REVIEWS
MULTIDRUG PERMEASES AND
LOW-DENSITY LIPOPROTEIN
RECEPTOR
(LDLR). A plasma-membrane
receptor found on most
mammalian cells. Responsible
for the salvaging of cholesterol
from circulation through the
endocytosis of LDL particles.
LIPOPROTEINS
Particles such as LDL and HDL,
found in the blood circulation,
which carry lipids from the liver
to peripheral tissues and back.
These particles have a
hydrophobic core containing
triglycerides and cholesterol
esters surrounded by a
phospholipid and protein coat,
composed of different
apolipoproteins.
Departments of Human
Genetics, Gene Therapy and
Molecular Medicine,
Box 1498, The Mount Sinai
School of Medicine,
1 Gustave L. Levy Place,
New York,
New York 10029, USA.
e-mail:
yiannis.ioannou@mssm.edu
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
SUBCELLULAR CHOLESTEROL
TRANSPORT
Yiannis A. Ioannou
Studies of NiemannPick C (NPC) and Tangier diseases have led to the identification of the
causative genes, NPC1 and ABCA1, respectively. Characterization of their protein products
shows that NPC1 and ABCA1 are permeases that belong to two different superfamilies of
efflux pumps, which might be important in subcellular lipid and cholesterol transport.
The discovery of the LOW-DENSITY LIPOPROTEIN RECEPTOR
(LDLR) over 20 years ago fuelled studies that resulted in
an extensive characterization of hypercholesterolaemia
and lipid transport. The intercellular transport of lipids
from their absorption at the digestive tract to their
processing and packaging by the liver and distribution
to peripheral tissues by the various LIPOPROTEIN PARTICLES
(FIG. 1) is well understood1. However, although much
is known about this process, the intracellular fate of
these molecules is only poorly understood and remains
an area of intense investigation2,3.
Cellular cholesterol homeostasis is maintained
through the orchestrated action of biosynthetic and
degradative enzymes, receptors, transcriptional regula-
tors and, presumably, subcellular transport proteins.
There are two ways in which most cells obtain choles-
terol: either by de novo synthesis using the acetyl-CoA
pathway; or by salvage through the LDLR pathway.
To maintain a balance between these two sources of
cholesterol, mammalian cells transcriptionally regu-
late specific points in each pathway and considerable
progress has been made in understanding how cells
regulate cholesterol at these key junctures of their
biosynthetic and salvage pathways. Several observa-
tions indicate the possible existence of specific sterol
transport and sorting pathways. First, cholesterol is
non-randomly distributed among the various subcel-
lular pools; second, transport of endogenously synthe-
sized cholesterol from the endoplasmic reticulum
(ER) to the plasma membrane seems to be distinct
from the movement of LDL-derived cholesterol to the
2001 Macmillan Magazines Ltd
plasma membrane; and third, single-gene defects can
disrupt normal subcellular transport. Characterization
of diseases that lead to aberrant subcellular lipid
and/or sterol transport has produced new insights into
the regulation of lipid transport and will be the main
focus of this review.
These studies have shown that multidrug-resistance
(MDR) proteins regulate cellular cholesterol and lipid
homeostasis. However, not all permeases that are dis-
cussed in this review are MDR proteins. The ATP-bind-
ing cassette (ABC) superfamily of permeases, for exam-
ple, contains both MDR and non-MDR transporters.
Whether some of these proteins show MDR activity
remains to be established.
Cholesterol biosynthesis and salvage
Biosynthesis. As mentioned above, a balance between the
de novo biosynthesis of cholesterol and its salvaging
through the LDLR pathway is maintained through the
transcriptional regulation of key points in each pathway.
The de novo pathway is regulated by tightly controlling
the enzyme that catalyses the first step in cholesterol
biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A
reductase (HMGCR)4. This committed step in choles-
terol synthesis is the reduction of HMGC to mevalonate.
The HMGCR enzyme contains eight transmembrane
domains, which lock the enzyme into the ER mem-
brane, followed by a catalytic domain, which resides in
the cytoplasm5. Studies have shown6 that the membrane
domains are responsible for the sterol-regulated degra-
dation of the enzyme; however, the mechanisms by
VOLUME 2 | SEPTEMBER 2001 | 6 5 7
REVIEWS
CLATHRIN-COATED VESICLE
Vesicles that bud off the plasma
membrane or the trans-Golgi
network. They have a
characteristic protein coat,
made up of clathrin triskelions.
658 | SEPTEMBER 2001 | VOLUME 2
B-100, 22%
LDL
2025 nm
Triglycerides 6%
Cholesterol esters 42%
Phospholipids 22%
Free cholesterol 8%
D
A-I
E
HDL
Triglycerides 5%
810 nm
Cholesterol esters 20%
C-III
A-II
Phospholipids 30%
Free cholesterol 8%
C-I C-II
Figure 1 | The structure of low- and high-density
lipoprotein particles. LDL particles are large with a cone
that is rich in cholesterol esters. Their protein coat is
composed predominantly of APOB-100. HDL particles are
similar and their protein coat contains a number of different
apolipoproteins.
which this sterol-sensing domain detects the level of
intracellular cholesterol remain obscure.
Salvage. Salvaging of extracellular cholesterol involves
receptor-mediated endocytosis of cholesterol-rich LDL
particles through the LDLRs1, an expanding gene family
that includes the LDLR-related protein 1 (LRP1), which
is responsible for binding and internalizing lipoprotein
particles rich in apolipoprotein E (APOE). On binding
of the LDL particles, the receptor is internalized, and
subsequently delivers its cargo to endosomes and lyso-
somes. After it leaves the lysosomes, cholesterol is trans-
ported to the ER and to the plasma membrane by
means of an intermediate step through the Golgi appa-
ratus. Excess free cholesterol is esterified by acyl-coen-
zyme A:cholesterol acyltransferase (ACAT)7, an enzyme
that localizes to the ER, and is stored as cytosolic
droplets of cholesterol esters.
Lipoproteins transport lipids such as fatty acids,
sterols and glycosphingolipids to cells, where their cargo
2001 Macmillan Magazines Ltd
is delivered to the endosomallysosomal system, which
eventually dismantles the particles and salvages their
core components. Numerous reviews have described the
events that take place during the formation of lipopro-
tein particles and the route taken to reach the plasma
membrane of a cell8. This review will focus on the events
that take place once the package has found its target
usually the LDLR on the plasma membrane of the
cell, and its subsequent fate.
The LDLR recognizes the protein coat of the LDL
particle, which is composed predominantly of APOB-
100. The particles contain mostly cholesterol esters and
phospholipids (~42% and ~22% of the particles dry
mass, respectively)8, in addition to the apolipoprotein
coat (~22%), and small amounts of triglycerides and free
cholesterol (~6% and ~8%, respectively) (FIG. 1). In addi-
tion, LDL particles also contain glycosphingolipids9. On
binding of the LDL particle to its receptor, the complex is
endocytosed through CLATHRIN-COATED VESICLES1. LDLRs
and their ligands are then transported to early endo-
somes, where LDL dissociates from its receptor and is
sorted to late endosomes and finally to lysosomes. LDL
particles are dismantled in the lysosome1 (FIG. 2), and very
little is known about the events that govern recovery of
the dismantled components and, in particular, the lipid
components of the particles. The LDLR is returned to
the plasma membrane from an early endosomal com-
partment to begin a new cycle of endocytosis.
Cholesterol transport
Free cellular cholesterol is found predominately at the
plasma membrane10, from where it must be shuttled to
other cellular organelles for esterification, the synthesis
of bile acids or the production of steroid hormones (FIG.
3). The mechanisms that control the sorting and move-
ment of cholesterol, whether salvaged or synthesized,
remain unresolved. Although most of the cholesterol
moves within the cell by vesicular transport, it also
seems able to move through the cytosol for example,
to reach the mitochondria and peroxisomes probably
by binding to cytosolic sterol acceptors, such as sterol
carrier proteins. Although cholesterol is moved rapidly
among the various cellular pools (FIG. 2), its asymmetric
distribution (high concentration at the plasma mem-
brane, low concentration at the ER) remains constant11.
There is evidence of distinct pathways for the intra-
cellular transport of endogenously synthesized and
exogenously salvaged cholesterol; however, it is not clear
whether they converge or remain separate during cho-
lesterol mobilization. A scheme depicting the various
routes that cholesterol can follow within the cell is
shown in FIG. 2.
Endogenously synthesized cholesterol seems to move
from the ER to the plasma membrane in a rapid, ener-
gy-requiring process12 that bypasses the Golgi13. This
transport mechanism remains functional in the pres-
ence of drugs that inhibit microtubule transport, lysoso-
mal function or protein synthesis14. Lysosomal LDL-
derived cholesterol is found quickly at the plasma
membrane, and there is evidence that it passes through
the Golgi en route15. Furthermore, it is also available for
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 REVIEWS

