustain eukaryotic life. Whereas glycosphingolipids are indispensable for the development of complex multicellular organisms, phosphosphingolipids fulfill a vital function at a more fundamental level, namely, in the growth and survival of individual cells. This requirement for phosphosphingolipids appears to be a conserved feature of eukaryotic cells, as it is found both in animal cells and in yeast. Hence, it is not unlikely that the different sphingolipids synthesized in the various eukaryotic cell types serve a common function. If so, one would expect sphingolipids to share essential features that determine their interactions with other cellular components. If there is such a unifying principle, what could it be?

This review serves to highlight some remarkable aspects of sphingolipids that we believe represent important guidelines for unraveling their vital function in eukaryotic cells. One of these is the striking observation that in yeast the requirement for sphingolipids can be bypassed by a suppressor mutation that enables yeast to synthesize a novel set of glycerolipids whose structural appearence and predicted biophysical properties closely mimic those of the sphingolipids (74, 193, 281). As discussed in section II, this would indicate that some structural function, rather than a signaling one, accounts for the sphingolipid requirement in cell growth. A comparison of sphingolipid structures from evolutionary distinct organisms (see sect. III) indicates that, despite considerable chemical differences, the biophysical properties held responsible for microdomain formation have been well preserved. Indeed, counterparts of the microdomains analyzed in mammalian cell membranes appear to exist in flies, worms, and even in yeast (see sect. IV).

A further striking aspect of sphingolipids is that their assembly in eukaryotic cells is spatially separated from that of glycerolipids and sterols. Whereas the latter are produced in the endoplasmic reticulum (ER), sphingolipid synthesis occurs primarily in the Golgi complex (see sect. V). The Golgi has been well established as a polarized sorting and processing station that is situated right at the intersection of two major circuits of intracellular membrane trafficking. One circuit interconnects the cis-side of the Golgi with the ER; the other one the trans-side with the plasma membrane. The ER and plasma membrane are engaged in fundamentally distinct cellular processes, as reflected by the dramatic differences found in the molecular composition and biophysical properties of their bilayers. In section VI we consider the possibility that sphingolipids evolved as an integral and essential part of the Golgi machinery responsible for establishing the compositional and functional differences between the ER and the plasma membrane.

	II. WHY ARE SPHINGOLIPIDS ESSENTIAL FOR EUKARYOTIC LIFE?

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The early steps in sphingolipid synthesis, up to the formation of sphingoid bases and ceramides, are essentially the same in all eukaryotes. It is in the subsequent reactions, when ceramide is converted to complex sphingolipids, that major species- and cell type-specific differences become apparent. Yeast, for example, is equipped with a single biosynthetic pathway that serves to convert ceramide into only a handful of inositol-containing phosphosphingolipids. Animals, on the other hand, evolved three independent pathways that can operate simultaneously to produce both phosphosphingolipids and hundreds of different types of glycosphingolipids.² The divergence in headgroup structures found in glycosphingolipids accompanies functional differences between the various cell types of an animal. Whereas the synthesis of at least some glycosphingolipid species appears essential for the viability of animal organisms (see sect. IIB), as a lipid class they are dispensable for the growth and survival of animal cells in culture. This is not the case for phosphosphingolipids. Phosphosphingolipid synthesis is required for cell growth and survival, in yeast as well as in mammals (the only organisms in which this has been studied so far). Yeast and Chinese hamster ovary cell mutants lacking the first committed enzyme in sphingolipid biosynthesis, serine palmitoyl-transferase (Fig. 2), die in the absence of externally added sphingoid base (123, 411). The mammalian mutant could be rescued by exogenous SM, but not by glucosylceramide (GlcCer), the precursor of higher glycosphingolipids (121, 123). It therefore appears that SM, and not GlcCer (or higher glycosphingolipids), fulfills a vital role in mammalian cells. Indeed, mammalian cell lines unable to synthesize GlcCer are viable and proliferate (145). The alternative monoglycosylceramide, galactosylceramide (GalCer), is not required for cell survival either, since it is absent from most mammalian cell types including the GlcCer-negative cells (145).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Pathways of ceramide synthesis. Known metabolic intermediates, substrates, and enzymes are indicated. Names of genes whose products are required for specific steps in yeast ceramide synthesis are shown in italics and discussed in section VA. Note that it remains to be established whether C4-hydroxylation occurs on the free sphingoid base (sphinganine) or on ceramide.

Collectively, these observations raise two major questions. What vital function do phosphosphingolipids serve in the cell? And for what purpose do animal cells create so many different types of glycosphingolipids in addition, given that, in principle, they can live without them? Complete answers to these questions are still lacking. However, experimental work over the last decade has indicated that cells exploit both chemical and biophysical characteristics of sphingolipids to accomplish some of their most fundamental tasks. Before exploring which of these characteristics account for the sphingolipid requirement in cell growth and survival, we will first turn our attention to what is known about the sphingolipid requirements of complex multicellular organisms.

Gene knock-out studies in mice are starting to reveal the physiological significance of sphingolipids in mammals. First of all, in addition to phosphosphingolipids being indispensable for cell proliferation (see sect. IIA), glycosphingolipid production is essential for development and differentiation. Mice lacking GlcCer and all complex glycosphingolipids due to disruption of the gene encoding ceramide:glucosyltransferase were found to be embryonically lethal at the gastrulation stage just after formation of the primitive germ layers (421). After ectopic transplantation, glycosphingolipid-deficient embryonic stem cells were able to differentiate into endodermal, mesodermal, and ectodermal derivatives but failed to give rise to mature, well-differentiated tissues.

Knock-out mice have also been generated without a functional ceramide:galactosyltransferase (27, 58), the enzyme that synthesizes GalCer, which is the precursor of only a few other lipids like sulfatide (HSO₃ -3 GalCer). These mice live, but male mice were unable to breed, which reflects a function of galactolipids in spermatogenesis (95). In addition, such mice displayed compromised nerve fuction (27, 58), a finding consistent with the putative insulatory function of the bulk quantities of GalCer and sulfatide found in myelin, the plasma membrane of oligodendrocytes, and Schwann cells. High concentrations of GlcCer (murine) and/or GalCer (bovine, human) and derivatives have also been found in the apical plasma membrane domain of epithelial cells lining the gastrointestinal and part of the urogenital tract (341). Here, the glycosphingolipids are thought to provide mechanical stability and, especially in the gut, protection against harmful, hydrolytic enzymes such as phospholipases.

