The Organizing Potential of Sphingolipids in Intracellular Membrane ...

PHYSIOLOGICAL REVIEWS 

Vol. 81, No. 4, October 2001 

Printed in U.S.A. 

The Organizing Potential of Sphingolipids 

in Intracellular Membrane Transport 

JOOST C. M. HOLTHUIS, THOMAS POMORSKI, RENÉ J. RAGGERS, HEIN SPRONG, 

AND GERRIT VAN MEER 

Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam; 

and Department of Cell Biology, Utrecht University School of Medicine, Utrecht, The Netherlands 

I. Introduction 1690 

II. Why Are Sphingolipids Essential for Eukaryotic Life? 1691 

A. Sphingolipid requirements at the cellular level 1691 

B. Sphingolipid requirements at the organismal level 1693 

C. Do sphingolipids exert a vital signaling function? 1694 

D. Sphingolipids and the spatial organization of cells 1694 

III. Sphingolipid Structure and Biophysical Properties 1695 

A. A concise inventory of sphingolipid structure in distinct eukaryotic life forms 1695 

B. Biophysical differences between sphingolipids and glycerolipids 1696 

IV. Sphingolipids and the Lateral Organization of Biological Membranes 1696 

A. Ordered sphingolipid domains in model membranes 1696 

B. Evidence for the existence of ordered sphingolipid domains in cellular membranes 1697 

V. Sphingolipid Assembly and Transport 1701 

A. Enzymes of sphingolipid metabolism and their topology 1701 

B. Subcellular distribution and topology of sphingolipids 1703 

C. Sphingolipid transport and sorting 1704 

VI. Sphingolipids and the Creation of Selectivity in Intracellular Membrane Transport 1706 

A. The Golgi: a central sorting device on the exocytic and endocytic pathways 1706 

B. A maturing view on Golgi organization 1707 

C. Sphingolipids: integral parts of the Golgi-based sorting machinery? 1709 

VII. Concluding Remarks 1712 

A. Sphingolipids and Golgi maturation 1712 

B. Domains of saturated lipids on the cytosolic surface 1713 

C. Sphingolipid domains as a general theme in cellular membrane traffic 1713 

Holthuis, Joost C. M., Thomas Pomorski, René J. Raggers, Hein Sprong, and Gerrit van Meer. The 

Organizing Potential of Sphingolipids in Intracellular Membrane Transport. Physiol Rev 81: 1689–1723, 2001.— 

Eukaryotes are characterized by endomembranes that are connected by vesicular transport along secretory and 

endocytic pathways. The compositional differences between the various cellular membranes are maintained by 

sorting events, and it has long been believed that sorting is based solely on protein-protein interactions. However, 

the central sorting station along the secretory pathway is the Golgi apparatus, and this is the site of synthesis of the 

sphingolipids. Sphingolipids are essential for eukaryotic life, and this review ascribes the sorting power of the Golgi 

to its capability to act as a distillation apparatus for sphingolipids and cholesterol. As Golgi cisternae mature, 

ongoing sphingolipid synthesis attracts endoplasmic reticulum-derived cholesterol and drives a fluid-fluid lipid phase 

separation that segregates sphingolipids and sterols from unsaturated glycerolipids into lateral domains. While 

sphingolipid domains move forward, unsaturated glycerolipids are retrieved by recycling vesicles budding from the 

sphingolipid-poor environment. We hypothesize that by this mechanism, the composition of the sphingolipid 

domains, and the surrounding membrane changes along the cis-trans axis. At the same time the membrane thickens. 

These features are recognized by a number of membrane proteins that as a consequence of partitioning between 

domain and environment follow the domains but can enter recycling vesicles at any stage of the pathway. The 

interplay between protein- and lipid-mediated sorting is discussed. 

www.prv.org 0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society 

1689

1690 HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER 

I. INTRODUCTION 

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. 

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). 

Physiol Rev • VOL 81 • OCTOBER 2001 • www.prv.org

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 

1 It should be noted that the name raft implies that this would be 

a small domain floating in a sea of other lipids. However, in the many 

cases where the relative sizes of the sphingolipid-rich and glycerophospholipid-rich 

environments are not known the term domain would be 

less suggestive. 

ORGANIZING POTENTIAL OF SPHINGOLIPIDS 1691 

Physiol Rev • VOL 81 • OCTOBER 2001 • www.prv.org 

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? 

A. Sphingolipid Requirements at the Cellular Level 

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 

2 Glycosphingolipid designation is according to the recommendations 

of the IUPAC-IUB Joint Commission on Biochemical Nomenclature 

(53), using the Svennerholm abbreviations for gangliosides and 

using sulfatide to indicate HSO 3 -3 Gal 1–1 Cer.


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, 


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 C 4-hydroxylation 

occurs on the free sphingoid 

base (sphinganine) or on ceramide. 

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). 

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 sur-

vival, we will first turn our attention to what is known 

about the sphingolipid requirements of complex multicellular 

organisms. 

B. Sphingolipid Requirements at the 

Organismal Level 

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 (HSO 3 -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 knockout 

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 



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 (Ga 2Cer), 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. 

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). 

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).


C. Do Sphingolipids Exert a Vital 

Signaling Function? 

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. 

D. Sphingolipids and the Spatial Organization 

of Cells 

Clearly, sphingolipids are not just a reservoir of signaling 

molecules; they also contribute to vital properties 

of cellular membranes. Studies of their physical behavior 

(see sect. III) have provided thorough insights in the basis 

of how sphingolipids and cholesterol induce lateral segregation 

of membrane components (see sect. IV). However, 

to understand the functional implications, we will 

have to define the consequences of this lateral organization 

for activities in and on the membrane. With what 

other molecules do sphingolipids interact, and for what 

processes are these interactions relevant? If we want to 

learn how sphingolipid-mediated processes are integrated 

in the physiology of the cell, we will also need to know 

how these processes are regulated at the level of the 

sphingolipids. What rules govern their interactions at the 

biophysical level, and what determines their concentration 

in the various cellular membranes in time? First 

insights have been obtained from the localization of the 

subcellular sites of sphingolipid synthesis and hydrolysis, 

and from studying their mechanisms of transport (see 

sect. V). Because metabolism and transport are mediated 

by enzymes and transporters, regulation of these processes 

must be exerted at the level of the proteins and the 

genes by which they are encoded. The available data 

suggest a pivotal role for sphingolipids in the operation of 

the Golgi complex, the central sorting station in the delivery 

of cargo, and membrane components to their 

proper destinations (see sect. VI). So far, it was believed 

that sorting processes were governed exclusively by information 

in the molecular structure of proteins. We now 

start to realize that sphingolipids produced in the Golgi

may well form indispensable parts of the organelle’s sorting 

machinery. Our challenge is to find out how the machine 

works. 

III. SPHINGOLIPID STRUCTURE AND 

BIOPHYSICAL PROPERTIES 

A. A Concise Inventory of Sphingolipid Structure 

in Distinct Eukaryotic Life Forms 

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 wildtype 

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 muta- 

3 Skin contains sphingolipids carrying very-long-chain (C28-C34) 

fatty acids that are hydroxylated at the -position. After having been 

esterified to linoleic acid and hydrolyzed, the -hydroxyl serves to 

couple the sphingolipid, first glucosylceramide and later ceramide, to 

proteins on the cell surface (79, 412) where they are essential for the 

permeability barrier of the skin (17).


tion, 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? 

B. Biophysical Differences Between Sphingolipids 

and Glycerolipids 

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 nm 2 (212), versus 

0.63 nm 2 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 

4 The distance between the head groups in a double layer of C18:0 

SM in the presence of cholesterol was reported to be 46–48 Å (232, 300), 

whereas this was 40 Å for C16:0-C18:1 PC in the presence and 35 Å in the 

absence of cholesterol (262, 300). 