HDL LDL these organelles, as the NPC-disease phenotype exem-

LDL receptor
Plasma membrane
Coated pit
ABCA1 Caveolin
Early endosome
TGN
NPC1 Lysosome
NPC2
Golgi MLN64
Late endosome
ACAT
ER
Nucleus
Figure 2 | Main routes of intracellular cholesterol movement. Cholesterol is salvaged
through the low-density lipoprotein receptor (LDLR) pathway at the plasma membrane. After
endocytosis of the LDL particles in clathrin-coated pits, cholesterol is released into the
endosomallysosomal system. The products of three genes in this system NiemannPick C CAV1 is activated in response to free cholesterol26.
1 (NPC1), NPC2 and MLN64 are thought to mediate cholesterol exit from the endosomal
system and its eventual transport to the plasma membrane. Caveolins are thought to facilitate
cholesterol movement to the plasma membrane where an ABC-type plasma membrane
transporter (ABCA1) can facilitate its efflux onto high-density lipoprotein (HDL) particles. Excess
cholesterol can be transported from the plasma membrane to the endoplasmic reticulum (ER)
where acyl-coenzyme A:cholesterol acetyltransferase (ACAT) facilitates esterification of
cholesterol for storage in lipid droplets in the cytosol. There is some evidence for a minor, direct
pathway of free cholesterol from endosomes to the ER (denoted by the small arrow).
Cholesterol transport to the mitochondria or peroxisomes is not shown. (ABCA1, ATP-binding
cassette transporter; MLN64, malignancy antigen 64; TGN, trans-Golgi network.)
esterification by ACAT in the ER, without cycling
through the plasma membrane16.
Recent work indicates17 that the rapid transport of
LDL-derived cholesterol to the plasma membrane
might be independent of the NPC1 protein, which
has been implicated in facilitating cholesterol exit
GLYCOSYLPHOSPHATIDYL-
from the endosomallysosomal system. The internal-
INOSITOL (GPI)-ANCHORED
PROTEINS
ization of plasma membrane cholesterol, however,
Proteins found predominantly and its distribution to various cellular compartments,
at the plasma membrane, does depend on the function of NPC117. These results
attached to the lipid bilayer are supported by other studies18 and suggest that
through a hydrophobic anchor,
consisting of the two-fatty-acid-
excess cholesterol is better stored at the plasma mem-
chain lipid, brane rather than in the endosomallysosomal sys-
glycosylphosphatidylinositol. tem, where it could eventually cause malfunction of
plifies (see below).
Transport of LDL-derived cholesterol is impervious
to energy poisons19, further suggesting distinct path-
ways for the movement of the two sources of cellular
cholesterol. Additional evidence for two separate cho-
lesterol-transport pathways is provided by studies of
NPC disease, in which cholesterol transport from lyso-
somes to the plasma membrane is impaired, although
the transport of newly synthesized cholesterol from the
ER to the plasma membrane remains functional20.
Caveolins and transport. Several studies have implicat-
ed caveolins in the transport of intracellular choles-
terol. Caveolin 1 (CAV1) and CAV2 are found in most
cells, whereas CAV3 is specific for muscle cells.
Caveolins are membrane proteins that are found pri-
marily in invaginations of the plasma membrane, but
also within the trans-Golgi network (TGN) and exo-
cytic vesicles21. Caveolae, which are highly enriched
with caveolin, are involved in endocytosis, pinocytosis,
transcytosis and signal transduction. Caveolin has been
shown to bind specifically to membrane cholesterol22
and also to facilitate the movement of newly synthe-
sized cholesterol from the ER to the plasma
membrane23. Furthermore, the concentration of plas-
ma-membrane cholesterol seems to be crucial for the
function of caveolae, as depletion of plasma membrane
cholesterol inhibits pinocytosis24 and causes GLYCO-
SYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS to
disperse24, leading to inappropriate activation of down-
stream signalling cascades25.
Analysis of the promoter region of the CAV1 gene
reveals two STEROL REGULATORY ELEMENT (SRE)-like
sequences, and it has been shown that transcription of
Interestingly, caveolin expression has been found to be
upregulated several fold in Npc1+/ heterozygotes27 and
Npc1-deficient mice28, supporting a possible role for
caveolin in the transport of lysosomal cholesterol. A
recent study29 has shown that expression of a domi-
nant-negative mutant of caveolin causes intracellular
accumulation of free cholesterol in late endosomes
and a decrease in surface cholesterol with a concomi-
tant decrease in cholesterol efflux and synthesis. This
and other studies30 indicate that caveolins might medi-
ate intracellular lipid and cholesterol homeostasis.
Cholesterol and other lipids are asymmetrically dis-
tributed within sphingolipid-rich microdomains in
both the exoplasmic and cytoplasmic leaflets of the
plasma membrane. A raft hypothesis has been pro-
posed31, according to which sphingolipids and choles-
terol assemble within the membrane to form a plat-
form, or raft, which recruits some proteins and excludes
others. Caveolins could function as stabilizers of the raft
domains within different membranes, including the
Golgi and the plasma membranes. In addition, there
might also be two routes for membrane transport
between the Golgi and the plasma membrane: a default
bulk-flow route for proteins without sorting signals,
and a route for raft-associated proteins.

