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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
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I. Introduction
II. Why Are Sphingolipids Essential for Eukaryotic Life?
A. Sphingolipid requirements at the cellular level
B. Sphingolipid requirements at the organismal level
C. Do sphingolipids exert a vital signaling function?
D. Sphingolipids and the spatial organization of cells
III. Sphingolipid Structure and Biophysical Properties
A. A concise inventory of sphingolipid structure in distinct eukaryotic life forms
B. Biophysical differences between sphingolipids and glycerolipids
IV. Sphingolipids and the Lateral Organization of Biological Membranes
A. Ordered sphingolipid domains in model membranes
B. Evidence for the existence of ordered sphingolipid domains in cellular membranes
V. Sphingolipid Assembly and Transport
A. Enzymes of sphingolipid metabolism and their topology
B. Subcellular distribution and topology of sphingolipids
C. Sphingolipid transport and sorting
VI. Sphingolipids and the Creation of Selectivity in Intracellular Membrane Transport
A. The Golgi: a central sorting device on the exocytic and endocytic pathways
B. A maturing view on Golgi organization
C. Sphingolipids: integral parts of the Golgi-based sorting machinery?
VII. Concluding Remarks
A. Sphingolipids and Golgi maturation
B. Domains of saturated lipids on the cytosolic surface
C. Sphingolipid domains as a general theme in cellular membrane traffic
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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.
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0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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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.
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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).
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ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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.
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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 HSO3 -3 Gal ␤1–1 Cer.
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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
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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,
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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-
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FIG. 2. Pathways of ceramide synthesis. Known metabolic intermediates, substrates, and enzymes are indicated.
Names of genes whose products are required for specific steps in yeast ceramide synthesis are shown in italics and
discussed in section VA. Note that it remains to be established whether C4-hydroxylation occurs on the free sphingoid
base (sphinganine) or on ceramide.
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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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
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FIG. 3. Major routes of sphingolipid synthesis in eukaryotic cells.
Ceramide serves as the immediate precursor for the phosphosphingolipids inositol phosphoceramide (IPC), ethanolamine phosphoceramide
(EPC), and sphingomyelin (SM), which obtain their phosphoinositol,
phosphoethanolamine, and phosphocholine headgroups by transfer
from the corresponding glycerophospholipids. In addition, ceramide can
be converted to the glycosphingolipids galactosylceramide (GalCer) and
glucosylceramide (GlcCer) by transfer of galactose or glucose from
UDP-Gal or UDP-Glc. GalCer can be further processed by the addition of
galactose (Ga2Cer), sialic acid (GM4), or sulfate (SGalCer). In contrast,
GlcCer can only be converted to LacCer, which in turn serves as the
precursor for a large number of glycosphingolipid series. The ganglioseries (GgCer) are conveniently described by the Svennerholm nomenclature (GA2, GM3, etc.). Although the term sulfatide is generally used
for SGalCer, often it is meant to include SLacCer.
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).
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Gene knock-out studies in mice are starting to reveal
the physiological significance of sphingolipids in mammals. First of all, in addition to phosphosphingolipids
being indispensable for cell proliferation (see sect. IIA),
glycosphingolipid production is essential for development
and differentiation. Mice lacking GlcCer and all complex
glycosphingolipids due to disruption of the gene encoding
ceramide:glucosyltransferase were found to be embryonically lethal at the gastrulation stage just after formation
of the primitive germ layers (421). After ectopic transplantation, glycosphingolipid-deficient embryonic stem cells
were able to differentiate into endodermal, mesodermal,
and ectodermal derivatives but failed to give rise to mature, well-differentiated tissues.
Knock-out mice have also been generated without
a functional ceramide:galactosyltransferase (27, 58),
the enzyme that synthesizes GalCer, which is the precursor of only a few other lipids like sulfatide (HSO3 -3
GalCer). These mice live, but male mice were unable to
breed, which reflects a function of galactolipids in spermatogenesis (95). In addition, such mice displayed
compromised nerve fuction (27, 58), a finding consistent with the putative insulatory function of the bulk
quantities of GalCer and sulfatide found in myelin, the
plasma membrane of oligodendrocytes, and Schwann
cells. High concentrations of GlcCer (murine) and/or
GalCer (bovine, human) and derivatives have also been
found in the apical plasma membrane domain of epithelial cells lining the gastrointestinal and part of the
urogenital tract (341). Here, the glycosphingolipids are
thought to provide mechanical stability and, especially
in the gut, protection against harmful, hydrolytic enzymes such as phospholipases.
In contrast to GlcCer and GalCer, complex glycosphingolipids are mostly present on cell surfaces in low
quantities only. Their importance for development and
differentiation is supported first of all by the observation that knock-out mice being unable to synthesize the
complex sialoglycosphingolipids. Of the double 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
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C. Do Sphingolipids Exert a Vital
Signaling Function?