In contrast to GlcCer and GalCer, complex glycosphingolipids are mostly present on cell surfaces in low quantities only. Their importance for development and differentiation is supported first of all by the observation that knock-out mice being unable to synthesize the complex sialoglycosphingolipids. Of the double knock-out mice being unable to synthesize glycosphingolipids beyond the simple ganglioside GM3 (Fig. 3), 50% died within 13 wk (158). Mice unable to synthesize glycosphingolipids beyond GM3 and GD3 displayed axonal degeneration and decreased myelination (332, 371). As a possible explanation, it has been proposed that glycosphingolipids are involved in specific recognition events between cells and between cells and matrix via their specific carbohydrate moiety, and in the modulation of plasma membrane signal processing. The latter may occur via specific glycosphingolipid-protein interactions (119) or via organizing functions of the glycosphingolipids in signaling domains (118, 202).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Major routes of sphingolipid synthesis in eukaryotic cells. Ceramide serves as the immediate precursor for the phosphosphingolipids inositol phosphoceramide (IPC), ethanolamine phosphoceramide (EPC), and sphingomyelin (SM), which obtain their phosphoinositol, phosphoethanolamine, and phosphocholine headgroups by transfer from the corresponding glycerophospholipids. In addition, ceramide can be converted to the glycosphingolipids galactosylceramide (GalCer) and glucosylceramide (GlcCer) by transfer of galactose or glucose from UDP-Gal or UDP-Glc. GalCer can be further processed by the addition of galactose (Ga2Cer), sialic acid (GM4), or sulfate (SGalCer). In contrast, GlcCer can only be converted to LacCer, which in turn serves as the precursor for a large number of glycosphingolipid series. The ganglioseries (GgCer) are conveniently described by the Svennerholm nomenclature (GA2, GM3, etc.). Although the term sulfatide is generally used for SGalCer, often it is meant to include SLacCer.

It should be noted that not only sphingolipid production, but also the proper removal of sphingolipids, is of physiological significance for an organism. The absence of hydrolytic enzymes down to ceramidase or cofactors like sphingolipid activator proteins from the lysosomes results in lysosomal storage of sphingolipids with characteristic and often severe pathologies in humans (167, 171, 186, 260). Strategies devised to cure the symptoms of these diseases now include enzyme replacement and gene therapy (22, 29, 323, 435) and prevention of accumulation by administrating an inhibitor of glycosphingolipid synthesis (153, 287).

An impressive number of studies have implicated sphingolipids in virtually all aspects of cellular signaling (reviewed in Refs. 75, 118, 127, 356). First, sphingolipids serve as ligands for receptors present on neighboring cells (or in the matrix) to trigger various types of cell behavior (growth, adhesion, differentiation, migration). Second, sphingolipids influence properties of receptors on the same cell via specific lipid-protein interactions, thereby changing the cellular responsiveness to external stimuli (119). Third, sphingolipids modulate signaling by their ability to assemble both receptors and their downstream effectors (e.g., Src family kinases, G proteins) in specialized plasma membrane microdomains, known as rafts and caveolae (7, 36, 118, 147, 202, 340). Finally, sphingoid bases, ceramides, and their phosphorylated derivatives act as signaling molecules in the regulation of membrane trafficking, cell growth, cell death, and the ability of cells to cope with environmental stress (13, 152, 346, 356). Especially this last paradigm has attracted much attention in the recent literature. Although ceramide-activated protein kinases and phosphatases have been implicated in transmitting sphingolipid-derived signals (126, 431), the mechanisms by which ceramide pathways operate have not been elucidated (138, 139, 170, 409). Recent work has shown that ongoing synthesis of sphingoid bases forms a prerequisite for the internalization step of endocytosis in yeast (429). It appears that sphingoid base levels help control the relative activities of specific protein kinases and phosphatases whose downstream targets are elements of the endocytic machinery and/or actin cytoskeleton (93). Another exciting development in the field is the emergence of sphingosine-1-phosphate as a prototype of a new class of lipid signaling molecules that function not only as intracellular second messengers, but also as extracellular ligands for cell surface receptors (356). In support of the extracellular ligand function, several closely related transmembrane receptors have recently been identified as putative sphingosine-1-phosphate receptors in mammals (6, 191).

Sphingolipid signaling pathways have been found to operate in many different cell types, from mammals down to yeast (74). The impressive array of cellular processes that appears to be regulated by these pathways would provide a logical explanation for the observed lethality of sphingolipid-deficient mutant cells and organisms. However, studies in yeast have demonstrated that the putative signaling function of its sphingolipids is dispensable for cell growth and survival, although only under nonstressed conditions. A mutant strain lacking sphingolipids has been isolated upon suppression of a genetic defect in sphingoid base synthesis. This suppression is due to a mutation in the SLC1 gene, believed to encode a fatty acyltransferase (261). The suppressor mutation enables cells to produce a novel set of glycerolipids that mimic sphingolipid structures, both with respect to their headgroup and fatty acyl chain composition (193). The novel lipids identified were phosphatidylinositol (PI), mannosyl-PI, and inositol-P-(mannosyl-PI), all containing a C26 fatty acid in the sn-2 position of the glycerol moiety. Normally the C26 fatty acid is not found in yeast glycerolipids, but only in the sphingolipids (Fig. 1) and in the lipid backbone of some glycosylphosphatidylinositol (GPI)-anchored proteins. When exposed to extremes of pH or temperature, the suppressor mutant fails to grow unless provided with externally added phytosphingosine (74, 193, 281). These and other observations (76) show that yeast requires sphingolipids to build up a proper stress response. In contrast, the essential function of sphingolipids in growth and survival under normal conditions can be taken over by the novel glycerolipids, and is, apparently, structural.

	III. SPHINGOLIPID STRUCTURE AND BIOPHYSICAL PROPERTIES

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The key structural features by which sphingolipids can be distinguished from glycerolipids and sterols are outlined in Figure 1. First of all, the backbone of sphingolipids is a sphingoid base. With respect to molecular conformation, the sphingoid base is structurally equivalent to the glycerol and the sn-1 acyl chain in a typical glycerolipid. Saturated sn-1 chains of 16 or 18 carbons (C16:0, C18:0) predominate in most eukaryotic glycerolipids. The prevalent sphingoid base found in mammalian sphingolipids is sphingosine with a chain length of 18 carbon atoms and a trans-double bond between carbons 4 and 5. However, over 60 different species of sphingoid bases have been described with alkyl chain lengths varying from 14 to 22 carbon atoms, different degrees of saturation [the saturated form of sphingosine is called sphinganine (53)] and hydroxylation (154). The saturated and 4-hydroxylated form of sphingosine is generally referred to as phytosphingosine because it is the common sphingoid base found in sphingolipids of plants and fungi. In vertebrates, high levels (even up to 70%) of phytosphingosine-based sphingolipids have been found in kidney and intestine (8, 28, 113, 155, 266). Sphingolipids in insects are based on a C14 sphingoid base rather than the C18 backbone generally found in mammals, plants, and fungi (192, 304, 414). Sphingolipids of nematodes are unusual in that they are based entirely on a C16 sphingosine methylated at C15 (55, 105).