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REVIEWS

20-hydroxylase Oxysterols
25-hydroxycholesterol,
26-hydroxycholesterol,
OH 3 27-hydroxycholesterol,
HO 20,22-hydroxycholesterol,
Cholesterol 4-hydroxycholesterol,
22(R)-hydroxycholesterol,
etc.

HO
20-hydroxycholesterol
O

22(R)-hydroxylase Bile acids O

OH OH
7
HO OH

O
Chenodeoxycholate
OH

12 O
HO
CoA~SH
20,22(R)-dihydroxycholesterol OH
O
3 7
HO OH 12 S-CoA
Lyase ATP
Cholate
O
ADP 3 7
HO OH
Cholyl CoA
Taurine
O
HO OH
Pregnenolone 12 O
CoA~SH

STEROL REGULATORY ELEMENT O

A consensus sequence found in
the promoter regions of several
genes. The element is
recognized by specific
transcription factors that
stimulate transcription when
cellular sterol or fatty acid levels
are low.
HIGH-DENSITY LIPOPROTEIN
PARTICLES
(HDLs). Differ from LDLs in
the composition of their
hydrophobic core and the
apolipoprotein composition of
their coat.
ABC-TYPE TRANSPORTER
A type of transport protein that
contains a consensus sequence
known as the ATP-binding
cassette.
WALKER A AND WALKER B
MOTIFS
Protein motifs that form the
nucleotide-binding site of an
ABC domain. Walker A has the
consensus GE-VALVGPSGSG
KSTLL and Walker B the
consensus ILLLDEPTSALD.
(bold amino acids are
invariant.)
PERMEASE
A membrane transporter, also
known as a carrier protein or a
transporter.
3
HO
Deoxycholate
Steroid hormones
Progesterone, cortisol,
corticosterone, aldosterone,
testosterone and oestrogens
Figure 3 | Cholesterol products. Cholesterol is the precursor during the synthesis of bile acids, steroid hormones and
oxysterols. Various specialized tissues can use cholesterol as the building block of steroid hormones, bile acids and oxysterol
synthesis.
High-density lipoproteins. Cholesterol efflux can occur
at the plasma membrane by a process that has recently
been uncovered. HIGH-DENSITY LIPOPROTEIN PARTICLES
(HDLs) (FIG. 1) have long been known to mediate the
reverse transport of cholesterol from peripheral tissues
to the liver8. In addition, a member of the scavenger
receptor family, SR-B1, is responsible for the selective
uptake of cholesterol esters out of HDL particles and
into cells. The recent elucidation of the molecular defect
in Tangier disease (see below) has provided the missing
link in this pathway a plasma-membrane transporter
that is responsible for the transfer of phospholipids and
cholesterol onto appropriate acceptors.
Multidrug-resistance proteins
Recently, multidrug transporters particularly the
ABC TYPE have come to be appreciated for their
involvement in lipid transport32,33 (see REF. 34 for an
excellent review of the human ABC superfamily). So
far, an inventory of human ABC proteins has identi-
fied 51 proteins that belong to various subfamilies35.
OH
12 NH
3 7
O S
HO OH
HO O
Taurocholate
These proteins are identified by the presence of the
ABC unit (~200250 amino acids), which contains the
WALKER A AND WALKER B MOTIFS.
On the basis of their topology and their number of
transmembrane domains, a scheme has been proposed
that places the ABC proteins into seven subfamilies35:
ATP-binding cassette 1 (ABC1); multidrug-
resistance/transporter associated with antigen process-
ing (MDR/TAP); multidrug resistance-associated pro-
tein/cystic fibrosis transmembrane conductance
regulator (MRP/CFTR); adrenoleukodystrophy (ALD);
RNase L inhibitor (OABP); GCN20 (from the yeast
Saccharomyces cerevisiae); and White (white gene of
Drosophila melanogaster). The ABC family is also found
in non-mammalian organisms. For example, characteri-
zation of open reading frames from S. cerevisiae has
revealed ~186 potential PERMEASES with several trans-
membrane domains, 2832 of which belong in the mul-
tidrug-resistance family36,37. The ubiquitous presence of
multidrug transporters is further supported by the fact
that, in Escherichia coli, there are 29 putative multidrug

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2001 Macmillan Magazines Ltd
 REVIEWS