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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
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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), mannosylPI, 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.
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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
Physiol Rev • VOL
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).
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The key structural features by which sphingolipids
can be distinguished from glycerolipids and sterols are
outlined in Figure 1. First of all, the backbone of sphingolipids is a sphingoid base. With respect to molecular
conformation, the sphingoid base is structurally equivalent to the glycerol and the sn-1 acyl chain in a typical
glycerolipid. Saturated sn-1 chains of 16 or 18 carbons
(C16:0, C18:0) predominate in most eukaryotic glycerolipids. The prevalent sphingoid base found in mammalian
sphingolipids is sphingosine with a chain length of 18
carbon atoms and a trans-double bond between carbons
4 and 5. However, over 60 different species of sphingoid
bases have been described with alkyl chain lengths varying from 14 to 22 carbon atoms, different degrees of
saturation [the saturated form of sphingosine is called
sphinganine (53)] and hydroxylation (154). The saturated
and 4-hydroxylated form of sphingosine is generally referred to as phytosphingosine because it is the common
sphingoid base found in sphingolipids of plants and fungi.
In vertebrates, high levels (even up to 70%) of phytosphingosine-based sphingolipids have been found in kidney and
intestine (8, 28, 113, 155, 266). Sphingolipids in insects are
based on a C14 sphingoid base rather than the C18 backbone generally found in mammals, plants, and fungi (192,
304, 414). Sphingolipids of nematodes are unusual in that
they are based entirely on a C16 sphingosine methylated
at C15 (55, 105).
The amino group at the second carbon of the sphingoid base serves to attach a fatty acid in amide linkage.
The N-acylated sphingoid base ceramide is the precursor
of all membrane sphingolipids. The trivial name of ceramides based on sphinganine is dihydroceramide. The
sphingoid C1 hydroxyl group in ceramide is used as the
attachment site for a polar head group. Based on their
head groups, sphingolipids are often grouped into phosphosphingolipids and glycosphingolipids. However, these
categories are not mutually exclusive; plants and fungi,
for example, add phosphoinositol to phytoceramide to
generate IPC, and this head group can be further decorated with one or more monosaccharides. Sphingolipids
with inositol phosphate-containing head groups are also
common in protozoans (192). The main phosphosphingolipid in mammals and nematodes, SM, carries a phosphocholine (12, 55). Mammals also produce small quantities
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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
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).
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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 (Tm) 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 (so), or lß 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.
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The interfacial region that links the nonpolar hydrocarbon region to the polar headgroup provides sphingolipids and glycerolipids with different biophysical properties. In sphingolipids, this region contains the 2-amide and
3-hydroxyl groups, often supplemented with additional
hydroxyls at the sphingoid C4 and fatty acid C2, and with
further hydroxyls on the carbohydrates. These groups can
function both as hydrogen bond donors and acceptors
and are thought to participate in extensive inter- and
intramolecular hydrogen bonding of the sphingolipids,
whereas glycerolipids have only hydrogen bond-accepting
properties in this part of the molecule (278). This property
allows sphingolipids but not glycerolipids to associate
with themselves in the plane of the membrane into a
highly flexible hydrogen-bonded network (23; see sect.
IIIB). In membranes exposed to physical and chemical
stress as in the apical membranes in kidney and intestine
and in microorganisms, the sphingolipids exhibit an increased number of hydroxyl groups. This property is believed to improve the stability and decrease the permeability of those surface membranes (278). Although this
may be generally true, under certain conditions the loss of
a specific hydroxyl was found to increase membrane stability in yeast (82).
The lipid tails of natural sphingolipids are more
tightly packed than those of the most abundant phosphoglycerolipid, monounsaturated PC. The surface area of
GlcCer and GalCer at a surface pressure of 30 mN/m,
typical of plasma membranes, is 0.40 nm2 (212), versus
0.63 nm2 for stearoyl-oleoyl PC (71). The fatty chains of
sphingolipids are therefore more extended, and consequently the thinner and taller sphingolipids form up to
30% thicker membranes than unsaturated phospholipids.4
Second, sphingolipids contact their neighbors along a
greater and flatter surface, which results in a dramatic
increase in the van der Waals attraction between neighboring sphingolipid molecules. Van der Waals interactions
have also been held responsible for the strong binding
between the sphingolipids and cholesterol with its rigid
and flat-cylindrical steroid backbone (235). A preferential
binding of cholesterol to sphingolipids, in particular to
SM, has been observed in many biophysical studies (23).5
Interestingly, the surface areas of disaturated PC (0.41–
0.44 nm2; Refs. 71, 212) and PS (0.40 – 0.44 nm2; Ref. 72)
are close to those of sphingolipids, and a preferential
interaction of cholesterol with diC16:0 PC (less than or
equal to that with SM; Refs. 181, 208) over mono-unsaturated PC has been observed (208). This illustrates the
possibility that disaturated phospholipids may resemble
sphingolipids in their hydrophobic interactions with other
membrane components. This is especially relevant for the
cholesterol-rich plasma membrane, where 40% of the PC
and 75% of the PS are disaturated, while disaturated species, sphingolipids, and cholesterol are virtually absent
from the ER (160).