SM, has been observed in many biophysical studies (23). 5 

Interestingly, the surface areas of disaturated PC (0.41– 

0.44 nm 2 ; Refs. 71, 212) and PS (0.40–0.44 nm 2 ; 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 

A. Ordered Sphingolipid Domains 

in Model Membranes 


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 

5 The cis double bond in C24:1, which in its 2-hydroxylated form or 

its nonhydroxylated form is found in a significant fraction (up to 40%) of 

the sphingolipids in mammalian myelin (89) and intestine (28), is located 

at C15, deep in the bilayer, where it does not disrupt the close packing 

arrangement of the sphingolipids (204) and the sphingolipid-cholesterol 

interaction (296). This contrasts with the C9 cis double bond in the C2 

fatty acids of the glycerolipids.

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 liquiddisordered 

(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 longrange 

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. 

B. Evidence for the Existence of Ordered 

Sphingolipid Domains in Cellular Membranes 

An overwhelming number of studies in artificial bilayers 

have demonstrated that lipids have a strong selforganizing 

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 

6 Phase separation will only occur above a critical concentration of 

a lipid in the membrane. In analogy to the aggregation of detergent into 

micelles above the critical micellar concentration (CMC), the concentration 

above which a lipid aggregates in an environment of other lipids 

may be called the critical domain concentration (CDC). Just as the CMC 

depends on the molecular characteristics of the detergent, the CDC 

depends on the molecular characteristics of the aggregating lipid. The 

CDC also depends on the lipid matrix within which the aggregate forms. 

Furthermore, it depends on the presence of similarly aggregating lipids, 

just like detergents in water coaggregate with other detergents.


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? 

1. Early evidence for lipid lateral heterogeneity 

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). 

2. From macrodomain in epithelial plasma membrane 

to microdomain in the trans-Golgi network 

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 lipidphase 

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). 

3. Resistance to detergent extraction 


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 mem- 

7 Although GM1 was found to be exclusively localized in axons of 

hippocampal neurons in culture (188), this was not confirmed in independent 

studies in the same system (335). Also, the presence of a lipid 

diffusion barrier between axon and dendrites (165) has been contested 

(99). 

8 With an SM content of 8–12% in the total Golgi (90, 160) and with 

all SM in the luminal leaflet, 16–24% of that surface might be covered by 

a sphingolipid microdomain. The expected enrichment of SM in the TGN 

to plasma membrane levels (16–19%), the presence of cholesterol, and 

participation of disaturated PC (9% of total Golgi lipids; Ref. 160) increases 

the relative area of the TGN luminal surface covered by the 

microdomain to numbers above 40%. In this sense, the term microdomain 

is misleading. 

9 A density of 1.081 g/ml equals 20% sucrose (wt/wt), 21.6% sucrose 

(wt/vol), or 0.63 M sucrose.

anes; 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 l 0 liquid-ordered 

phase before the addition of the detergent (1), supporting 

the idea that DRMs are derived from preexisting l 0 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 

10 The term DRM describes the remnant left after detergent extraction. 

Formally, this term cannot be used to describe a domain that 

existed in the original membrane before detergent addition, since this 

would assume that detergent has no effect on the properties of the 

domain. The latter must be proven for each property studied. 

11 Although also hemagglutinin became detergent insoluble after 

20 min (344), this may have a different reason. The insolubility of 

hemagglutinin requires its triple palmitoylation (sites), and the organelle 

where palmitoylation takes place has not been established (237). So 

insolubility 20 min after synthesis might be due either to contact with 

sphingolipids in the Golgi or to palmitoylation in the Golgi. 

12 However, 60–80% of the GalCer and cholesterol in myelin were 

resistant to each detergent, showing that at least part of the GalCer was 

resistant to both detergents. One possible explanation is that there were 

three different domains: one domain containing TX-100-resistant proteins, 

one containing CHAPS-resistant proteins, and a third domain 

resistant to both detergents and containing some 40% of the GalCer. 

Alternatively, there was only one type of domain, but TX-100 and CHAPS 

selectively extracted certain proteins. 



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, cholesterolbinding 

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. 

13 According to common cell fractionation experience, the 35%/5% 

sucrose interface should contain most of the Golgi, early endosomes, 

and plasma membrane. The fraction at the 45%/35% sucrose interface 

typically contains ER and lysosomes, while the alkaline carbonate used 

for homogenization should cause a large part of the ER to float up to the 

35%/5% interface as well. Without any determination of the flotation of 

these membranes, the use of the term purified caveolae membranes is 

optimistic, if not naive.


4. Microscopy on fixed cells 

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 

(Gb 4Cer) or Forssman glycosphingolipid (IV 3 -- 

GalNAc-Gb 4Cer) 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-biotingold 

(246). The latter studies were performed on freezesubstituted 

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 antibodygold 

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 lipidlinked 

molecules is the use of multimeric reporter ligands 

without proper controls. 

14 When sections of freeze-substituted A431 cells were labeled 

with cholera toxin B-gold, clusters were observed in uncoated invaginations 

of the plasma membrane. It seems unlikely that clustering was 

induced by the multimeric ligand as lipids most likely do not diffuse on 

the surface of the section which in this case consists of Lowicryl 

polymer. This is different in frozen sections, where during the labeling of 

the thawed sections lipids are free to diffuse in the membranes. In the 

freeze-substituted cells, GM1 was also found in small vesicles in the 

cytosol in close approximation with the patches on the plasma membrane. 

Because the vesicle profiles were not in contact with the plasma 

membrane during the labeling of the section, a higher concentration of 

GM1 in these vesicles must have been present already in the living cell. 

In the cell, the vesicle membranes are thought to be continuous with the 

plasma membrane or to be derived from the plasma membrane by 

endocytosis. In both cases, the results demonstrate that GM1 is enriched 

in subdomains of the plasma membrane of these A431 cells. 


5. Optical studies on living cells 

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 lipidlipid 

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 m (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 choles- 

15 It should be noted that each gold particle possessed many 

binding sites against the GPI protein (antibodies conjugated to gold) and 

to GM1 (cholera toxin B subunits conjugated to gold). In the case of the 

GPI protein, the number of binding sites did not affect the results. 

Concerning GM1, cholera toxin has been shown to reduce its solubility 

in detergent, which implies that binding of the pentavalent toxin changes 

the phase behavior of GM1 (117).

terol 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 m, 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 

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 4,5trans-double 

bond is introduced in the sphinganine moiety 

to form ceramide containing sphingosine as its longchain 

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). 

2. Synthesis of sphingomyelin and glycosphingolipids 

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, Glc- 

NAcß1–3: 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, wellknown 

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). 


3. Synthesis of inositol sphingolipids 

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 ERderived 

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. 

4. Sphingolipid hydrolysis and signaling 

sphingolipids 

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. Glc- 

Cer 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-1phosphate 

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). 

B. Subcellular Distribution and Topology 

of Sphingolipids 

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 gly- 

16 A similar SM hydrolysis of 50–70% was observed when sphingomyelinase 

was applied after glutaraldehyde fixation (5) or at 15°C 

(334, 389), conditions under which there is no vesicular traffic between 

the plasma membrane and the endosomes/Golgi. Still, it should be 

realized that the sphingomyelinase method is an invasive technique; it 

modifies the membrane under study and may not be free from artifacts 

(336).


cosphingolipids. 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, 

17 Because the cells had been treated with acetone and/or TX-100 

before antibody addition, it is possible that these lipids were originally 

present on the luminal side of cytosolic vesicles or vacuoles. 

18 Diffusion into the opposite leaflet of the bilayer without a 

change in the longitudinal orientation of the molecule, by which the 

molecule merely dips deeper into the membrane, is followed by movement 

back to its original position, without consequences for transport. 

Only transversal diffusion plus rotation around one of its short axes, by 

which the head group changes orientation, flips the molecule into the 


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. 

2. Monomeric transport through the cytosol 

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 phosphory- 

opposite membrane leaflet. The degree of wobble experienced by a lipid 

in a certain membrane is clearly one parameter that determines the flip 

rate. Other parameters are the size and polarity of the polar head group 

and the dielectric constant of the membrane interior. 