Table 1 | Multidrug resistance proteins and associated diseases

Family Subfamily Name Chromosomal Associated disease Substrate OMIM
location
ABC A ABCA1 9q2131 Tangier disease, Cholesterol, 600004
familial HDL deficiency phospholipids
ABC A ABCA4 1p22 Stargardt disease Retinoids 601691
ABC B ABCB4 7q21 Progressive familial Phosphatidylcholine 171060
intrahepatic cholestasis
type 3
ABC B ABCB11 2q24 Progressive familial Bile acids 603201
intrahepatic cholestasis
type 2
ABC C ABCC2 10q2323 DubinJohnson syndrome Glutathione conjugates, 603201
leukotriene C4
ABC C ABCC6 16p13.1 Pseudoxanthoma elasticum ? 264800
ABC D ABCD1 Xq28 Adrenoleukodystrophy Very long chain 300100
fatty acids
ABC D ABCD2 12q11 Adrenoleukodystrophy-like Very long chain 601801
fatty acids
ABC G ABCG5 2p21 Sitosterolaemia Sterols 210250
ABC G ABCG8 2p21 Sitosterolaemia Sterols 210250
RND NPC1 18q1112 NiemannPick C1 disease Fatty acids, ? 257220
RND NPCL1 7p13 ? Fatty acids, ?
RND PTCH1 9q22.3 Various tumours ? 601309
RND PTCH2 1p32 Various tumours ? 603673

ANTIPORTS, SYMPORTS AND
UNIPORTS
Uniports transport their
substrate across a membrane.
Coupled transporters couple
the transport of their substrate
to the transfer of a second
solute, either in the same
direction (symports) or in the
opposite direction (antiports).
PROTON-MOTIVE FORCE
(PMF). The force generated
across a membrane by the
unidirectional transport of
protons across a membrane.
Both the membrane potential
and the pH gradient pH
can contribute to this force.
APOLIPOPROTEIN A-1 (APOA1)
One of the apolipoproteins
found predominantly in the
coat of HDL particles.
transporters, at least two of which are of the ABC type38.
ABC transporters are characterized by the ABC cas-
sette, which allows the protein to function by using ATP
as an energy source. A second family of multidrug
transporters, composed of ANTIPORTS, SYMPORTS AND UNI-
39
PORTS, has been divided into five superfamilies , includ-
ing the resistancenodulationdivision family of per-
meases (see below with respect to NPC1). Unlike the
ABC transporters, these permeases cannot use ATP, and
instead use a PROTON-MOTIVE FORCE (PMF) to derive energy
for transport. Although this type of transporter is com-
mon in prokaryotes, there are few examples in eukary-
otes the mouse multidrug endosomal transporter
(MTP)40, for example, and the rat and bovine
monoamine transporters VMAT1 and VMAT2, respec-
tively41. In line with their requirement for a PMF, which
is usually provided by the acidic pH of the
endosomallysosomal system (see below), the function
of these transporters is inhibited by drugs that disrupt
PMF gradients42.
Identification of a sterol-regulated ABC protein
(ABCA7)43 and elucidation of the molecular defect in
Tangier disease have fuelled interest in these types of
transport protein. As shown in TABLE 1, ABC trans-
porters are important contributors to cellular lipid
transport, and future studies should reveal other mem-
bers of this family that are involved in lipid homeosta-
sis. Furthermore, members of the PMF-dependent fam-
ily also seem to be involved in lipid homeostasis,
especially in subcellular compartments that can sustain
PMF gradients. So, multidrug-resistance proteins from
different subfamilies seem to be the gatekeepers of
intracellular sterol and lipid homeostasis.
Tangier disease
Studies of a rare autosomal-recessive condition, Tangier
disease, have led to the resolution of an important puz-
zle about lipid transport. Tangier disease and familial
HDL deficiency (FHD) are characterized by the accu-
mulation of sterol deposits in tissue macrophages and a
severe deficiency of HDL44. Patients have no APOLIPOPRO-
TEIN A-1 (APOA1) and accumulate cholesterol esters in
reticulo-endothelial cells of various tissues, including
the liver, spleen, bone marrow, tonsils, thymus and
lymph nodes44.
Both Tangier disease and FHD were recently shown
to be caused by defects in an ABC-type plasma mem-
brane transporter, ABCA14547. Furthermore, mutations
in the ABCA1 gene have been identified in patients with
FHD, indicating that FHD is a heterozygous form of
Tangier disease48,49. Expression of ABCA1 can be
induced in cultured cells by the addition of 22(R)-
hydroxycholesterol and 9-cis-retinoic acid, indicating
the possible involvement of nuclear receptors of the
liver X receptor (LXR) and retinoid X receptor (RXR)
families50. The sterol-dependent transactivation of
ABCA1 has been shown to occur through a 25-bp ele-
ment in its promoter region50. This upregulation of
ABCA1 by sterols (up to sevenfold induction) lends fur-
ther support to the idea that this transporter is responsi-
ble for sterol transport across the plasma membrane.
Recently, two new members of the ABC superfamily,
ABCG5 and ABCG8, were shown to be mutated in
patients with sitosterolaemia, an autosomal-recessive
disorder, which is characterized by increased intestinal
absorption and decreased biliary excretion of dietary
sterols including the plant sterol, sitosterol51. So, these