The amino group at the second carbon of the sphingoid base serves to attach a fatty acid in amide linkage. The N-acylated sphingoid base ceramide is the precursor of all membrane sphingolipids. The trivial name of ceramides based on sphinganine is dihydroceramide. The sphingoid C1 hydroxyl group in ceramide is used as the attachment site for a polar head group. Based on their head groups, sphingolipids are often grouped into phosphosphingolipids and glycosphingolipids. However, these categories are not mutually exclusive; plants and fungi, for example, add phosphoinositol to phytoceramide to generate IPC, and this head group can be further decorated with one or more monosaccharides. Sphingolipids with inositol phosphate-containing head groups are also common in protozoans (192). The main phosphosphingolipid in mammals and nematodes, SM, carries a phosphocholine (12, 55). Mammals also produce small quantities of ethanolaminephosphorylceramide (213). The latter is the principal phosphosphingolipid found in insects (304, 414). Tremendous diversity exists among the carbohydrate head groups of glycosphingolipids (Fig. 3), which may contain as many as 30 glycosyl residues per lipid. A glycosphingolipid is termed ganglioside if one or more of its sugar residues is a sialic acid. Gangliosides are particularly abundant in the central nervous system of higher organisms (240). Whereas sphingolipids display a striking organism- and cell type-dependent variation in head group composition, this is generally not the case for the glycerolipids. In most eukaryotic cells, glycerolipids utilize phosphate ester-linked choline, serine, ethanolamine, and inositol to achieve diversity.

One additional feature that makes sphingolipids different from glycerolipids is their fatty acyl chain. Typically, the amide-linked fatty acyl chains in sphingolipids are long and saturated. Often they are hydroxylated at the -position.³ In mammals, SM typically consists of two types, roughly half containing C16:0 and C18:0 and the other half C22:0, C24:0, and C24:1 (12, 156). The glycosphingolipids have similar fatty acids, but, depending on the tissue, a large fraction (up to 70%) may consist of the -hydroxylated form of each of these fatty acids (28, 268). The acyl chains in the sn-2 position of mammalian glycerophospholipids, on the other hand, are mostly shorter, (poly)unsaturated (e.g., C18:1, C18:2, or C20:4) and not hydroxylated. As illustrated in Figure 1, this striking difference in length, saturation, and hydroxylation status between fatty acyl chains of sphingolipids and glycerolipids has also been observed in Caenorhabditis elegans (55), Drosophila (304), and yeast. In yeast sphingolipids, C26:0 and hydroxylated C26:0 are the major fatty acids (192, 411). Interestingly, the prevalence of monounsaturated species of aminophospholipids at the expense of diunsaturated species in the plasma membrane of wild-type yeast is reversed in elo3 mutant cells that accumulate C22:0 fatty acid-containing sphingolipids instead of the normal C26:0 fatty acid-containing ones (269, 325). This observation indicates that the structural differences between the fatty acid chains of sphingolipids and glycerolipids serve important biological functions.

We conclude that, despite the considerable variation in chemical composition between lipids of distinct organisms, the structural features by which sphingolipids can be discriminated from glycerolipids have been preserved from vertebrates down to flies, worms, and fungi. Moreover, when these differences are undermined by mutation, organisms will exploit lipid-remodeling mechanisms to recreate structural diversity (193, 325). This presents us with the question, What would be the functional significance of these structural differences for eukaryotic life?

The interfacial region that links the nonpolar hydrocarbon region to the polar headgroup provides sphingolipids and glycerolipids with different biophysical properties. In sphingolipids, this region contains the 2-amide and 3-hydroxyl groups, often supplemented with additional hydroxyls at the sphingoid C4 and fatty acid C2, and with further hydroxyls on the carbohydrates. These groups can function both as hydrogen bond donors and acceptors and are thought to participate in extensive inter- and intramolecular hydrogen bonding of the sphingolipids, whereas glycerolipids have only hydrogen bond-accepting properties in this part of the molecule (278). This property allows sphingolipids but not glycerolipids to associate with themselves in the plane of the membrane into a highly flexible hydrogen-bonded network (23; see sect. IIIB). In membranes exposed to physical and chemical stress as in the apical membranes in kidney and intestine and in microorganisms, the sphingolipids exhibit an increased number of hydroxyl groups. This property is believed to improve the stability and decrease the permeability of those surface membranes (278). Although this may be generally true, under certain conditions the loss of a specific hydroxyl was found to increase membrane stability in yeast (82).

The lipid tails of natural sphingolipids are more tightly packed than those of the most abundant phosphoglycerolipid, monounsaturated PC. The surface area of GlcCer and GalCer at a surface pressure of 30 mN/m, typical of plasma membranes, is 0.40 nm² (212), versus 0.63 nm² for stearoyl-oleoyl PC (71). The fatty chains of sphingolipids are therefore more extended, and consequently the thinner and taller sphingolipids form up to 30% thicker membranes than unsaturated phospholipids.⁴ Second, sphingolipids contact their neighbors along a greater and flatter surface, which results in a dramatic increase in the van der Waals attraction between neighboring sphingolipid molecules. Van der Waals interactions have also been held responsible for the strong binding between the sphingolipids and cholesterol with its rigid and flat-cylindrical steroid backbone (235). A preferential binding of cholesterol to sphingolipids, in particular to SM, has been observed in many biophysical studies (23).⁵ Interestingly, the surface areas of disaturated PC (0.41-0.44 nm²; Refs. 71, 212) and PS (0.40-0.44 nm²; Ref. 72) are close to those of sphingolipids, and a preferential interaction of cholesterol with diC16:0 PC (less than or equal to that with SM; Refs. 181, 208) over mono-unsaturated PC has been observed (208). This illustrates the possibility that disaturated phospholipids may resemble sphingolipids in their hydrophobic interactions with other membrane components. This is especially relevant for the cholesterol-rich plasma membrane, where 40% of the PC and 75% of the PS are disaturated, while disaturated species, sphingolipids, and cholesterol are virtually absent from the ER (160).

The sphingoid chain extends less far into the membrane core than the N-linked fatty acid, a disparity not found in glycerolipids (Fig. 1). It has been argued that the difference in length between the two chains leads to packing defects (voids) in the membrane interior and that these defects may be complemented via interdigitation of two sphingolipids in the opposed bilayer leaflets (23, 44, 324). This may be an example of how lipids in one bilayer leaflet may couple to lipids in the opposite leaflet (see sect. VIIB). Interestingly, also cholesterol molecules in the two opposed bilayer leaflets may interact by forming transmembrane cholesterol dimers (129, 205, 250). Whether these transmembrane lipid interactions are of physiological relevance depends first of all on whether sphingolipids and cholesterol are present in both bilayer leaflets, and second on the relative affinities involved in transmembrane interactions and in lateral interactions of the lipids.