19 Membranes may come into close proximity, which stimulates 

monomeric exchange though the aqueous phase by enhancing the desorption 

from the membrane (treated in detail in Ref. 39). The cytosolic 

monolayers of the two organelles may transiently become continuous 

(hemi-fusion), or transient fusion between the apposed membranes may 

occur. The last possibility can be considered a special case of vesicular 

traffic. 

20 In an in vitro study in a mixture of ER and plasma membrane, 

cholesterol preferentially partitioned into the plasma membrane, which 

is thought to be due to its high affinity for the sphingolipids abundant in 

plasma membranes (408; for a review see Ref. 203). On the other hand, 

transfer proteins might pick up a lipid from one membrane and deliver 

it to another membrane after being modified by, e.g., phosphorylation at 

that target membrane. The activated protein would then leave the membrane 

empty, be inactivated at the donor membrane, and start a new 

round of delivery. No experimental support for this mechanism exists.

lated 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 results obtained with such lipids must take this 

difference in properties into account (reviewed in Ref. 

397). In cells, transfer of sphingolipids may be stimulated 

by protein factors. One protein has been identified that in 

vitro stimulates transfer of glycosphingolipids between 

membranes (198), whereas proteins that can transfer SM 

have been identified as well (73, 83). Whether these proteins 

actually function as transfer proteins in the cell 

remains to be demonstrated, as is the case for the phospholipid 

transfer proteins first identified over 30 years ago 

(388, 417). 

For monomeric transport of a sphingolipid to another 

cellular membrane to occur, the sphingolipid must 

be present on the cytosolic surface of the donor compartment. 

As discussed above, this is the case for sphingolipids 

synthesized on the cytosolic surface like the sphingoid 

bases and their phosphates. Little is known concerning 

their intracellular distribution and transport and concerning 

the site of action of the sphingoid long-chain base 

phosphates in intracellular signaling. Also, ceramide is 

synthesized on the cytosolic surface. Evidence has been 

presented to suggest that ceramide, newly synthesized in 

the ER and destined for GlcCer synthesis in the Golgi, can 

be transported from the ER to the Golgi by nonvesicular 

traffic (62, 97, 98, 169, 249). However, new evidence raises 

the alternative interpretation that some ceramide glucosyltransferase 

is located in the ER, while the pathway 

of ceramide transport from the ER to the Golgi site of SM 

synthesis is ATP and cytosol dependent, and therefore 

likely mediated by vesicles (K. Hanada, personal communication). 

Finally, also GlcCer is synthesized on a cytosolic 

surface, that of the Golgi. Indeed, GlcCer transport 

from the Golgi to the plasma membrane proceeded in the 

presence of brefeldin A when vesicular traffic was inhibited 

(406), suggesting that it may have equilibrated via the 

aqueous phase. In view of its very low rate of spontaneous 

exchange, such transport would most likely be mediated 

by a transfer protein. 

Sphingolipids could also become available on the 

cytosolic surface by a transbilayer translocation event. 

This has been suggested to occur for GalCer after synthesis 

on the luminal aspect of the ER (45). It is unclear 

whether GalCer exchanges between membranes through 

the aqueous phase. Both GlcCer and GalCer can be re- 



moved from the cytosolic surface of the Golgi to the Golgi 

lumen where they can be utilized for higher glycosphingolipid 

synthesis. This process is apparently mediated by 

a non-energy-consuming translocator (45, 183). In addition, 

GlcCer is actively translocated from the cytosolic 

surface of the plasma membrane to the outer bilayer 

leaflet by the multidrug transporter MDR1 P-glycoprotein 

(390; Raggers and van Meer, personal communication). 

Translocation across the ER membrane may not be limited 

to GalCer, since this event is probably mediated by a 

nonspecific translocator (46, 133). Although only a small 

fraction of the sphingolipids is in the ER, these molecules 

may have access to the cytosolic surface and transfer 

through the aqueous phase. It is presently unclear 

whether the cell contains translocators to flip them back 

to the noncytoplasmic surface (295). Finally, also a small 

fraction of the SM may reside in the cytosolic leaflet (see 

above). Under some conditions SM may flow into the 

cytosolic leaflet of the plasma membrane due to the activation 

of a scramblase (295, 434). It is unclear what is its 

fate: transfer to other organelles via a transfer protein in 

the cytosol (73), removal by a translocator like MDR1 

P-glycoprotein (390), or hydrolysis by a cytosolic neutral 

sphingomyelinase. In cancer cells, a remarkable increase 

in SM content has been observed in microsomes (ER) and 

mitochondria (20, 21, 142, 301). Because mitochondria are 

thought to receive lipids from the ER by a monomeric 

transfer process only, SM may have reached the mitochondria 

via translocation across the ER membrane followed 

by monomeric transport, maybe via sites of ERmitochondrial 

contact (see Ref. 66). 

3. Vesicular traffic 

The organelles along the exocytic and endocytic 

routes are connected through a series of fusion and fission 

events. Essentially, membrane vesicles bud from one 

compartment and fuse to the next, while at the same time 

there is a vesicular pathway in the opposite direction. As 

a variation on this theme, both at the level of the Golgi 

and that of the endosomes, the complete organelle may 

move forward in a maturation process, while generating 

vesicles that follow the opposite direction (discussed in 

detail in sect. VI). The rate of the process is sufficiently 

high that, if each budding vesicle would randomly reflect 

the composition of the membrane of origin, complete 

mixing of all membrane components could be expected 

on a time scale of hours. 21 Such mixing is not observed, 

and the different cellular membranes are capable of main- 

21 Calculations on fibroblasts suggested that per hour a surface 

area is endocytosed equivalent to the plasma membrane (112). An area 

equivalent to five times their surface area would pass through the 

endosomes per hour. Endocytic uptake of 50% of the plasma membrane 

surface area per hour was measured for the apical and the basolateral 

plasma membrane domain of kidney epithelial MDCKII cells (386).


taining their unique protein and lipid compositions. For 

proteins, the underlying process is clearly not ongoing 

local synthesis and hydrolysis, and this is also true for 

lipids. The enrichment of sphingolipids in the plasma 

membrane compared with the ER is not due to synthesis 

of these lipids in the plasma membrane nor to hydrolysis 

of sphingolipids in the ER. As a consequence, there must 

be specificity in vesicular transport; membrane components 

must be sorted. As defined above, this implies that 

at each budding event, specific membrane components 

must be included or excluded from the budding vesicle. In 

the exocytic pathway, the sphingolipids are preferentially 

included in membranes that travel in the forward direction 

and/or excluded from transport in the retrograde direction. 

Because SM and all glycosphingolipids with the exception 

of GlcCer are synthesized on the luminal surface 

of the Golgi, sphingolipid domains would form in the 

luminal leaflet of the Golgi membrane (398). 22 From the 

preferential interaction between cholesterol and sphingolipids 

(see sect. IIIB), it was then suggested that sorting of 

the sphingolipids could be the driving force for sorting of 

cholesterol along the exocytic pathway (395). Obviously, 

the presence of cholesterol may be a prerequisite for 

segregation to occur. To convert the lateral segregation 

into a sorting event whereby sphingolipids and cholesterol 

are sorted to the plasma membrane, minimally one 

of the two domains must be selectively included into a 

budding vesicle. Vesicle budding along the exocytic pathway 

is mediated by protein coats on the cytosolic surface. 

Therefore, sorting via lipid domains requires the recruitment 

of a specific set of coat components to one domain. 

Because the coat proteins are recruited on the cytosolic 

surface while the domains reside on the opposite, luminal 

surface, there must be components that recognize both. 