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REVIEWS
LIPIDOSIS
Storage of various lipids in the
lysosomal system is the
common phenotype for this
group of lysosomal storage
diseases.
HEPATOSPLENOMEGALY
An enlargement of the liver and
spleen seen in several lysosomal
storage diseases.
662 | SEPTEMBER 2001 | VOLUME 2
a directly to ABCA1 and, in addition, that the efflux of
ABCA
ABC ABC
b acterized by the accumulation of unesterified cholesterol
RND
RND signature
c TGN, and its relocation to and from the plasma mem-
NPC1
SSD
Figure 4 | Topology of ABCA and RND-type permeases.
a | The ABCA subfamily of transporters is composed of a six-
transmembrane domain plus the ABC cassette motif,
repeated twice. b | The resistancenodulationdivision
(RND)-type permeases have a 5-plus-1-transmembrane
domain, separated by a large hydrophilic loop, repeated
twice. This motif is known as the RND signature. c | The
topology of NiemannPick C 1 (NPC1) is also shown for
comparison. NPC1 contains the RND signature. The five-
transmembrane-domain motif shows homology to the sterol-
sensing domain (SSD) of HMG-CoA reductase and SCAP.
transporters are members of the expanding list of sterol
and lipid transporters (TABLE 1).
The ABCA1 protein. ABCA1 is a typical ABC trans-
porter with six transmembrane domains and a
nucleotide-binding domain, repeated twice (FIG. 4).
The nucleotide domains consist of two Walker A and
Walker B motifs52, and members of the ABCA subfam-
ily (TABLE 1) also have a hydrophobic domain that sepa-
rates the two repeat units of six transmembrane
helices. Because of its hydrophobicity, this domain
could interact with the membrane and facilitate sub-
strate transport, although the interaction remains to
be shown experimentally.
The severe impairment of reverse cholesterol trans-
port in Tangier disease has led to the hypothesis that the
causative protein is involved in this process. Recently,
ABCA1 was shown to transfer lipids to APOA1 rather
than to HDL through a direct binding of APOA1 to
ABCA1 at the plasma membrane53. This transfer
involves the simultaneous movement of phospholipids
and cholesterol from the outer leaflet of the membrane
onto APOA1. Despite their high concentration of cho-
lesterol, lipid-raft domains are not involved in the
ABCA1-mediated lipid-efflux pathway54. Interestingly, a
recent study55 has shown that cholesterol does not bind
2001 Macmillan Magazines Ltd
phospholipids by ABCA1 does not require cholesterol
and can proceed normally in its absence. The authors
conclude that the binding of APOA1 to ABCA1 leads to
the formation of apolipoproteinphospholipid com-
plexes, which in turn promote cholesterol movement55.
NiemannPick C disease
NPC disease is a rare autosomal-recessive LIPIDOSIS, char-
in lysosomes20,56. Patients show progressive neurodegen-
eration and HEPATOSPLENOMEGALY, which leads to death
during early childhood57. The most prominent bio-
chemical feature is the accumulation of LDL-derived
unesterified cholesterol in the endosomallysosomal
system56. In addition, cholesterol accumulates in the
brane is delayed57. In fibroblasts, the defect in cholesterol
exit from lysosomes is accompanied by attenuation of
the downregulation of two key components of choles-
terol homeostasis HMGCR and LDLR58.
The NPC1 gene. The gene that is mutated in most NPC
patients, NPC1, maps to chromosome 18q1112 (REF. 59)
and encodes a messenger RNA of ~4.9 kb, which is pre-
dicted to produce a protein consisting of 1,278 amino
acids. The NPC1 gene spans ~47 kb and contains 25
exons (ranging in size from 74 to 788 nucleotides) and
introns (ranging from 0.097 to 7 kb)60. More than 80
mutations have been described59,6166 in patients lacking
NPC1, including nonsense and missense mutations, and
insertions, deletions and duplications. These mutations
are spread throughout the NPC1 gene and do not indi-
cate any functionally crucial protein domains; however,
there is a small cluster of mutations in the carboxy-ter-
minal third of the protein, in a region that has cysteine
residues that are conserved between the various NPC1
orthologues63.
The effects of reduced or absent NPC1 in both
humans67,68 and animal models20,57 have been well stud-
ied, establishing this protein as an essential component
of intracellular cholesterol transport. In addition, several
mutant Chinese hamster ovary cell (CHO) lines that
show the NPC1-disease phenotype have been character-
ized69,70, and these have been shown to have a defective
NPC1 gene17. The function of the NPC1 protein is still
unknown, although recent evidence indicates that it
might be a lipid permease71.
The NPC1 protein. Analysis of the NPC1 sequence does
not reveal any significant homologies to other proteins.
However, transmembrane domains three to seven (FIG.
4) show homology to a protein called Patched (PTC).
PTC is a membrane-bound receptor for Sonic
Hedgehog (SHH)72,73, which is a developmental sig-
nalling molecule that, in its active state, contains a cova-
lently attached cholesterol moiety74,75. This sequence
similarity is also shared by the sterol-sensing domains
of HMGCR and the sterol-regulated-element cleavage-
activating protein (SCAP). In HMGCR, the sterol-sens-
ing domain is involved in enzyme degradation when
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LAMP-POSITIVE ORGANELLES
Organelles that contain the
lysosome-associated membrane
protein. Labels lysosomes.
RAB7-POSITIVE
Organelles that contain Rab7, a
small GTPase found
predominantly in late
endosomes.
BODIPY
Trade name for a family of
fluorophores that span the
visible spectrum, and are used
to label proteins, nucleotides,
lipids and other molecules.
PERINUCLEAR VESICLES
Vesicular structures that are
seen surrounding the nucleus.
Usually indicative of lysosomes.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
High sterols NH2
COOH COOH
NH2 SCAP
SREBP
WD domains
ER
Low sterols
HOOC NH2
NH2
COOH
ER
NH2
HOOC
COOH Nucleus
NH2
S2P
S1P
Golgi
Figure 5 | Sterol-regulatory-element-binding proteins.
SREBPs are sterol- or fatty-acid-regulated transcription
factors that reside in the membrane of the endoplasmic
reticulum (ER). There are two SREBP genes, SREPB1 and
SREBP2. SREBP2 produces the factor SREBP2, the
maturation of which is repressed by sterols. SREBP1
encodes two proteins, SREBP1a and SREBP1c. Their
maturation is repressed by polyunsaturated fatty acids. In the
absence of sterols or fatty acids, SREBPs interact with
another ER membrane protein, the SREBP-cleavage-
activating protein (SCAP). The complex subsequently moves
to the Golgi where two proteases, S1P and S2P, release the
active nuclear form of SREBPs. Activated SREBPs enter the
nucleus and turn on the expression of genes that contain
SRE elements in their promoters, such as the low-density
lipoprotein receptor (LDLR), HMG-CoA synthase, squalene
synthase and fatty acid synthase.
the cell senses adequate levels of cholesterol, whereas
in SCAP, it might function during the activation of the
sterol-regulatory-element-binding proteins (SREBPs),
a family of transcription factors that regulate several
crucial enzymes in the salvage and de novo cholesterol
synthesis pathways76 (FIG. 5). In addition, a human gene
homologous to NPC1, NiemannPick C1-like 1
(NPC1L1)77, has also been found to contain a sterol-
sensing domain.
NPC1 is a membrane glycoprotein that localizes to
LAMP-POSITIVE ORGANELLES, presumably endosomes and
lysosomes7880. Further studies have shown that NPC1
2001 Macmillan Magazines Ltd
REVIEWS
resides primarily in RAB7-POSITIVE late endosomes and