The sphingoid chain extends less far into the membrane core than the N-linked fatty acid, a disparity not
found in glycerolipids (Fig. 1). It has been argued that the
difference in length between the two chains leads to
packing defects (voids) in the membrane interior and that
these defects may be complemented via interdigitation of
two sphingolipids in the opposed bilayer leaflets (23, 44,
324). This may be an example of how lipids in one bilayer
leaflet may couple to lipids in the opposite leaflet (see
sect. VIIB). Interestingly, also cholesterol molecules in the
two opposed bilayer leaflets may interact by forming
transmembrane cholesterol dimers (129, 205, 250).
Whether these transmembrane lipid interactions are of
physiological relevance depends first of all on whether
sphingolipids and cholesterol are present in both bilayer
leaflets, and second on the relative affinities involved in
transmembrane interactions and in lateral interactions of
the lipids.
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
Physiol Rev • VOL
glycerolipids into an lo 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 ld and lo 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 lo or in an ld phase. Still, individual
molecules will rapidly exchange positions, whereby the
rate of exchange, and thus lateral diffusion, will be higher
in the ld 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.
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termed the liquid-crystalline, liquid disordered (ld), or l␣
phase (173, 319). Sphingolipids (especially glycosphingolipids) display a much higher Tm 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 Tm revealed that at temperatures between the Tm of the two lipids, a cooperative
phase separation can occur, resulting in the coexistence
of gel and ld phase. The physiological significance of this
type of phase separation in eukaryotes is questionable,
mainly because membranes that contain high amounts of
lipids with Tm 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-Tm lipid (saturated-chain
lipid) and cholesterol (299). At temperatures above the Tm
of the lipid, with increasing cholesterol concentrations a
liquid-ordered (lo) phase can separate from the liquiddisordered (ld) phase. Above a threshold concentration of
cholesterol, only the lo phase exists, independent of the
temperature. For diC16:0 PC, this occurs at 30 mol%
cholesterol (319). Acyl chains of lipids in the lo phase have
properties that are intermediate between those of the gel
and ld phases; they are extended and ordered as in the gel
phase but are laterally mobile in the bilayer as in the ld
phase. Also in bilayers of more complex composition the
coexistence of lo and ld phases depends on the cholesterol
concentration (reviewed in detail by Refs. 37, 40), and
three fluid phases can coexist, of which two are lo phases
(294). The phase separation is especially pronounced
when the Tm 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 lo phase, which as a consequence would
contain more cholesterol, whereas (poly)unsaturated lipids would prefer the ld phase. Using a fluorescence
quenching assay, Ahmed et al. (1) found that cholesterol
induces the formation of a SM-enriched lo 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
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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
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
Physiol Rev • VOL
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.
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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).
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).
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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.
Physiol Rev • VOL
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.
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branes; DRMs).10 The low-density vesicles contained
some 10% of the total cellular lipids: all of the SM, 50% of
the glycosphingolipids, 25% of the cellular cholesterol, but
only 5% of the glycerolipids (38). Subsequent work on
model membranes has suggested that SM is detergent
insoluble only when it was present in an l0 liquid-ordered
phase before the addition of the detergent (1), supporting
the idea that DRMs are derived from preexisting l0 phase
domains. The power of the DRM isolation method to study
the structure and function of domains was demonstrated by
the finding in the original paper (38) that the GPI proteins
became detergent insoluble at 20 min after synthesis only,
which suggested that the proteins became insoluble upon
entering the Golgi where they are thought to come into
contact with the sphingolipids (see sect. VI).11 The method
has been applied to identify proteins involved in the transport pathway from the trans-Golgi network (TGN) to the
apical domain (180, 304). Evidence has been reported for
the coexistence in the same membrane of domains with
different (GPI) protein compositions and different physical properties (211, 307). The same was reported for
myelin where TX-100 and CHAPS yielded DRMs of different protein compositions (342).12 SM-enriched DRMs
have been isolated from nuclei, where they were assumed
to represent the inner nuclear membrane (18).