	IV. SPHINGOLIPIDS AND THE LATERAL ORGANIZATION OF BIOLOGICAL MEMBRANES

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At low temperatures, bilayers of a single phospholipid or sphingolipid exist in a frozen state. Above a melting temperature (T_m) characteristic of each lipid, the bilayer enters a phase in which the lipid acyl chains are fluid and disordered (50). The frozen state is referred to as the gel, solid-ordered (s_o), or l_ß phase. The fluid phase is termed the liquid-crystalline, liquid disordered (l_d), or l phase (173, 319). Sphingolipids (especially glycosphingolipids) display a much higher T_m than glycerolipids, due to their denser packing, which in turn is due to the saturation of their fatty chains and to the fact that they are more prone to intermolecular hydrogen bonding. While the phase transition for the typical mono-unsaturated PCs C16:0-C18:1 PC and C18:0-C18:1 PC occurs at 3 and 7°C, this temperature is 42°C for diC16:0 PC, 45°C for C18:0 SM, but 85°C for C18:0 GlcCer and GalCer (see Refs. 172, 173). Studies with model membranes consisting of binary mixtures of lipids with different T_m revealed that at temperatures between the T_m of the two lipids, a cooperative phase separation can occur, resulting in the coexistence of gel and l_d phase. The physiological significance of this type of phase separation in eukaryotes is questionable, mainly because membranes that contain high amounts of lipids with T_m values at or above room temperature, like the plasma membrane (160), also contain high levels of cholesterol. Cholesterol at concentrations of 30-50 mol% abolishes the gel-liquid crystalline phase transition (179, 319).

Remarkably, a phase separation between two fluid phases has been described in model membranes containing binary mixtures of a high-T_m lipid (saturated-chain lipid) and cholesterol (299). At temperatures above the T_m of the lipid, with increasing cholesterol concentrations a liquid-ordered (l_o) phase can separate from the liquid-disordered (l_d) phase. Above a threshold concentration of cholesterol, only the l_o phase exists, independent of the temperature. For diC16:0 PC, this occurs at 30 mol% cholesterol (319). Acyl chains of lipids in the l_o phase have properties that are intermediate between those of the gel and l_d phases; they are extended and ordered as in the gel phase but are laterally mobile in the bilayer as in the l_d phase. Also in bilayers of more complex composition the coexistence of l_o and l_d phases depends on the cholesterol concentration (reviewed in detail by Refs. 37, 40), and three fluid phases can coexist, of which two are l_o phases (294). The phase separation is especially pronounced when the T_m of the lipids is greatly different, like in the case of PCs of different chain length (338). Disaturated phospholipids and sphingolipids would preferentially partition into the l_o phase, which as a consequence would contain more cholesterol, whereas (poly)unsaturated lipids would prefer the l_d phase. Using a fluorescence quenching assay, Ahmed et al. (1) found that cholesterol induces the formation of a SM-enriched l_o phase at 37°C in a mixture of SM and monounsaturated PC. Mixtures of GlcCer, GalCer, and monounsaturated PC were found to be significantly inhomogeneous at 37°C, regardless of whether cholesterol was absent or present at concentrations at which the lateral separation in mixtures of SM and PC is abolished. Hence, glycosphingolipids appear to have a stronger tendency than SM to segregate from glycerolipids into an l_o phase (337).⁶ Ceramides at low concentrations occur as single monomers in a membrane, whereas at high concentrations they partition into hydrophobic ceramide droplets in between the two leaflets of the bilayer (404). At intermediate concentrations (~10 mol%; Ref. 141), the ceramides can aggregate to form a ceramide domain. The higher the affinity between ceramides, e.g., long acyl chain containing hydroxylated ceramides of yeast versus short acyl chain and sphingosine containing ceramides of mammals (204), the lower the ceramide concentration needed for segregation.

In a membrane with coexisting l_d and l_o phases, the distribution of each lipid species over the two phases is not governed solely by its relative affinity for the lipids in either phase. A different energy parameter is the long-range order of the lipids. According to the superlattice view (350, 372), lipids tend to be regularly distributed. Cholesterol with its different shape and smaller surface area imposes a steric strain on the alkyl chain matrix. To minimize the strain, cholesterol will adopt a regular lateral distribution, which yields a so-called superlattice. In a particular phospholipid mixture, only a defined set of cholesterol concentrations fits a superlattice, and this has been experimentally confirmed (349). At any given cholesterol concentration, a membrane would thus consist of domains displaying one of the allowed, minimum-energy superlattice organizations. A particular lipid will be arranged according to some superlattice pattern whether it is present in an l_o or in an l_d phase. Still, individual molecules will rapidly exchange positions, whereby the rate of exchange, and thus lateral diffusion, will be higher in the l_d phase. The degree to which each lipid type fits the superlattice of one phase will contribute to its partitioning between the two phases.

An overwhelming number of studies in artificial bilayers have demonstrated that lipids have a strong self-organizing capacity; lipid immiscibility can drive phase separation and give rise to domains (used in the meaning of "environments") with unique lipid compositions and biophysical properties. The current evidence does support the existence of such phase-separated lipid domains in cellular membranes. However, many uncertainties remain concerning their composition, size, and dynamics and concerning their location. In which of the subcellular membranes do they occur? Do they exist on both sides of the membrane, and if so, do domains on the one surface colocalize with domains on the opposite surface? Are domains the cause of variations in biomembrane thickness? Finally, a most relevant question: by what molecular interactions can membrane proteins discriminate between different lipid environments (69), and what is the contribution of membrane proteins to the properties of the domains?

The fluid-mosaic models of biomembranes in which proteins freely float around in an oily lipid bilayer (148, 343) marked a breakthrough in the thinking about membrane structure and function. However, already at that time, evidence became available suggesting that lipids and proteins were not randomly distributed in the membrane. For the lipids, phase separations had been demonstrated in the plasma membrane of Acholeplasma by calorimetry (363) and by freeze-fracture electron microscopy (403). Electron spin resonance studies suggested lipid phase heterogeneity in the ER (234, 364), and subsequently, a variety of techniques suggested the same for the plasma membrane of mammalian cells (157). Ideas on long-range order in the organization of lipids and proteins and the occurrence of microdomains were then formulated (148).

A unique type of sphingolipid (macro)domain organization exists in the plasma membrane of epithelial cells, where originally an accumulation of glycosphingolipids was reported for the apical plasma membrane domain of intestinal epithelial cells (92). Later work (30, 81, 159) demonstrated a two- to fourfold enrichment of glycosphingolipids in the apical versus the basolateral domain of the continuous plasma membrane of these cells. Similar differences have been reported for urinary bladder epithelium (369) and, indirectly, for Madin-Darby canine kidney (MDCK) cells derived from the distal nephron (341). Apical membranes from kidney cortex, which contain low levels of glycosphingolipids (358), contained an exceptionally high level of SM (35% compared with 13% in basolateral membranes; Refs. 48, 137, 247, 330, 402). These differences reside in the outer leaflet of the plasma membrane where they are maintained by the presence of a barrier to lipid diffusion, the tight junctions (reviewed in Ref. 341). Similar surface domains have been proposed to exist in sperm (reviewed in Refs. 14, 102), and a lipid-phase separation has been held responsible for their maintenance.⁷ Based on sphingolipid transport experiments (398), it was subsequently postulated that the sphingolipid-rich apical macrodomains originate from sphingolipid- and cholesterol-enriched microdomains in trans-Golgi membranes (339, 341, 395).⁸ These would then act as a sorting device for the delivery of apically directed cell surface components in polarized epithelial cells (see sect. VI).