only secondarily in lysosomes and the TGN80. This is an
important distinction in view of data81 indicating that
cholesterol accumulation in NPC1/ cells might occur
primarily in late endosomes, which are sorting sites for
various cellular components. In addition, NPC1/ cells
cannot efflux endocytosed sucrose or sort the mannose
6-phosphate receptor, indicating that the retrograde
movement of proteins and cargo from late endosomes
to the TGN might be perturbed78,81.
Further characterization of the effects of absent or
mutated NPC1 protein indicate that NPC1/ cells
might have a generalized block in lipid recycling from
late endosomes to the Golgi and plasma membrane. A
role for cholesterol and its potential function in modu-
lating lipid transport has been proposed82. In these
studies, BODIPY-labelled lactosyl ceramide (BLC; a gly-
cosphingolipid) was used to probe the distribution of
the glycosphingolipid in normal and NPC1/ cells after
endocytosis from the plasma membrane, and was
found to localize predominantly in the Golgi apparatus
in normal cells82. However, in cells from patients with
sphingolipid storage disorders, including those with
NPC1 disease, BLC is found in PERINUCLEAR VESICLES that
are characteristic of endosomes and lysosomes82.
When normal cells are grown in the presence of high
levels of cholesterol (to induce an accumulation of cho-
lesterol in the endosomallysosomal system), BLC is
found in perinuclear vesicles, similar to those seen in
NPC1/ cells, indicating that cholesterol might modu-
late the movement of other lipids within the cell82.
Subsequent studies have implicated NPC1 in regulating
the movement of various lipids late in the endocytic
pathway, presumably from late endosomes8385. Studies
of NPC1 overexpression in CHO cells86 indicate that
ectopic expression of NPC1 (about 15-fold above
endogenous levels) results in an increase in the trans-
port of LDLcholesterol to the plasma membrane,
lending further support to the idea that NPC1 is
involved in subcellular lipid transport.
Analysis of its topological arrangement within
membranes indicates that NPC1 contains 13 trans-
membrane domains (FIG. 4), three large lumenal
hydrophilic loops and a cytoplasmic tail77. Within the
cytoplasmic tail is a dileucine motif that has been
shown to direct the delivery of other membrane pro-
teins to the endosomallysosomal system87. Several gly-
cosylation consensus sequences are scattered through-
out the protein, most of which seem to be used in vivo77.
Interestingly, several unique consensus sequences for
a prokaryotic lipoprotein-attachment site, usually
found in prokaryotic multi-transmembrane proteins,
are found within the NPC1 protein. With the exception
of one site at amino-acid position 904, the sites are all
found within the predicted transmembrane domains 2,
4, 10 and 11. The existence of these sites and their
potential use in vivo might explain some of the prob-
lems encountered in the attempts to solubilize this large
glycoprotein. More importantly, however, these sites are
found in prokaryotic permeases of the RND family.
Further characterization of this relationship has
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MANNOSE 6-PHOSPHATE
MODIFICATION
A phosphate modification of
the carbohydrate moieties of
proteins destined for the
endosomallysosomal system.
This modification is recognized
by the mannose 6-phosphate
receptor in the trans-Golgi
network, which captures these
proteins and transports them to
late endosomes.
MLN64
Acceptor
Acceptor
shown71 that NPC1 and members of the RND family
share the same RND signature: six transmembrane
domains, separated by a large hydrophilic loop between
transmembrane domains 1 and 2. This domain is
repeated twice (FIG. 4).
Expression of human NPC1 in E. coli has shown that
NPC1 functions as a multidrug permease, similarly to
its prokaryotic relatives88, positing NPC1 as the first
mammalian member of this ancient family71. Transport
studies in E. coli that express NPC1 show that NPC1 can
transport fatty acids efficiently, but not cholesterol or
cholesterol esters. The lack of detectable NPC1 choles-
terol-transport activity in E. coli might be due to the
lack of necessary accessory proteins or appropriate cho-
lesterol acceptors in the prokaryotic membrane, or it
might simply reflect the fact that NPC1 does not trans-
port cholesterol. In fact, on the basis of data from the
Tangier-disease transporter ABCA1, NPC1 might trans-
port fatty acids or phospholipids as its primary sub-
strates and only indirectly facilitate cholesterol move-
ment55. Alternatively, NPC1 might transport a group of
lipids, such as sphingolipids and gangliosides, en masse.
A hypothetical model for the function of NPC1 is
shown online in animation 1.
New cholesterol-transport candidates
In addition to NPC1 and ABC-type transporters, there
Acid lipase
NPC2
NPC1
are other proteins that potentially regulate or contribute
to subcellular lipid or sterol movement.
NPC2. Mutations in a gene encoding a small soluble
protein, originally identified as an important secreted
protein from human epididymis (HE1)89, were found
to be responsible for the second complementation
group of NPC disease, NPC2, which is responsible for
~5% of patients90. After its identification as an epi-
didymis-secreted protein, the pig homologue of HE1
was shown to bind cholesterol specifically91. The HE1 or
NPC2 gene is located on 14q24.3 and contains five
exons90. Contrary to its original characterization, NPC2
(HE1) is expressed in all tissues analysed. The protein
receives the classic MANNOSE 6-PHOSPHATE MODIFICATION for
soluble lysosomal proteins, and can reach the lysosome
even when added exogenously onto cells in culture.
Subcellular fractionation studies have confirmed that
this small ~18-kDa soluble glycoprotein resides in the
lysosome lumen90.
Similarly to the case in NPC1 disease, patients with
NPC2 disease are characterized by an accumulation of
free cholesterol in their endosomal lysosomal system,
indicating that NPC2 is involved in the exit of choles-
terol and/or other lipids from the endosomal or lysoso-
mal membranes. However, it is difficult to imagine how
this small, cholesterol-binding protein is involved in this
pathway. Furthermore, as NPC1 resides predominantly
in late endosomes80, where cholesterol is found to accu-
mulate81, how can a protein that is resident in the lyso-
some90 be involved in cholesterol exit from the endoso-
mal membrane?
Their locations indicate that NPC1 and NPC2 might
not interact directly, but that they function at two steps
of the same pathway. A model could be proposed in
which NPC2 acts as a bridge to allow free cholesterol,
released from its fatty-acid moiety by the action of lyso-
somal acid lipase, to insert into the membrane of the
organelle (FIG. 6). In the absence of NPC2, free choles-
terol might form structures that are refractive to such
incorporation into the membrane or, alternatively, it
might crystallize.
It is well accepted that free cholesterol readily crystal-
lizes at physiological temperatures92. In fact, cholesterol
crystallization in lysosomes has been reported after
uptake of cholesteryl-ester lipid droplets93, apparently
due to bombardment of the system with free cholesterol
after hydrolysis of the cholesterol esters by acid lipase.
Similar crystals have been found in the lysosomes of
NPC-deficient mice94. So, the function of NPC2 might
be to facilitate cholesterol insertion into the endosomal
or lysosomal membrane or, alternatively, to keep free
cholesterol soluble until such insertion occurs (FIG. 6).