DRMs have been isolated from the plasma membrane, suggesting the existence of phase-separated sphingolipid/cholesterol domains on the cell surface where
they would compartmentalize, modulate, and integrate
signaling events by providing sites for the assembly of
components involved in signal transduction (202, 340).
Exciting new developments in the field are the regulation,
by ligand binding, of the association with the domain of
signaling receptors and of the Src family kinases that
transduce the signals into the cell. The latter kinases are
anchored to the cytoplasmic bilayer leaflet by two saturated acyl chains, whereas in contrast proteins anchored
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
5. Optical studies on living 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 (Gb4Cer) or Forssman glycosphingolipid (IV3-␣GalNAc-Gb4Cer) was followed by labeling with a dimeric
secondary antibody, tetrameric protein A, or when multimeric complexes were used of the primary or secondary
ligand to ferritin or colloidal gold (47, 96, 377). Clustering
of the ganglioside GM1 was observed when it was labeled
with pentameric cholera toxin B subunit by itself (9) or
conjugated with gold into a multimeric complex (276), or
when a biotinylated GM1 was labeled with anti-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.
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-
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.
Physiol Rev • VOL
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).
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4. Microscopy on fixed cells
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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
Physiol Rev • VOL
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
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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).
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Physiol Rev • VOL
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 inositol1-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
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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.,
Gal␣1– 4: globo- or Gb, Gal␣1–3: isoglobo or iGb, GlcNAcß1–3: lacto or Lc (Fig. 3). The ganglio series (or Gg)
is based on GalNAcß1– 4LacCer. The ganglio series also
comprises the simple gangliosides NeuAc1–3-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).
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
B. Subcellular Distribution and Topology
of Sphingolipids
A sphingolipid gradient exists along the organelles of
the secretory pathway. Although primarily assembled in
Physiol Rev • VOL
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 ⱕ20% 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).
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complex cascade of glycosidases and activator proteins
or saposins in the lysosomes, and defects in any of the
steps result in lysosomal storage diseases (171). A nonlysosomal glucocerebrosidase activity has been found
(400). It appears to be active on the cytosolic surface of a
cellular membrane, possibly the plasma membrane. GlcCer is the only glycosphingolipid that is synthesized on
the cytosolic surface of the Golgi. The enzyme may be
involved in regulating GlcCer availability on cytosolic
surfaces (R. Raggers and G. van Meer, personal communication). SM is degraded by a lysosomal acid sphingomyelinase (327) in the lysosomal lumen. Alternatively, the
acid sphingomyelinase and neutral sphingomyelinases
(51, 85, 140, 378) have been invoked as the enzymes that
generate ceramide in signal transduction on the cytosolic
surface of cellular membranes (see sect. IIC). Sphingomyelinase activity has also been reported for yeast (84) and
requires ISC1, a gene whose product is structurally related to neutral sphingomyelinases (320). The ISC1-encoded protein displays phospholipase C activity toward
SM, IPC, MIPC, and M(IP)2C, but not to PI, PC, or lyso-PC,
indicating that it functions as a inositol phosphosphingolipid-specific phospholipase C in yeast (320). Where in the
cell and on which side of the membrane Isc1p-mediated
hydrolysis of sphingolipids occurs is unclear. Whether
yeast contains additional enzymes for breaking down its
sphingolipids remains to be established. Ceramides are
broken down by a number of ceramidase activities that
are classified as acidic, neutral, or alkaline, based on their
pH optimum. Acidic ceramidase is localized in lysosomes
and its gene has been cloned from human (166). Two
alkaline ceramidases associated with the ER have been
identified in yeast (222, 223).
Although the bulk of sphingoid bases and ceramides
is incorporated into sphingolipids, cells do contain small
quantities of free sphingoid bases and ceramides, including some phosphorylated derivatives; these are newly
synthesized or derived from sphingolipid breakdown and
are generally (re)utilized for sphingolipid synthesis in ER
and Golgi. Sphingosine-1-phosphate and sphinganine-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).
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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
Physiol Rev • VOL
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.
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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).
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
Physiol Rev • VOL
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 main21
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).
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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-
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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,
Physiol Rev • VOL
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.
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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
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
1707
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.
Physiol Rev • VOL
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
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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.
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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.
Physiol Rev • VOL
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.
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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 intraGolgi 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
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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).
Physiol Rev • VOL
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).
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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.
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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).
Physiol Rev • VOL
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.
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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
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
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).
Physiol Rev • VOL
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- or O-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
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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,
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HOLTHUIS, POMORSKI, RAGGERS, SPRONG, AND VAN MEER
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
Physiol Rev • VOL
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.
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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).
ORGANIZING POTENTIAL OF SPHINGOLIPIDS
B. Domains of Saturated Lipids on the
Cytosolic Surface
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.
Physiol Rev • VOL
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: [email protected]).
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