The first candidate proteins of sphingolipid membrane domains were the proteins that are anchored in the noncytoplasmic bilayer leaflet of apical membranes by a GPI anchor (201). These proteins were found to be targeted to the apical cell surface, obviously via some interaction in the luminal leaflet of the Golgi membrane (34, 200). At the same time it was observed that influenza virus hemagglutinin, which is targeted to the apical membrane of infected epithelial cells, became partially resistant to extraction by Triton X-100 (TX-100) in the cold during transport to the surface (344), and it was proposed that the protein may complex with glycosphingolipids, as glycosphingolipids had been shown to be TX-100 insoluble as well. Progress in the field was tremendously accelerated when it was realized that not only hemagglutinin and sphingolipids but also the GPI proteins show resistance to extraction by detergent at low temperature, that this behavior might reflect the presence of these components in GPI protein/sphingolipid microdomains in the membrane, and that detergent insolubility might be an experimental tool to study the domains (38). The method consisted of the addition of 1% TX-100 to cells on ice for 20 min, homogenization of the cells, addition of sucrose to 40%, and flotation in a 30%-5% linear sucrose gradient without detergent. A GPI protein was found to float to a density of 1.081 g/ml,⁹ and closer inspection showed that the GPI protein was present in closed membrane vesicles that were now resistant to TX-100 (detergent-resistant membranes; DRMs).¹⁰ The low-density vesicles contained some 10% of the total cellular lipids: all of the SM, 50% of the glycosphingolipids, 25% of the cellular cholesterol, but only 5% of the glycerolipids (38). Subsequent work on model membranes has suggested that SM is detergent insoluble only when it was present in an l₀ liquid-ordered phase before the addition of the detergent (1), supporting the idea that DRMs are derived from preexisting l₀ phase domains. The power of the DRM isolation method to study the structure and function of domains was demonstrated by the finding in the original paper (38) that the GPI proteins became detergent insoluble at 20 min after synthesis only, which suggested that the proteins became insoluble upon entering the Golgi where they are thought to come into contact with the sphingolipids (see sect. VI).¹¹ The method has been applied to identify proteins involved in the transport pathway from the trans-Golgi network (TGN) to the apical domain (180, 304). Evidence has been reported for the coexistence in the same membrane of domains with different (GPI) protein compositions and different physical properties (211, 307). The same was reported for myelin where TX-100 and CHAPS yielded DRMs of different protein compositions (342).¹² SM-enriched DRMs have been isolated from nuclei, where they were assumed to represent the inner nuclear membrane (18).

DRMs have been isolated from the plasma membrane, suggesting the existence of phase-separated sphingolipid/cholesterol domains on the cell surface where they would compartmentalize, modulate, and integrate signaling events by providing sites for the assembly of components involved in signal transduction (202, 340). Exciting new developments in the field are the regulation, by ligand binding, of the association with the domain of signaling receptors and of the Src family kinases that transduce the signals into the cell. The latter kinases are anchored to the cytoplasmic bilayer leaflet by two saturated acyl chains, whereas in contrast proteins anchored by the branched isoprenyl groups are not recovered in DRMs (237). This supports the idea that an ordered lipid environment is present on the cytoplasmic surface of the sphingolipid rafts. Some of the DRMs from the plasma membrane were derived from caveolae, morphologically well-established flasklike invaginations of the plasma membrane (7, 37). Caveolae are defined by the presence on the cytosolic surface of the palmitoylated, cholesterol-binding protein caveolin (7, 256). Multiple palmitoylation of caveolin was found to be required for its interaction with dually acylated G proteins (103), but not for binding to caveolae (78). Caveolin (VIP21) was also localized to the Golgi (177, 207), where it is supposed to be involved in membrane transport. DRMs have been isolated from many different mammalian cell types, but also from Drosophila (304), Dictyostelium (419), yeast (11, 174), and protozoans (430).

Obviously, the detergent extraction method has its limitations. It cannot be concluded that the organization of the lipids (and proteins) in the resulting DRMs reflects their organization in the cellular membrane of origin in full detail, because some rearrangement may have occurred as a consequence of low temperature, detergent addition, and detergent dilution. Low temperature favors formation of ordered domains, while detergent will insert into the membrane and influence the partitioning of the various lipids. Notably, the size of the original sphingolipid domains cannot be assessed by detergent extraction, because separate domains within the same membrane may coalesce due to removal of the surrounding glycerolipids. Because the bulk of the sphingolipids has been localized to the noncytoplasmic surface of cellular membranes (see sect. VB), one other uncertainty is the composition of the lipids on the cytosolic surface facing the sphingolipid domain and their behavior during detergent extraction (discussed in detail in Ref. 36). Various groups have published methods to isolate caveolae without the use of detergent (326, 348, 351, 361). Whereas three groups set out by isolating plasma membranes, the fourth group (351) followed the detergent extraction method but without detergent. Cells were homogenized and sonicated, and the membranes were floated up in a sucrose gradient. Membranes at the 35/5% sucrose interface were separated from membranes at the 45/35% sucrose interface and were designated "purified caveolae membranes."¹³ It will be interesting to see how these protocols will complement the DRM method.

Independent evidence for clustering of glycosphingolipids on the cell surface has been obtained by microscopy. A first approach has utilized glycosphingolipid-binding proteins, which were visualized by a fluorescent or electron-dense tag or by a secondary labeled protein. The major problem in such studies is that lipids cannot be fixed at their original location. Artificial clustering was induced when the binding of a primary antibody to globoside (Gb₄Cer) or Forssman glycosphingolipid (IV³--GalNAc-Gb₄Cer) was followed by labeling with a dimeric secondary antibody, tetrameric protein A, or when multimeric complexes were used of the primary or secondary ligand to ferritin or colloidal gold (47, 96, 377). Clustering of the ganglioside GM1 was observed when it was labeled with pentameric cholera toxin B subunit by itself (9) or conjugated with gold into a multimeric complex (276), or when a biotinylated GM1 was labeled with anti-biotin-gold (246). The latter studies were performed on freeze-substituted samples in which redistribution seems rather unlikely. In an independent approach, labeling of glycosphingolipids with a primary antibody was followed by fixation before the addition of the secondary antibody-gold complex, a condition that had been shown to prevent redistribution of Forssman glycosphingolipid (47, 96). Clusters of GM3 were still observed in one study (354), while clustering of a number of sphingolipids in caveolae was no longer observed under these stringent conditions (96). Still, a local enrichment of the ganglioside GM1 in caveolae was found by a postembedding labeling protocol using cholera toxin where redistribution could be excluded (276).¹⁴ In cells transfected with influenza virus hemagglutinin or GPI proteins, the distribution of these proteins overlapped with that of GM1 (128). Clustering of GPI proteins has been observed by microscopy under a variety of conditions (7). However, in many cases clustering was induced by the protocol used. A major point of concern in most microscopic studies on lipid and lipid-linked molecules is the use of multimeric reporter ligands without proper controls.