MLN64. Another potential regulator of subcellular lipid

Figure 6 | Cholesterol movement facilitators. NPC1, MLN64 and NPC2 reside in the
endosomallysosomal system. The proposed function of each is described in the text. NPC1
may function as a permease to allow phospholipids and cholesterol to exit the endosomal
system. This activity may depend on NPC2s activity, which may act as a chaperone for sterol
insertion into the endosomal/lysosomal membrane. MLN64 could act independently of NPC1
and facilitate the shuttling of cholesterol between the endosomal membrane and an acceptor.
(MLN64, malignancy antigen 64; NPC1 and 2, NiemannPick disease 1 and 2.)
movement is MLN64. This 50-kDa protein was initially
identified as an upregulated transcript in malignant
(MLN) cells, and it maps to chromosome 17q1221
(REF. 95). The MLN64 protein contains a domain that is
homologous to the steroidogenic acute-regulatory pro-
tein (STAR)96,97, which regulates cholesterol uptake by

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2001 Macmillan Magazines Ltd

YINYANG-1-BINDING SITE
A consensus sequence found in
the promoter region of several
genes. In the context of a sterol
regulatory element, it acts as a
negative regulator of
transcription.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
the mitochondria by facilitating cholesterol transfer
from the outer to the inner mitochondrial membrane
and, ultimately, the production of steroids in the adren-
al glands and gonads. In addition to MLN64 and STAR,
several other proteins contain the START (STAR-related
lipid transfer) domain, including the signal-transducing
protein p122-RhoGAP and the phosphatidylcholine
transfer protein.
The crystal structure of STAR shows that the START
domain binds a single cholesterol molecule98. It has
been proposed that STAR functions by shuttling choles-
terol through the intermembrane space of the mito-
chondria, one molecule at a time98. These observations
can be extended to MLN64, which is predicted to con-
tain four transmembrane domains at its amino termi-
nus and a carboxy-terminal START domain that is
completely cytosolic99.
MLN64 has been colocalized with NPC1 in the
membrane of the late endosome99, indicating that it
might have a role in cholesterol egress together with
NPC1. However, it is not clear how MLN64 would fit
into this pathway; it is puzzling that the START domain,
which binds cholesterol, is predicted to be located in the
cytosol. It is tempting to speculate that, similar to the
mitochondrial STAR protein, MLN64 facilitates the
movement of cholesterol between two membranes, pre-
sumably between two vesicles. But what, then, is the
function of NPC1? It seems more plausible that MLN64
might facilitate cholesterol efflux from late endosomes
independently of NPC1, which might be involved in
more generalized lipid efflux (see animation 1 online).
Alternatively, MLN64-derived cholesterol might be
directed to a different cellular compartment than NPC1-
derived cholesterol. Finally, no patients with cholesterol
accumulation in their endosomal system have been
identified who have mutations in their MLN64 gene.
NPC1L1. The strong sequence and structural homolo-
gies of NPC1L1 and NPC1 indicate that NPC1L1 might
have a similar role to NPC1. NPC1L1 was identified on
the basis of its homology to NPC1, and it maps77 to the
short arm of chromosome 7 at 7p13. The predicted
protein shares 42% identity and 51% similarity to
NPC1. Unlike NPC1, however, NPC1L1 localizes to the
TGN, and NPC1L1 might have a similar function to
NPC1 at this location. In contrast to NPC1, the pro-
moter region of NPC1L1 contains both sterol-regulated
elements and YINYANG-1-BINDING SITES100102, indicating that
its expression might be sterol or lipid regulated77.
NPC1L1 also contains a sterol-sensing domain, further
supporting the idea that it is involved in sterol or lipid
transport. Further insights into the potential function
of NPC1L1 must await the generation of a mouse
knockout for Npc1l1, which is now in progress (Y.A.I.
and J. P. Davies, unpublished data).
Ubiquitous mammalian permeases?
The identification of NPC1 as the first mammalian
RND-type transporter raises the question of whether
other mammalian proteins might belong to this family.
Because they require a PMF to function, other mem-
2001 Macmillan Magazines Ltd
REVIEWS
Patched
NH2
Smoothened
NH2
COOH
COOH
Hedgehog
NH2
NH2
HOOC
COOH
Gene expression
Figure 7 | The HedgehogPatchedSmoothened
pathway. Hedgehog molecules are secreted, cholesterol-
modified proteins. Target cells express the Hedgehog
receptor, a 12-transmembrane-domain protein called
Patched. In the absence of Hedgehog, Patched acts as a
repressor of the seven-transmembrane-domain protein,
Smoothened. Smoothened is a signalling molecule that can
transmit signals to turn on the expression of downstream
target genes, including Patched. When Hedgehog binds to
Patched, Smoothened is released and is allowed to induce
the expression of its target genes.
bers of this family might regulate different steps of the
endocytic or exocytic pathway, as the lumens of
organelles in this system have lower pH values than that
of the cellular cytosol. A second favourable environ-
ment for such proteins can be provided by the potential
across the inner mitochondrial membrane. So, other
mammalian members of the RND family will probably
be identified and shown to function at these locations.
There are several candidate members for the mam-
malian RND family. First, the predicted topology of the
mammalian NPC1 homologue, NPC1L1, reveals an
RND signature (FIG. 4), so this protein might also func-
tion as an RND permease. Its subcellular location at the
TGN, a considerably acidic compartment (pH ~6.0),
would fit the second prerequisite for a functional RND
permease that of an available PMF.
Second, the morphogen receptor PTC also shows
strong homology to NPC1 (REF. 59). Its predicted topolo-
gy matches that of NPC1, and it also contains the RND
signature. Although this receptor has been extensively
characterized, its precise function remains elusive.
Hedgehog proteins a class of secreted proteins
including SHH, Indian hedgehog, Desert hedgehog and
Tiggy-winkle hedgehog are ligands for the receptor
and are necessary for Drosophila, mouse and human
development73,103. They are modified by the covalent
attachment of a cholesterol molecule to their carboxyl
terminus following an autoproteolytic event in the
ER104, which causes their secretion and eventual binding
to neighbouring cells through the PTC receptor.
PTC, in turn, interacts with the seven-transmem-
brane-domain protein Smoothened (SMO)105 (FIG. 7).
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REVIEWS