These could be proteins that span the membrane like 

SNAREs (discussed in sect. VI). Alternatively, or in addition, 

lipid segregation may also occur in the cytosolic 

surface, and a liquid-ordered domain on the luminal surface 

may colocalize with a similar domain on the cytosolic 

surface. Evidence for such a mechanism may be found in 

the fact that at least one type of lipid on the cytosolic 

surface of the plasma membrane, phosphatidylserine 

(PS), is highly saturated compared with the same lipid 

class in the ER of rat liver (160) 23 and yeast (325). One 

22 In principle, domains of GlcCer may also exist on the cytosolic 

surface of cellular membranes. In cells that synthesize GalCer, sphingolipid 

domains may already form in the luminal leaflet of the ER, where 

GalCer is synthesized (328, 359, 360). As discussed in section IIIB, the 

possibility exists that ceramide in the ER forms a domain, especially in 

yeast with its long-chain hydroxylated ceramides (252). Because shortchain 

analogs of Cer and GalCer readily translocate across the ER 

membrane (45), domains of these lipids may be situated in either membrane 

leaflet. 

23 While of the PS in the plasma membrane, which is exclusively 

present in the cytosolic leaflet, at least 75% was found to be disaturated, 


additional indication that the lipid environment on the 

cytosolic leaflet opposed to a sphingolipid domain in the 

noncytoplasmic leaflet may be special comes from studies 

on sphingolipid domains involved in signaling in the 

plasma membrane (see sect. IVB). By various techniques, 

the coat protein caveolin and signaling proteins that are 

anchored to the cytosolic surface via saturated fatty acids 

were found to colocalize with the domains in the opposite 

noncytoplasmic leaflet (202, 237). 

VI. SPHINGOLIPIDS AND THE CREATION 

OF SELECTIVITY IN INTRACELLULAR 

MEMBRANE TRANSPORT 

A. The Golgi: a Central Sorting Device on the 

Exocytic and Endocytic Pathways 

Remarkably, sphingolipid synthesis in eukaryotic 

cells primarily takes place in the Golgi, spatially separated 

from glycerolipid and sterol production in the ER (see 

sect. VA). To better understand the biological significance 

of this arrangement, the first two parts of this section 

serve to describe the elementary features of the Golgi and 

to discuss how the organelle is organized to perform its 

vital function in the cell. 

The Golgi occupies a key position in the secretory 

membrane system of cells. Arguably its most fundamental 

task is to act as a filter between the glycerolipid-rich ER 

and sterol/sphingolipid-rich plasma membrane. A filter 

function is reflected by the organelle’s unique morphological 

appearance: a stack of flattened cisternae flanked by 

two tubular networks that serve as the polarized entry 

and exit faces (Fig. 4). The number of cisternae within the 

stack can vary from 3 to 20, depending on cell type 

(248). The multicisternal nature of the Golgi is thought to 

allow repeated opportunities for sorting out ER-based 

and Golgi-resident components from constituents to be 

delivered to the plasma membrane or elsewhere in the cell. 

The cis-Golgi network, also called ER-Golgi intermediate 

compartment, forms the site where ER-derived 

cargo is received in a process that involves the formation 

and fusion of vesicular tubular clusters. It also provides a 

major site for the budding of vesicles mediating retrograde 

transport of selected proteins and lipids from the 

Golgi back to the ER. This retrograde flow serves to 

maintain the surface area of the ER in the face of extensive 

membrane outflow into the secretory pathway (415), 

to return escaped ER resident proteins (284), and to 

recycle membrane machinery involved in ER to Golgi 

in the ER at least 50% of the PS contained two unsaturated fatty acids. 

For comparison, the major species of cellular PC contains a saturated 

C16 or C18 at the sn-1 position and an unsaturated fatty acid at the sn-2 

position of the glycerol.

transport (196, 310). As cargo moves through the stack, it 

is modified by Golgi-associated processing enzymes. 

These enzymes, which include numerous glycosidases 

and glycosyltransferases, are generally not distributed 

evenly between the cisternae, but often found in the order 

in which they act on their substrates (see sect. VA). 24 An 

advantage of this compartmental organization is that 

cargo can be exposed to an ordered array of processing 

steps, allowing the cell to generate highly complex glycoproteins 

and glycosphingolipids. Positioned at the transside 

of the Golgi stack is the TGN. Here, processed cargo 

24 It should be noted that this intra-Golgi separation is not precise 

because the enzymes are generally spread over several cisternae, displaying 

considerable overlap in their distributions (reviewed in Ref. 

109). Therefore, different Golgi cisternae have unique mixtures of enzymes 

rather than different sets of enzymes. Moreover, the distribution 

of a given enzyme can vary between different cell types. The basis for 

this plasticity is unknown but may reflect an underlying dynamic behavior 

of Golgi membranes, as discussed further below. 


FIG. 4. Model depicting how sphingolipid synthesis may contribute to the sorting power of the Golgi. As Golgi 

cisternae mature (large gray arrow), sphingolipids are synthesized and gradually accumulate by being specifically 

excluded from the tightly curved membrane in percolating COPI vesicles or tubular connections. By attracting endoplasmic 

reticulum (ER)-synthesized cholesterol, the flat sphingolipid-rich regions in the cisternal bilayer grow thicker 

(see footnote 4). Due to their short transmembrane segments, Golgi enzymes and escaped ER-resident proteins are 

excluded from the thick, sphingolipid/sterol-rich membrane regions where transport intermediates depart for the cell 

surface. Instead, they will partition preferentially into earlier cisternae. Upon thickening of the bilayer, basolateral (or 

lysosomal) membrane proteins adopt a conformation recognized by adaptor protein complexes (AP1/AP3; see footnote 

30). AP-dependent removal of basolateral (lysosomal) material represents the terminal step in the cisternal maturation 

process, leaving behind an apical membrane carrier whose content is determined by a “sorting by retention” principle 

based on coaggregative properties of apical sphingolipids and proteins. Note that although the model implies a gradual 

increase in membrane thickness across the Golgi stack, the shape of this thickness gradient is not known (see footnote 

28). See text for further details. VTC, vesicular-tubular clusters; PGC, post-Golgi containers; PM, plasma membrane. 


is sorted, packaged into distinct vesicles (or larger membrane 

carriers), and shipped to various destinations. 

These post-Golgi destinations include the cell surface 

(either the apical or basolateral surface of epithelial 

cells), secretory storage granules, and the various compartments 

of the endosomal/lysosomal system. The TGN 

not only serves as a major branching point in the secretory 

pathway but also forms the major site where the 

secretory and endocytic pathways become interconnected. 

This interconnection enables cells to balance membrane 

flow between the pathways and to maintain the proper 

composition of their surfaces and intracellular organelles. 

B. A Maturing View on Golgi Organization 

A fundamental problem currently being addressed in 

cell biology is how the Golgi is organized to perform its 

vital tasks and how its functional integrity is maintained


despite the constant flow of membrane that enters and 

leaves the organelle on both sides. Until recently, it was 

widely held that the structural elements of the Golgi, the 

cisternae, have a stable existence and that anterograde 

movement of cargo is achieved exclusively by transport 

vesicles that pinch off from one cisternae and fuse with 

the next, while Golgi resident enzymes stay put in the 

appropriate cisternae. This idea was largely based on 

results obtained with a cell-free system in which isolated 

Golgi membranes were used to measure the sequential 

processing of cargo by Golgi-associated enzymes (309, 

312). These studies offered key insights into the intra- 

Golgi transport machinery. They allowed the identification 

of a cytoplasmic coat protein complex, termed COPI, 

whose regulated assembly on Golgi membranes was 

found to promote the formation of fusion-competent vesicles 

containing secretory cargo (270, 275). 25 In the light 

of these data, it was assumed that COPI-coated vesicles 

mediate transport of secretory cargo through sequential 

Golgi compartments in a cis-to-trans (or anterograde) 

direction. 