Already in the early 1980s, measurements on the behavior of fluorescent probes in biomembranes supported the concept of lipid domains in membranes (157), and microscopy was performed on living cells using fluorescent lipids (357). In the latter study, the redistribution of GM1 by cholera toxin caused cocapping of the unrelated ganglioside GM3, which suggested that the headgroup-labeled GM1 and GM3 were associated by lipid-lipid interactions. Much more recently, a major breakthrough in the field has been the application to living cells of novel high-resolution optical techniques, like resonance energy transfer between fluorescent membrane molecules, single particle tracking, two-dimension scanning resistance, and single dye tracing. A number of these studies support the existence of locations on the cell surface that are enriched in GPI proteins and glycosphingolipids and, in addition, of areas with enhanced resistance to lateral diffusion that are preferred by some but not by other probes. One controversial issue is the size of the domains. What is their diameter? Whereas the detergent-extraction studies yielded DRM vesicles with a diameter of 0.1-1 µm (39), suggesting a diameter size of 200-2,000 nm, this was a few hundred nanometers for the small regions to which a GPI protein and GM1 were found to be confined in particle tracking studies (290, 331).¹⁵ The newest single particle tracking measurements on these domains suggest that they are even smaller with a diameter of 50 nm (roughly 3,500 lipids), exist for more than 1 min, and comprise <50 proteins (289). Chemical cross-linking and fluorescence resonance energy transfer to measure GPI-protein interactions led to estimates of 70 nm (94, 401). In contrast to these data, no clustering of a GPI protein was observed on the apical surface of MDCK cells (162). In addition, a comparison between various GPI proteins on various cell surfaces did not provide evidence for the occurrence of a sizeable fraction of the GPI proteins as stable clusters (163). The authors explained the discrepancies with the earlier work, by concluding that lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

Here, it becomes relevant to discuss the area of the membrane covered by rafts. SM constitutes ~20% of the plasma membrane phospholipids. If, as generally believed, SM is located in the outer leaflet, it covers 40% of the surface. When saturated phospholipids and cholesterol are added to this figure, one would expect most of the plasma membrane to be covered by a liquid-ordered domain. In a special case, the apical surface of epithelial cells of kidney and intestine, the surface is completely covered by sphingolipids (341). This supports the idea that the apical membrane of kidney cells, like MDCK, is one big raft (162). Clustering on apical surfaces may therefore not occur (162). When it occurs, it may imply that there are different types of rafts (211, 307, 342). Interestingly, when beads attached to a GPI protein (major histocompatibility complex class I) were scanned over the surface of hepatoma cells by laser tweezers, the beads experienced areas of increased resistance of 100 nm diameter, that were independent of the actin skeleton and covered an area of <10% of the surface, which is similar to caveolae (370). The physicochemical basis of the existence of multiple types of rafts remains to be resolved but may involve cholesterol-induced domains (294) and caveolae (7), both of which are very stable. In contrast to measurements on beads attached to diC16:0 phosphatidylethanolamine (PE) where no indications for areas of higher resistance were observed (370), in tracings of single fluorescent molecules on muscle cells, the lipid analog Cy5-diC14:0-PE, but not Cy5-diC18:1-PE (which were assumed to represent lipids preferring areas of lower and higher fluidity, respectively), was 100-fold enriched in domains of ~0.7 µm, that were stable for several minutes (329). No ultrastructural correlate for such domains has been found.

In line with the model of GPI protein sorting in epithelial cells via sphingolipid domains (201, 341), a GPI protein was found by various techniques to be clustered during transport to the apical MDCK cell surface, after which it diffused over the apical surface (125). However, in contrast to inhibition of sphingolipid synthesis (233), cholesterol depletion did not change polarity of cell surface delivery of the GPI protein (124). Cholesterol depletion did inhibit transport of hemagglutinin to the apical membrane (161).

	V. SPHINGOLIPID ASSEMBLY AND TRANSPORT

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A.  Enzymes of Sphingolipid Metabolism and Their Topology

1.  Ceramide synthesis

Sphingolipid synthesis in all eukaryotes starts in the ER with the condensation of L-serine with palmitoyl coenzyme A, a reaction catalyzed by serine palmitoyl-transferase (SPT) and yielding 3-ketosphinganine (Fig. 2). SPT forms the target of several potent natural inhibitors that include sphingofungin (436), lipoxamycin (214), myriocin (245), and viridiofungins (217). Genetic studies in yeast revealed that at least two genes, LCB1 and LCB2, are required for SPT activity, suggesting that the enzyme is composed of at least two distinct proteins (43, 258). This was confirmed when the proteins encoded by mammalian homologs of these genes were characterized and found to be part of a functional enzyme complex (121, 122). The product of SPT, 3-ketosphinganine, is reduced to sphinganine (also called dihydrosphingosine), which in yeast requires the TSC10 gene product (15). Reactions up to the formation of sphinganine appear to be the same from mammals down to yeast. Sphinganine in mammals is N-acylated by ceramide synthase to form dihydroceramide, which is rapidly desaturated to give ceramide; a 4,5-trans-double bond is introduced in the sphinganine moiety to form ceramide containing sphingosine as its long-chain base (241). In yeast, but probably also in mammals, sphinganine is hydroxylated to form 4-hydroxy-sphinganine, commonly named phytosphingosine. This reaction requires the SYR2 gene product and, unlike earlier enzymatic steps in the biosynthetic pathway, is not essential for cell growth in yeast (116). Amide linkage of a fatty acid to phytosphingosine yields phytoceramide, the ceramide found in most fungal and plant sphingolipids and abundant in some mammalian tissues (see sect. IIIA). Ceramide synthesis is believed to take place on the cytosolic face of the ER (220). In mammals, this reaction can be inhibited by fumonisins (405), while australifungin effectively blocks ceramide synthesis in yeast (216). So far, no single acyl CoA-dependent ceramide synthase has been purified or cloned. However, the alkaline ceramidases encoded by the yeast genes YPC1 and YDC1 possess a reverse, acyl CoA-independent ceramide synthase activity that is insensitive to classical ceramide synthesis inhibitors (222, 223). The very-long-chain (20+ carbon) fatty acids found in (phyto)ceramides are formed by ER membrane-bound fatty acid elongation systems whose components belong to a evolutionary conserved family of transmembrane proteins (269, 382, 383). Disruption of the corresponding genes in yeast reduces the cellular sphingolipid levels (269) and induces pleiotropic phenotypes such as bud site localization defects, changes in plasma membrane H⁺-ATPase levels, and resistance to sterol synthesis inhibitors (104, 303). The fatty acid moiety in ceramides can be mono- or dihydroxylated. In yeast, the first hydroxylation step occurs in the ER and requires SCS7, a gene structurally related to the SUR2 gene (116). The second hydroxylation takes place in the Golgi and requires the Golgi copper transporter encoded by the CCC2 gene (16).