Although the events in this pathway have not been
completely worked out, it is clear that SHH binds to
PTC and prevents its interaction with SMO, which in
turn allows SMO to initiate a cascade of events that lead
to specific gene expression. Mutations that inactivate
PTC or activate SMO have been shown to result in
basal-cell carcinoma, medulloblastoma, rhabdomyosar-
coma and other human tumours106109.
The sterol-sensing domain of PTC has recently been
shown110,111 to mediate its vesicular transport and the
regulation of SMO, implying a relationship between
SMO and sterol regulation by PTC. Although purely
speculative, the fact that PTC contains the RND signa-
ture, and the fact that it is located within the endosomal
system, strongly implicate PTC as another eukaryotic
member of the RND family of permeases. It remains to
be established whether PTC has permease activity and
what its specific substrates might be.
Conclusions and future directions
Cholesterol and other lipids. Cholesterol has historically
been viewed as an actively transported molecule within
various cellular membranes, so has received the most
attention. However, fatty acids, glycosphingolipids and
gangliosides are also found within membranes, and
there must be mechanisms for their targeting and distri-
bution. Interestingly, data on the fate of the fatty-acid
moiety, after its release from cholesterol, through the
action of acid lipase are scarce (FIG. 6). It is accepted that
fatty acids are transported by an active mechanism, but
virtually nothing is known about the proteins involved
in this pathway112.
Most attempts to understand fatty-acid transport
have focused on plasma-membrane fatty-acid transport
proteins and small cytoplasmic fatty-acid carrier pro-
teins113116. The importance of fatty acids is just begin-
ning to be appreciated, especially in light of observa-
tions that the levels of fatty acids rather than cholesterol
regulate maturation of SREBPs in certain tissues117120.
Do multidrug proteins regulate lipid transport? There is
no question that multidrug proteins, especially the ABC
type, are involved in lipid transport and homeostasis32
(TABLE 1). The classification of NPC1 as a multidrug per-
mease adds a new family to this group of lipid-trans-
port regulators. The identification of NPC1L1 and PTC
as potential members of this family should provide new
avenues for investigation in addition to raising ques-
tions about cellular lipid homeostasis.
This review has focused mainly on the involvement
of multi-transmembrane proteins in lipid homeostasis.
The involvement of soluble proteins (with the exception
of NPC2) has not been addressed, although proteins
such as the small sterol-carrier proteins, fatty-acid-carri-
er proteins and annexins have been shown to be
involved in lipid transport. In support of this concept,
the transfer rate of cholesterol from purified lysosomal
membranes is >100 fold slower than the rate reported in
intact cells121, indicating that additional soluble factors
might be necessary for this process to occur efficiently.
From the point of view of NPC disease and the
implications of a generalized block in lipid transport, as
discussed above, the importance of understanding how
glycosphingolipids, gangliosides and fatty acids exit the
endosomallysosomal system cannot be overempha-
sized. Further support for this conclusion is provided by
studies that elegantly show the accumulation of other
lipids in NPC1/ cells in addition to cholesterol, such as
lysobisphosphatidic acid81, GM2 and GM3 ganglio-
sides122, the glycosphingolipid globotriaosylceramide123,
and even amyloid- protein124. So, NPC1 seems to be an
important regulator of transport, the absence of which
can produce pronounced cellular traffic jams125.
Conclusion. The identification of the genes causing NPC
and Tangier diseases and characterization of their pro-
tein products has created a new model of cellular lipid
homeostasis. We are just beginning to understand the
function of these and related proteins, and future studies
should unravel the complexities of subcellular lipid
movement. These proteins belong to large families of
transporters, and new members will undoubtedly be
added, as the complete genomes of various organisms,
including human, begin to be reported. The large num-
ber of these proteins and their differences in structure,
topology and substrate recognition requires the devel-
opment of new naming schemes to reflect correctly their
properties and function. An important challenge is the
development of labelled substrates and methodology to
allow for the characterization of their properties and
functions within a living cell.
Links
DATABASE LINKS LDLR | HMGCR | LRP1 | APOE | ACAT
| APOB | NPC1 | NPC | CAV1 | CAV2 | CAV3 | SR-B1 |
Tangier disease | inventory of ABC proteins | ABC1 | TAP
| CFTR | GCN20 | White | VMAT1 | VMAT2 | ABCA7 |
APOA1 | ABCA1 | LXR | RXR | ABCG5 | ABCG8 |
sitosterolaemia | PTC | SHH | NiemannPick C1-like 1 |
HE1 | NPC2 | MLN64 | STAR | Indian hedgehog | Desert
hedgehog
FURTHER INFORMATION Ioannou lab

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Acknowledgements
I apologize for the omission of many outstanding papers that could
not be cited or discussed owing to space limitations.
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