However, this view was challenged by the discovery 

that COPI plays a crucial role in retrograde vesicular 

transport. This notion emerged when COPI subunits were 

found to bind the dilysine retrieval signal on the cytoplasmic 

tails of ER membrane proteins (64). Moreover, mutations 

in COPI blocked retrieval of these proteins from 

the Golgi complex (194). Consistent with a function of 

COPI in retrograde transport, quantitative immunoelectron 

microscopy revealed that COPI-coated tips at the 

CGN are enriched for recycling membrane proteins 

whereas secretory cargo prevailed at COPI-negative regions 

(228). The finding that COPI vesicles contain Golgi 

enzymes and serve as their transport intermediates in 

vitro (185, 197, 206) offered an alternative explanation for 

the results previously obtained with the Golgi cell-free 

transport assay; instead of moving secretory cargo forward, 

COPI may serve to retrieve transport machinery 

and Golgi enzymes to cisternae containing cargo to be 

modified. These novel data predicted a prominent role for 

COPI-mediated retrograde traffic in Golgi function and 

maintenance. Support for this prediction came when 

25 Coat proteins are believed to have a dual function in membrane 

trafficking, serving both to shape a transport vesicle and to select, by 

direct or indirect interactions, the desired set of cargo molecules. 

Among the cargo molecules recruited by these vesicle coats are components 

that specify the docking and fusion of transport vesicles with 

their appropriate target organelles. These include members of the 

SNARE family of membrane proteins (311). Coat recruitment, cargo 

selection, and the subsequent fission and fusion of transport vesicles is 

coordinated by distinct families of GTPases. Hence, coat proteins function 

together with targeting and fusion components to form so-called 

“vesicle sorting machines” by which specific types of cargo can be 

moved from one destination to the next. Cells have a variety of coat 

proteins, allowing different species of vesicles to depart from various 

subcellular locations. 


movement of green fluorescent protein (GFP)-tagged 

Golgi enzymes was followed with fluorescence video microscopy. 

In contrast to previous assumptions (265), it 

was found that Golgi enzymes are highly mobile (61), 

undergo substantial recycling within Golgi stacks (418), 

and redistribute into the ER when export from this compartment 

is blocked (60, 368, 428). 

The existence of recycling pathways both within and 

from the Golgi stack together with the unsolved problem 

of how COPI vesicles, the only type of vesicles known to 

mediate intra-Golgi transport, would contribute to vectorial 

transport of secretory cargo led to a renewed interest 

into an old idea, namely, that the Golgi is a collection of 

maturing compartments (111). 26 This “cisternal progression” 

or “maturation” model arose from previous observations 

that large cargo complexes produced by certain 

cell types (e.g., algal scales, procollagen) move through 

the Golgi stack without entering transport vesicles or 

leaving the cisternae (25, 238). According to the model, 

cisternae are continuously formed de novo by fusion of 

ER-derived vesicles (314), pass through the stack as they 

mature, and eventually disintegrate at the level of the 

TGN. While secretory cargo stays put in cisternae, Golgi 

enzymes are delivered at the appropriate time and in the 

appropriate order by recycling vesicles so that each cisterna 

matures into the next (reviewed in Refs. 4, 110, 285). 

On the basis of the currently available data, evidence 

in support of cisternal maturation cannot preclude the 

possibility of coexisting anterograde vesicular transport, 

and vice versa. In fact, there is reason to believe that both 

mechanisms operate simultaneously (286). First, procollagen 

aggregates appear to traverse the Golgi stack at a 

much slower pace (hours) than other cargo proteins such 

as VSV-G (10–20 min; Refs. 24, 25). This finding suggests 

that cisternal maturation is too slow to account for all 

anterograde cargo transport. Second, Golgi proteins involved 

in vesicle targeting, like SNAREs, are typically 

distributed over multiple cisternae across the stack (130, 

272). Because there are no known components of the 

targeting machinery that can specify a single cisterna, it is 

hard to envision how an exclusive unidirectional movement 

of COPI vesicles, whether forward or backward, can 

be achieved. On the basis of these notions, it has been 

postulated that COPI vesicles may “percolate” up and 

down the stack in a bidirectional fashion, allowing a rapid 

26 A role for COPI in bidirectional vesicular transport has been 

postulated based on the observation that pancreatic -cells contain two 

distinct populations of Golgi-associated COPI vesicles: one containing 

recycling components and the other one secretory cargo (274). However, 

because the concentration of secretory cargo found in the second 

vesicle population was comparable to that in the cisternae, it is unclear 

how these vesicles would contribute to vectorial transport through the 

stack. Moreover, the model leaves unresolved by what mechanism proteins 

bearing dilysine retrieval signals can be excluded from anterograde 

COPI vesicles while being included in retrograde ones.

equilibration of plasma membrane and soluble secretory 

cargo between adjacent cisternae (272, 286). Percolating 

vesicles (or transient tubular connections; Ref. 410) 

would give rise to a “fast track” by which cargo can flow 

across the entire Golgi stack. Larger cargo structures and 

other molecules that cannot enter COPI vesicles (or tubules) 

would then transit the stack at the slower pace of 

cisternal maturation. 

In view of these coexisting modes of transport, how 

does the Golgi ensure efficient vectorial movement of cell 

surface-destined proteins and lipids, while maintaining at 

the same time an asymmetric arrangement of its processing 

enzymes? Without a mechanism that regulates the 

partitioning of cargo molecules and Golgi residents into 

COPI vesicles, a rapid interchange would result in complete 

uniformity and undermine directionality of transport. 

In the next section, we discuss how the assembly of 

sphingolipids in Golgi cisternae and their impact on the 

lateral organization of other membrane molecules may 

provide a physical basis exploited by the Golgi to segregate 

forward-moving cargo from recycling components, 

and once this is accomplished, to organize specialized 

TGN export sites used for polarized secretion. 

C. Sphingolipids: Integral Parts of the Golgi-Based 

Sorting Machinery? 

1. Segregating forward-moving cargo from 

recycling components 

After assembly in the Golgi, sphingolipids accumulate 

in the plasma membrane (see sect. VB). Their long 

and saturated fatty acyl chains, capacity for hydrogen 

bonding, and association with sterols is thought to 

promote the compactness and thickness, and hence the 

impermeability of the cell’s outer bilayer (see sect. IIIB). 

Compared with the plasma membrane, the ER contains 

much lower concentrations of sphingolipids (396), despite 

extensive bidirectional membrane trafficking at 

the ER-Golgi interface (415). Hence, a mechanism must 

exist by which the Golgi prevents backflow of sphingolipids 

to the ER and promotes their concentration toward 

the trans-site where membrane carriers depart 

for the plasma membrane. This could in principle be 

achieved if sphingolipids were prevented from leaving 

the cisternae in which they are made. The result would 

be a gradual rise in their concentration as cisternae 

mature. Such a scenario is in line with the recent 

finding that COPI vesicles contain significantly less 

sphingolipids and cholesterol than their parental Golgi 

membranes (41). Indeed, a segregation of sphingolipids 

and cholesterol from COPI vesicles could explain why 

the interleaflet clear space of these vesicles appears 

thinner than that of the cisternal membranes from 

which they bud (273). 



How are sphingolipids and sterols segregated from 

COPI vesicles? Two distinct mechanisms can be envisaged: 

1) segregation may occur because the coat machinery 

influences the lipid composition at the bud site. 

According to this scheme, sorting could result from 

preferential interactions of certain lipids with the membrane-proximal 

surfaces of the coat proteins and/or the 

transmembrane segments of selected proteins (e.g., 

COPI coat receptors; Ref. 31). Alternatively, the COPI 

coat machinery might influence the lipid composition at 

the bud site by imposing a high curvature onto the 

vesicle membrane. This would result in a preferential 

exclusion of those lipid species that contribute to the 

rigidity of flat bilayers (e.g., sphingolipids, sterols, and 

saturated glycerolipids). In this respect, it is of interest 

to note that COPI assembly on liposomes is negatively 

influenced by acyl chain saturation. 27 2) Segregation 

may occur because the coat machinery is specifically 

recruited to lipid domains in the Golgi bilayer. 