In animal cells, ceramide is used to produce SM and GlcCer, the precursor for the higher glycosphingolipids (Fig. 3). SM production occurs in the lumen of the cis and medial Golgi and involves the transfer of phosphocholine from PC to ceramide, yielding diacylglycerol as a (potentially important) side product (101, 150). A second SM synthase activity has been located on the cell surface (392). Two SM synthases with different properties have also been found in the intra-erythrocyte stage of Plasmodium (120). The SM synthases remain to be identified. The ceramide:glucosyltransferase involved in GlcCer production is located on the cytosolic surface of the early Golgi (45, 65, 100, 151, 224). Whereas the GlcCer synthase in mammals is encoded by a single gene, three GlcCer synthase analogs have been identified in the C. elegans genome (144). The physiological significance of this multiplicity remains to be solved. By an unknown mechanism, GlcCer is translocated to the Golgi lumen where it can be trapped by galactosylation to Galß1-4GlcCer (LacCer; Refs. 45, 183, 267). Various series of complex glycosphingolipids can then be generated by stepwise addition of sugars to LacCer, whereby the first sugar and its glycosidic linkage determine the name of the series (53), e.g., Gal1-4: globo- or Gb, Gal1-3: isoglobo or iGb, GlcNAcß1-3: lacto or Lc (Fig. 3). The ganglio series (or Gg) is based on GalNAcß1-4LacCer. The ganglio series also comprises the simple gangliosides NeuAc_1-3-LacCer, well-known under the Svennerholm nomenclature as GM3, GD3, and GT3, respectively (53). Glycosphingolipids in the various series may be fucosylated or sulfated. LacCer synthesis and all further conversions occur in the lumen of the Golgi and require import of the necessary sugar nucleotides (45, 183; reviewed in Ref. 209). With the use of subcellular fractionation, a sequential distribution over the Golgi was observed for glycosphingolipid glycosyltransferases (143, 380, 381), with a significant overlap in distributions. A later paper (184) assigned the synthesis of LacCer, GM3, and GM2 to the trans-Golgi and the TGN, whereby a considerable fraction of the LacCer and GM3 synthases localized to the cis-Golgi. Pharmacological studies, using the drugs brefeldin A (308, 333, 384, 427) and monensin (244, 316, 317, 385) to discriminate enzymes of the Golgi stack from those in the TGN, localized GM2 synthase (GalNAc-transferase) and two galactosyltransferases of complex glycosphingolipid synthesis to the TGN (see, however, Ref. 426). The same conclusion was reached in a study on mitotic cells (62).

Arthropods and mollusks transfer a mannose onto GlcCer and further extend this chain in the arthro (At) or the mollu (Mu) series. In some mammalian tissues, notably myelin, but in humans also the epithelia of the gastrointestinal and urogenital tracts, ceramide is mainly glycosylated to GalCer by the ceramide:galactosyltransferase, an enzyme situated on the luminal face of the ER (328, 359, 360). The enzyme displays a preference for ceramides containing a 2-hydroxylated fatty acid which are abundant in these tissues. GalCer can be further galactosylated and/or sulfated (or sialylated) in the Golgi lumen (45, 183, 373).

In contrast to animals, all fungi and plants studied sofar, as well as several protozoa, add inositol phosphate to phytoceramide to form IPC (192). The biosynthetic route of inositol sphingolipids in yeast has been studied in great detail. Yeast produces only three types of inositol sphingolipids: IPC, mannose 1-2IPC: MIPC, and inositol-1-P-6 mannose 1-2IPC or mannose-(inositol-P)₂-ceramide: M(IP)₂C. The IPC synthase, or an essential subunit thereof, is encoded by the AUR1 gene (259). AUR1 homologs have been identified in a wide variety of fungi (132, 176). IPC activity is effectively blocked by the antifungal agents aureobasidin A (259), khafrefungin (219), and rustmycin (218). Because IPC synthase activity is essential and not found in mammals, it provides an ideal target for therapeutic drugs to fight pathogenic fungi in immunocompromised individuals. IPC synthesis in yeast was previously thought to occur in the ER. This idea was based on the fact that the formation of IPC from ER-derived ceramide and PI continues under conditions when ER-to-Golgi vesicular transport is blocked in temperature-sensitive secretion (sec) mutants (292). However, fluorescence microscopy and membrane fractionation experiments have recently shown that both the AUR1 gene product and IPC synthase activity are located in the Golgi (195). This discrepancy in results remains to be clarified but could be explained if the IPC synthase would constitutively cycle between the ER and the Golgi, a feature inherent of several Golgi-based proteins (60, 146, 210). An alternative explanation could be that ceramide made in the ER can reach the Golgi by a vesicle-independent transport mechanism (see sect. VC).

Whereas the sidedness of IPC production is unknown, mannosylation to form MIPC most likely occurs in the lumen of the Golgi. The latter reaction requires at least three genes: SUR1, VRG4, and CSG2. SUR1 most likely encodes a mannosyltransferase (16), whereas VRG4 is required for GDP-mannose transport into the Golgi lumen (68). CSG2 encodes a member of the major facilitator superfamily, and its role in MIPC production is unclear (432). The final and most abundant sphingolipid in yeast, M(IP)₂C, is formed by transfer of inositol phosphate from PI onto MIPC and requires a protein encoded by the IPT1 gene (77). This reaction resembles the one that yields IPC. Accordingly, the IPT1 and AUR1 gene products exhibit a striking structural similarity.

The major pathway along which sphingolipids are degraded is removal of the head group and subsequent hydrolysis of ceramide to sphingoid base and free fatty acid. The breakdown products are then further metabolized or reutilized. Glycosphingolipids are degraded via a complex cascade of glycosidases and activator proteins or saposins in the lysosomes, and defects in any of the steps result in lysosomal storage diseases (171). A nonlysosomal glucocerebrosidase activity has been found (400). It appears to be active on the cytosolic surface of a cellular membrane, possibly the plasma membrane. GlcCer is the only glycosphingolipid that is synthesized on the cytosolic surface of the Golgi. The enzyme may be involved in regulating GlcCer availability on cytosolic surfaces (R. Raggers and G. van Meer, personal communication). SM is degraded by a lysosomal acid sphingomyelinase (327) in the lysosomal lumen. Alternatively, the acid sphingomyelinase and neutral sphingomyelinases (51, 85, 140, 378) have been invoked as the enzymes that generate ceramide in signal transduction on the cytosolic surface of cellular membranes (see sect. IIC). Sphingomyelinase activity has also been reported for yeast (84) and requires ISC1, a gene whose product is structurally related to neutral sphingomyelinases (320). The ISC1-encoded protein displays phospholipase C activity toward SM, IPC, MIPC, and M(IP)₂C, but not to PI, PC, or lyso-PC, indicating that it functions as a inositol phosphosphingolipid-specific phospholipase C in yeast (320). Where in the cell and on which side of the membrane Isc1p-mediated hydrolysis of sphingolipids occurs is unclear. Whether yeast contains additional enzymes for breaking down its sphingolipids remains to be established. Ceramides are broken down by a number of ceramidase activities that are classified as acidic, neutral, or alkaline, based on their pH optimum. Acidic ceramidase is localized in lysosomes and its gene has been cloned from human (166). Two alkaline ceramidases associated with the ER have been identified in yeast (222, 223).