Sphingolipids have a strong intrinsic tendency to segregate 

from unsaturated glycerolipids and display a 

high affinity for cholesterol (see sect. IV). Hence, sphingolipids 

synthesized in Golgi cisternae may trigger a 

phase separation, thus creating domains enriched in 

unsaturated glycerolipids that function as donor sites 

for COPI vesicle biogenesis versus sphingolipid- and 

sterol-enriched domains that do not. There is evidence 

that such lipid subdomains exist in early Golgi cisternae 

(108). 

Of the two lipid-sorting mechanisms described 

above, the latter has the particular appeal that it is inherently 

self-organizing, thus escaping the requirement for 

proteins that to trigger a lateral segregation of lipids need 

themselves to be retained in place. It is tempting to speculate 

that ongoing sphingolipid synthesis in the Golgi acts 

as a sink for ER-synthesized cholesterol and that a phase 

separation is nucleated once these lipids have reached a 

critical concentration in the cisternal bilayer (see sect. IV). 

Regardless of the actual mechanism of lipid segregation, 

the exclusion of sphingolipids and sterols from COPI 

vesicles would cause their progressive accumulation in 

the maturing cisternae. As a result, anterograde transport 

of sphingolipids and cholesterol would be coupled to a 

gradual depletion of unsaturated glycerolipids along the 

secretory pathway. A sphingolipid/sterol concentration 

gradient would form across the Golgi stack (26, 56, 57, 

271), causing an increase in bilayer thickness toward the 

27 In a chemically defined in vitro assay, the assembly of COPI 

coats on synthetic liposomes was found to be strongly affected by acyl 

chain saturation; diC18:1 PC/diC18:1 PE vesicles, which contain unsaturated 

fatty acyl chains, allowed coat recruitment, whereas C16:0-C18:1 

PC/C16:0-C18:1 PE vesicles, carrying one saturated and one unsaturated 

fatty acid, did not (355).


TGN (114; Fig. 4). 28 Preferential interactions between lipids 

and transmembrane proteins with the best matching hydrophobic 

length has been recognized as a potential mechanism 

for protein sorting in the Golgi (32, 255). This idea emerged 

with the observation that the transmembrane domains 

(TMDs) of Golgi-resident proteins are on average five residues 

shorter than those of plasma membrane proteins 

(32). 29 This phenomenon is well conserved among eukaryotic 

cells, from mammals (32) to yeast (195). It has been 

postulated that due to their shorter TMDs, Golgi-resident 

proteins would be excluded from the thicker sphingolipid/ 

sterol-enriched membrane regions destined for the cell surface 

and hence be retained in the Golgi (32, 255). 30 In support 

of this idea, lengthening the TMDs of several Golgi 

enzymes results in their movement to the plasma membrane 

(229, 253, 254), while shortening the TMD of a plasma membrane 

protein causes its accumulation in the Golgi (60). In 

addition, the ER residency of several membrane proteins 

can be disrupted by elongating their TMDs (282, 422). TMD 

length has also been recognized as a critical parameter in the 

sorting of SNAREs (298), membrane proteins whose specific 

interactions contribute significantly to the specificity of vesicle 

docking and fusion (236, 311). Particularly intruiging in 

this respect is the finding that the requirement of exocytic 

v-SNAREs (vesicular SNAREs) for secretion in yeast can be 

28 The shape of the thickness gradient is not known. In one extreme 

model, the phase separation between sphingolipids/cholesterol 

and unsaturated glycerophospholipids gives rise to a thick domain that 

is transported to the plasma membrane and a thin domain that is 

transported back to the ER. However, it is clear from the literature that 

glycerolipid membranes become thicker upon addition of cholesterol 

(see footnote 4). Also, it is clear that sphingolipid-rich membranes of 

different thickness can leave the TGN (like the apical and basolateral 

transport vesicles in epithelial cells). We therefore hypothesize that the 

membrane thickness increases gradually across the Golgi stack. Reasons 

for this may be the presence in cis-Golgi cisternae of sphingolipids 

at a concentration that is insufficient to cause phase separation, a 

gradient in sphingolipid synthesis, and a cholesterol gradient. However, 

a proper understanding of this phenomenon will require a quantitative 

analysis of these parameters. 

29 Previous studies had revealed that the steady-state localization 

of intra-Golgi processing enzymes is largely determined not by coatbinding 

motifs but by their TMDs. In all cases examined sofar, the TMDs 

of glycosyltransferases are sufficient to direct localization of hybrid 

proteins to the Golgi (reviewed in Ref. 109). These TMDs lack obvious 

sequence motifs that could serve as sorting determinants, and attempts 

to identify such motifs by mutagenesis have failed. 

30 It is important to note that the extent to which lipids in a bilayer 

can distort to match the length of a transmembrane -helix in a protein 

is limited. Although a short transmembrane -helix can incorporate into 

a thin bilayer, it cannot incorporate into a bilayer that is too thick, and 

instead will form aggregates (164, 190). A long transmembrane -helix, 

on the other hand, can incorporate into either a thick or a thin bilayer, 

in the latter case by tilting. Tilting of a transmembrane protein may alter 

its conformation and function. The effect of bilayer thickness on protein 

function will probably be stronger in the case of proteins containing a 

bundle of multiple transmembrane helices (e.g., an ion channel) as a 

sliding of helices within a bundle may be neccessary to achieve the most 

optimal hydrophobic match. Interestingly, a coupling between hydrophobic 

membrane thickness and protein activity has been observed for 

several transporter proteins, including ion pumps (63, 362). 


bypassed by genetic mutations in enzymes responsible for 

the elongation of the long-chain fatty acids found in ceramide 

and sphingolipids (67). 31 

Apart from being short, some Golgi TMDs display an 

unusual high content of the bulky residue phenylalanine, a 

feature that may well promote a segregation from the ordered 

sphingolipid/sterol-rich membrane regions (32). Oligomerization 

into multi-enzyme complexes (265) provides 

another factor that could influence phase behavior of Golgi 

enzymes. Collectively, the variation in such properties would 

offer considerable scope for establishing different steadystate 

distributions of enzymes within the Golgi stack. Bidirectional 

transport mediated by percolating COPI vesicles or 

transient tubular connections would allow an enzyme to 

transiently explore the entire stack until it finds a cisterna 

whose lipid composition suits its TMD (283). Ongoing sphingolipid 

synthesis and depletion of unsaturated glycerolipids 

will gradually remodel the lipid composition of the cisternal 

bilayer to that of the plasma membrane, and eventually drive 

all Golgi-resident proteins, including the sphingolipid-synthesizing 

enzymes themselves, into recycling COPI vesicles. 

Likewise, plasma membrane proteins whose rapid 

diffusion through the stack is mediated by percolating 

vesicles or tubules could achieve the desired directionality 

in transport if their membrane anchors would favor 

cisternal bilayers that have become thicker and more 

organized due to a progressive accumulation of sphingolipids 

and sterols. Indeed, GPI-anchored proteins have a 

high affinity for glycosphingolipid/cholesterol-rich domains 

(see sect. IVB), a feature thought to be required for 

their efficient transport from the Golgi to the cell surface 

(233, 345). The same has been reported for influenza virus 

hemagglutinin (see sect. IVB; Ref. 161). 

The notions that sorting in the Golgi depends on transitions 

in bilayer thickness and that these transitions are the 

result of sphingolipid-induced phase separations provide a 

compelling explanation for why structural features render 

sphingolipids essential for the viabilty of cells; they form an 

integral part of the mechanisms by which cells generate the 

compositional and functional differences between their 

plasma membrane and internal organelles. 

2. Organizing specialized export sites 

for polarized secretion 

The TGN is a major branching point in secretory 

traffic where apical and basolateral cell surface compo- 

31 Membrane fusion reconstitution experiments have revealed that 

v-SNAREs active in the lysosomal system of yeast can replace the 

exocytic v-SNAREs in a reaction that mimics fusion of secretory vesicles 

with the plasma membrane (236). With respect to the observed redundancy 

of exocytic v-SNAREs in a fatty acid elongation mutant (67), it is 

tempting to speculate that a shortening of sphingolipid-associated fatty 

acids would cause lysosomal v-SNAREs to leak into secretory vesicles 

and take over the function of the exocytic v-SNAREs.

nents are segregated and delivered via separate transport 

routes. Although previously viewed as a special feature of 

epithelial cells, it has become clear that apical and basolateral 

delivery pathways operate in both polarized and 

nonpolarized cell types (257, 425). However, only in epithelial 

cells, they would result in macroscopic domains 

laterally separated by a diffusion barrier at the tight junction. 

Recent studies have indicated that the establishment 

of cell polarity is initiated by signaling pathways that are 

responsive to cell-cell and cell-matrix contacts (423). This 

would allow a molecular definition of contacting and 

noncontacting cell surfaces (the precursors of the basolateral 

and apical membrane domains, respectively). The 

next step would then involve a localized assembly of 

cytoskeletal elements and a positioning of the TGN relative 

to the spatial cues. Finally, the cell surface polarity 

that was initially defined by the spatial cues would be 

reinforced by targeted delivery of apical and basolateral 

cell surface components that are constitutively sorted in 

the TGN. 32 

As discussed in the previous section, the enrichment 

of sphingolipids and saturated glycerolipids along the 

secretory pathway could be explained if COPI vesicles 

budding from the Golgi cisternae would select thinner, 

unsaturated lipid-enriched membrane regions. Still an additional 

level of lipid sorting must be invoked to explain 

the difference in lipid composition between the apical and 

basolateral surface of epithelial cells. The basolateral cell 

surface has a composition very similar to that of the 

plasma membrane of nonpolarized cells, but the apical 

surface is highly enriched in sphingolipids, either glycosphingolipids 

or SM (see sect. IVB; Ref. 341). It therefore 

appears that superimposed on the lateral segregation of 

sphingolipids from unsaturated glycerolipids throughout 

the Golgi stack, a second lateral segregation in the TGN 

membrane would occur. This would give rise to domains 

highly enriched in glycosphingolipids or SM and with 

targeting information for the apical surface, and domains 

with a more moderate enrichment in sphingolipids that 

would contain basolateral information (395). The segregation 

of apical from basolateral lipids could imply the 

existence of three phases in the TGN membrane. However, 

a simpler mechanism would be extension to the 

TGN of the segregation of the two fluid lipid phases 

occurring in the Golgi stack. Depletion of unsaturated 

lipids and ongoing sphingolipid synthesis would result in 

a gradual change in the composition of both subdomains, 

32 It should be noted that a fraction of the apical proteins are 

transported to the apical surface via the basolateral surface in some 

cells (187, 230), but not in other cells (413). Interestingly, the polarity of 

apical and basolateral proteins may alter with the physiological state of 

a cell (2). In retinal pigmented epithelial cells, developmental switches 

control apical and basolateral trafficking of a number of proteins (225). 



culminating in an apical domain and a basolateral domain 

at the TGN. 

It is believed that apical protein sorting relies on the 

formation of glycosphingolipid and cholesterol-enriched 

domains in the luminal leaflet of the TGN (341, 398). This 

idea is supported by the following observations: 1) GPI 

proteins use their lipid anchors as apical sorting determinants 

(34, 200), while sorting information in at least two 

apically expressed transmembrane proteins (influenza 

hemagglutinin and neuraminidase) resides in their transmembrane 

segments (175, 322); 2) influenza hemagglutinin 

and a GPI protein acquire detergent resistance in the 

Golgi during biosynthetic transport (38, 86); and 3) inhibition 

of sphingolipid synthesis or deprivation of cholesterol 

causes a reduction in detergent insolubility and randomizes 

the cell surface distribution of influenza 

hemagglutinin and a GPI protein without affecting sorting 

of basolateral proteins (161, 233). A lipid-based sorting 

mechanism similar to that reported for the apical delivery 

of proteins in epithelial cells has been suggested to mediate 

axonal delivery of proteins in neurons (80, 188, 189). 

In addition, it appears that carbohydrates in glycoproteins 

also provide apical sorting information (88). Several 

secretory and membrane proteins have been reported 

to rely on their N- orO-glycans for apical delivery (3, 115, 

321), including GPI-anchored proteins (19). The mechanism 

of carbohydrate-mediated apical sorting is unclear 

(see Ref. 306). One potential mechanism involves recognition 

of N-glycans by sorting lectins such as VIP36 (87, 

420), which is thought to partition into glycosphingolipid/ 

cholesterol-rich domains in the TGN along with other 

apical membrane proteins. In summary, protein sorting to 

the apical membrane is variable and largely depends on 

targeting signals present in the luminal domain or membrane 

anchor. 

In contrast, basolateral sorting is typically mediated 

by discrete targeting signals that are confined to the cytoplasmic 

tails of transmembrane proteins. These signals 

often consist of tyrosine- or dileucine-based amino acid 

motifs and resemble the signals that mediate endocytosis 

and protein sorting to endosomes and lysosomes. It has 

been well established that interactions between tyrosineand 

leucine-based sorting signals and AP coats are responsible 

for selective cargo uptake into TGN- and 

plasma membrane-derived clathrin-coated vesicles (239). 

Basolateral sorting is most likely based on a similar coatmediated 

mechanism. An epithelial cell type-specific isoform 

of the AP-1 coat complex, designated AP-1B, has 

recently been identified and found to play a critical role in 

the basolateral targeting of a number of membrane proteins 

(91). Still, AP-1B-independent mechanisms for basolateral 

sorting must exist as epithelial cells are capable of 

correctly delivering some basolateral membrane proteins 

in the absence of AP-1B (313). Moreover, AP-1B is not 

expressed in hepatocytes and neurons, yet these cell


types rely on basolateral-type targeting signals for transport 

to their sinusoidal and somatodendritic surfaces, 

respectively (416, 424). A novel, ubiquitously expressed 

adaptor protein complex, AP-4, localizes to the TGN (70) 

and may be involved in basolateral sorting as well, but 

this remains to be shown. 

Interestingly, there appears to be a hierarchical readout 

of basolateral and apical sorting signals. Basolateral 

sorting signals are usually dominant; influenza hemagglutinin 

equipped with a tyrosine-based sorting signal is converted 

from an apical to a basolateral protein (33). On the 

other hand, inactivation of basolateral signals does not 

always lead to apical delivery, even if the protein is glycosylated. 

It has been suggested that some basolateral 

membrane proteins are sorted by exclusion from glycosphingolipid 

rafts; such a scenario may apply to a number 

of proteins whose efficient basolateral distribution is 

achieved in the absence of an active basolateral sorting 

signal (233, 423). 

3. Apical sorting by retention? 

In contrast to the high-affinity protein-protein interactions 

involved in the formation of basolateral vesicles, 

apical sorting seems largely based on a cooperativity of 

weak interactions between lipids, proteins, and sugars 

that occur predominantly within the bilayer domain or 

lumen of the TGN. Therefore, a role for vesicular coats in 

the formation of apical transport carriers has remained 

rather elusive. The production of apical transport vesicles 

has been proposed to involve VIP21/caveolin, a membrane 

protein with high affinity for cholesterol (256). It 

was suggested that oligomerization and intercalation of 

VIP21/caveolin into the glycosphingolipid/cholesterol-enriched 

domains would cause the bilayer to bend into 

apical transport vesicles (277). However, evidence in support 

of this hypothesis remains indirect. An alternative 

possibility is that the inclusion of basolateral molecules 

into coated vesicles budding from the TGN provides the 

primary means by which apical and basolateral components 

are physically segregated into different membrane 

carriers. Interestingly, correlative light and electron microscopic 

studies have indicated that the membrane carriers 

involved in transport from the Golgi to the plasma 

membrane are not only small vesicles, but also larger 

tubulovesicular structures (136, 379), sometimes even as 

large as half a Golgi cisternea (288). It appears that these 

structures arise by a controlled drawing out and progressive 

breakdown of TGN material from the Golgi complex 

according to a mechanism that bears similarities with the 

formation and maturation of secretory storage granules in 

neuroendocrine cells (10, 288). An intruiging possibility 

would be that apical transport intermediates are formed 

by means of a progressive maturation of TGN membranes 

via AP-dependent removal of endosomal/lysosomal and 

basolateral material (Fig. 4). Such a mechanism would 

escape the requirement of a specific apical coat complex 

as it would represent a terminal step in the cisternal 

maturation process. Analogous to the mechanism of 

cargo sorting in secretory storage granules (374), sorting 

of apical cargo may rely on a “sorting by retention” principle, 

based on the coaggregative properties of the apical 

proteins and membrane lipids. To become a functional 

transport intermediate, a maturing apical container must 

recruit components of the docking and fusion machinery. 