Although the bulk of sphingoid bases and ceramides is incorporated into sphingolipids, cells do contain small quantities of free sphingoid bases and ceramides, including some phosphorylated derivatives; these are newly synthesized or derived from sphingolipid breakdown and are generally (re)utilized for sphingolipid synthesis in ER and Golgi. Sphingosine-1-phosphate and sphinganine-1-phosphate are special in the sense that they are the final substrates in sphingolipid hydrolysis, being converted by a lyase to ethanolamine phosphate and a C16 aldehyde (315, 399, 433). At the same time, sphingoid long-chain base-1-phosphates are important intra- and intercellular second messengers (see sect. IIC), and their concentration is tightly regulated by the combination of kinases (167, 186, 260), the lyase, and a phosphatase (149, 215, 221, 399).

A sphingolipid gradient exists along the organelles of the secretory pathway. Although primarily assembled in the Golgi, sphingolipids are enriched in the plasma membrane and endocytic membranes of cells, whereas only low amounts are found in the ER. This has been demonstrated both in yeast (131, 280) and in animal cells (59, 90, 160, 302). Although over 90% of the cellular SM was assigned to the plasma membrane by a cell fractionation approach (182), some 60% of the cellular SM has been routinely found in the plasma membrane by an assay based on exogenous sphingomyelinase (5, 297, 334, 347, 389). Very high concentrations of glycosphingolipids (30-40% of total membrane lipids) have been found in the apical plasma membrane domain of epithelial cells (see sect. VB) and in myelin, a specialized plasma membrane domain of Schwann cells and oligodendrocytes (366). By electron microscopy, the complex glycosphingolipid Forssman antigen was found absent from mitochondria and peroxisomes, low in ER and Golgi and enriched in plasma membrane and endocytic structures (387). Although generally enriched in the plasma membrane and related membranes, there are differences with respect to the distribution of individual sphingolipid classes between organelles (231) or between apical and basolateral plasma membrane domains (159, 358). The intracellular distribution of ceramides, sphingoid bases, and their 1-phosphates is less clear.

The accessibility of glycosphingolipids and SM to reagents, antibodies, and enzymes on the cell surface has led to the general belief that sphingolipids are primarily situated in the noncytoplasmic leaflet of cellular membranes. This is in line with their site of synthesis that is on the luminal aspect of the Golgi (for GalCer on the luminal aspect of the ER). Only GlcCer is synthesized on the cytosolic surface of the Golgi (see sect. VA). The best evidence seems available for SM. In the original studies on erythrocytes, bacterial sphingomyelinase hydrolyzed 80-85% of the SM (404), suggesting that the bulk of the SM is situated in the outer, noncytoplasmic leaflet. A similar conclusion can be drawn from the accessibility of 60% of the SM to sphingomyelinase in intact nucleated cells (5, 297, 334, 347, 389), where a significant fraction of the remaining 40% can be expected to be present in intracellular membranes like endosomes and Golgi.¹⁶ Indeed, no SM was found accessible to sphingomyelinase in microsomal membranes (263, 264), and 20% was hydrolyzed in isolated chromaffin granules (42), in which cases the cytosolic surface is exposed. Only little quantitative data are available on the transbilayer distribution of glycosphingolipids. From studies on GalCer in myelin (199), GM3 in membrane viruses (365, 367), and GM3 and GD1a in a number of cells (242) it has been concluded that the bulk of these lipids is situated in the noncytoplasmic surface of the plasma membrane (for discussion of the methodology, see Ref. 336).

Still, pools of sphingolipids may exist on the cytosolic surface of membranes. This is especially true for GlcCer, which after synthesis is initially present in the cytosolic leaflet of the Golgi membrane (65). A cytosolic protein that can interact with GlcCer has been isolated from cytosol and its gene cloned (198). Other cytosolic proteins have been found to be capable of interacting with complex glycosphingolipids (49, 54, 134, 135, 243, 352, 353), whereas also glycosphingolipids have been colocalized with cytoskeletal elements (106, 107, 318).¹⁷ This may suggest that also complex glycosphingolipids are present in cytosolic surfaces. This could be a consequence of lipid mixing caused by fission and fusion events during membrane traffic, or of transbilayer equilibration of a small sphingolipid fraction reaching the ER (see below).

C.  Sphingolipid Transport and Sorting

1.  Concepts

After synthesis, sphingolipids can move around the cell in various ways. Intracellular transport processes are fast (minutes) compared with sphingolipid turnover (many hours). So, to maintain the differences in sphingolipid concentration between cellular membranes, there must be specificity in sphingolipid transport. We recently discussed sphingolipid transport in a separate review (397). The present paper focuses on the specificity in sphingolipid transport and the involvement of sphingolipids in sorting other membrane components.

When situated in a membrane, sphingolipids first of all can diffuse as monomers in four directions. If we do not take into account the motions of the entire molecule that do not result in transport, like the rotation around their longitudinal axis and the wobble (279), sphingolipids can diffuse laterally in the two-dimensional plane of the membrane; they can diffuse out of the membrane into the aqueous phase, and they can flip across the membrane into the opposite lipid monolayer.¹⁸ Of these movements, only diffusion out of the membrane may result in transport between cellular organelles. The second mechanism of lipid transfer in cells is by the vesicular transport pathways that connect most cellular organelles. Finally, lipids may be transported between organelles via transient contacts between the membranes of the two organelles.¹⁹

The word sorting is used to indicate the process by which the cell generates the differences in protein and lipid composition between two membranes, starting from a membrane where these components were mixed. Because the intracellular traffic is practically a closed system for membrane components, the term generates is equivalent to the term maintains. Where transport between membranes occurs by aqueous diffusion of a certain component as monomers, sorting requires a different affinity of this component for the two membranes. This may concern the affinity of the component for other membrane components, or, theoretically, the aqueous diffusion could be made unidirectional by transfer proteins.²⁰ In vesicular transport pathways, bidirectional transport of vesicles of random composition would result in mixing of all components and dissipation of differences between the two compartments. In this case sorting requires preferential inclusion of a specific component in the budding vesicle in at least one of the two compartments. This means lateral concentration of this component and locating the site of higher concentration to the site of vesicle budding.

The rate of monomeric diffusion of a sphingolipid between two membranes strongly depends on its physical structure. The smaller the hydrophobic part, and the larger or more polar the hydrophilic part, the higher the rate of exchange. Sphingoid bases and their phosphorylated derivatives can therefore be expected to readily equilibrate between membranes with half times of seconds, whereas ceramide is on the other end of the spectrum. For SM and glycosphingolipids, the rate of spontaneous exchange is very low with half times of days, even when they are present as monomers in a liquid-disordered membrane (39, 375). Over the last decade, many studies have utilized analogs of SM or GlcCer carrying a shortened radiolabeled or fluorescent fatty acyl chain. In contrast to the natural lipids, these lipids display greatly enhanced rates of intermembrane exchange, and interpretations of result