Specific members of the SNARE family (TI-VAMP and 

syntaxin 3) have been identified on apical membrane 

carriers and were shown to play a critical role in membrane 

trafficking from the TGN to the apical cell surface 

(180). The MAL/VIP17 proteolipid may represent another 

part of the apical transport machinery (227, 291), but its 

mode of action remains to be clarified. All these components 

occurred in detergent-resistent membrane fractions 

along with protein cargo destined for the apical cell surface, 

suggesting that both apical cargo and transport machinery 

are subject to the same lipid-based sorting mechanism. 

VII. CONCLUDING REMARKS 


A. Sphingolipids and Golgi Maturation 

Above, we have worked out the concept that a separation 

between two fluid phases of lipids lies at the heart 

of the sorting potential of the Golgi complex. This phase 

separation would be based on the differential miscibility 

of sphingolipids, glycerolipids, and sterols. An essential 

feature of the model is that the Golgi membrane matures 

along the cis-trans axis, based on cisternal progression, a 

gradual change in sphingolipid composition by biosynthetic 

reactions, and continuous retrieval of the most fluid 

lipids. As a consequence, the Golgi membrane as a whole 

becomes less fluid and thickens, resulting in a sequential 

loss of Golgi-resident enzymes and transport machinery 

to recycling pathways that run from the cis-Golgi to the 

ER and from within the Golgi stack to more proximal 

cisternae. After distillation, a membrane persists that is 

highly enriched in sphingolipids, cholesterol, and selected 

proteins and forms the prototype “apical” membrane carrier. 

The ordered array of sphingolipid synthetic enzymes 

that extends from the ER to the trans-Golgi seems an 

ideal device to gradually convert the highly fluid and 

flexible ER bilayer into a rigid and robust plasma membrane. 

In fact, it may form an integral part of the mechanistic 

framework by which the compartmental organization 

of the secretory pathway is established.

B. Domains of Saturated Lipids on the 

Cytosolic Surface 

Because SM and all glycosphingolipids with the exception 

of GlcCer are synthesized on the luminal surface 

of the Golgi, sphingolipid domains would form in the 

luminal leaflet of the Golgi membrane (398). We have 

recently obtained evidence that the pool of GlcCer in the 

cytosolic leaflet is regulated by the combination of MDR1 

P-glycoprotein, a GlcCer translocator, and a nonlysosomal 

glucoceramidase (390). In addition, our studies show 

that glycosphingolipid synthesis is required for an intact 

AP-3 pathway to melanosomes (H. Sprong, P. van der 

Sluijs, and G. van Meer, personal communication). The 

data suggest that GlcCer on the cytosolic surface is involved 

in the recruitment of specific coat proteins at the 

level of the TGN. This could be by a direct interaction of 

GlcCer with a cytosolic protein (complex). Alternatively, 

GlcCer could form (or be part of) a domain on the cytosolic 

surface. 

Evidence for lipid phase separation in the cytosolic 

bilayer leaflet of Golgi membranes may be found in the 

fact that at least one type of lipid on the cytosolic surface 

of the plasma membrane is highly saturated compared 

with the same lipid class in the ER of rat liver (160) and 

yeast (325). Such a domain would mature along with the 

luminal sphingolipid/cholesterol domain. One additional 

indication that the lipid environment on the cytosolic 

leaflet opposed to a sphingolipid domain in the noncytoplasmic 

leaflet may be special comes from studies on 

sphingolipid domains involved in signaling in the plasma 

membrane (see sect. IVB). By various techniques, the coat 

protein caveolin and signaling proteins that are anchored 

to the cytosolic surface via saturated fatty acids colocalize 

with the domains in the opposite noncytoplasmic leaflet 

(202, 237). Whether the opposed domains would be 

connected via transmembrane lipid-lipid interactions (see 

sect. IIIB) or via membrane-spanning proteins is an open 

issue. 

C. Sphingolipid Domains as a General Theme 

in Cellular Membrane Traffic 

Lipid segregation does not necessarily start in the 

cis-Golgi. As discussed in section IIIB, the possibility exists 

that ceramides in the ER, especially in yeast with its 

long-chain hydroxylated ceramides, occur in domains 

(252). In cells that synthesize GalCer, this sphingolipid 

may organize in domains already in the luminal leaflet of 

the ER, where it is synthesized (328, 359, 360). Short-chain 

analogs of Cer and GalCer (45) and other (sphingo)lipids 

(133) readily translocate across the ER membrane. If true 

for natural lipids, domains of these lipids may be situated 

in either membrane leaflet. 


Moreover, the ultimate formation of an “apical” 

sphingolipid/cholesterol remnant in the TGN is a final 

stage in Golgi maturation but clearly is not a final stage in 

cellular membrane transport. Membrane recycles from 

the plasma membrane via endosomes to the TGN, and 

evidence for lipid-based sorting has been found in the 

endocytic recycling pathway (reviewed in Ref. 397). Although, 

unfortunately, essentially all evidence has been 

obtained using fluorescent analogs of membrane lipids, 

still it leads to the conclusion that certain lipidic molecules 

can be sorted in this pathway. In short, fluorescent 

sphingolipids are concentrated during the first step(s) of 

endocytosis (52, 407). In some cells, fluorescent GlcCer 

segregates from fluorescent SM during recycling from the 

plasma membrane (168, 393, 394). However, such sorting 

was not observed in different cell types (386), and it 

cannot be assumed that these lipid analogs strictly monitor 

vesicular transport processes (226). Independent evidence 

for lipid sorting via phase separation was obtained 

by studying transport of lipid probes of different chain 

length and saturation (251). The data in the latter study 

suggest that lipids with longer or more saturated chains 

are preferentially transported to late endosomes versus 

the recycling endosome. Furthermore, cholesterol had a 

dramatic effect on endocytic lipid sorting (293). Because 

endosome maturation has many parallels with the maturation 

of Golgi cisternae, a sphingolipid-driven sorting 

device may also be central to the endocytic pathway. 

Because sphingolipid synthesis is limited to the Golgi (see 

sect. VA; Ref. 391), and all components are present at the 

start of the endosomal maturation process, in the plasma 

membrane, the endosomal lipid maturation can be considered 

to be an extension of the Golgi system. 

We thank the other members of our lipid cell biology group 

for many useful discussions. In addition, we are grateful to Rick 

Brown, Ken Hanada, Richard Pagano, Rob Parton, Rick Proia, 

Peter Slotte, Pentti Somerharju, Paul van Veldhoven, and Rob 

Zwaal for discussing distinct issues in the review and for sending 

preprints of unpublished work. 

We thank the following organizations for financial support: 

the Royal Netherlands Academy of Arts and Sciences (to J. C. M. 

Holthuis), the Netherlands Foundations for Chemical Research 

and for Life Sciences with financial aid from the Netherlands 

Organization for Scientific Research (to R. J. Raggers and H. 

Sprong), and the European Molecular Biology Organization (to 

T. Pomorski). 

Address for reprint requests and other correspondence: G. 

van Meer, Dept. of Cell Biology and Histology, Academic Medical 

Center L3, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands 

(E-mail: g.vanmeer@amc.uva.nl). 

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