Biochimica et Biophysica Acta 1733 (2005) 29 – 52
http://www.elsevier.com/locate/bba
Review
Function of prokaryotic and eukaryotic ABC proteins in lipid transport
Antje Pohla,b, Philippe F. Devauxb, Andreas Herrmanna,*
a
b
Humboldt-University Berlin, Institute of Biology, Invalidenstr. 42, D-10115 Berlin, Germany
Institut de Biologie Physico-Chimique, UMR CNRS 7099, 13 rue Pierre et Marie Curie, 75005 Paris, France
Received 2 August 2004; received in revised form 8 November 2004; accepted 16 December 2004
Available online 31 December 2004
Abstract
ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans,
members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier
disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or
sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of
endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved
in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites.
Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides,
arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about
the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a
better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and
signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating
transbilayer lipid transport in various membranes of cells and organelles.
It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins
interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are
needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry.
D 2004 Elsevier B.V. All rights reserved.
Keywords: ABC protein superfamily; Flippase; Cholesterol; Lipid asymmetry; Lipid exposure; Molecular mechanism
1. Introduction
The ATP binding cassette (ABC) protein superfamily
comprises transporters for a whole variety of organic and
inorganic compounds. In 1992, Higgins and Gottesmann [1]
Abbreviations: ABC, ATP binding cassette; APLT, aminophospholipid
translocase; Cer, ceramide; FA, fatty acid; GlcCer, glucosylceramide; HDL,
high density lipoprotein; LTC, leukotriene C; nbd, [N-(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino]; NBD, nucleotide binding domain; PAF, platelet
activating factor; PC, phosphatidylcholine; PE, phosphatidylethanolamine;
PS, phosphatidylserine; PXE, pseudoxanthoma elasticum; SL, spin labeled;
SM, sphingomyelin; TM, TMD, transmembrane (domain); VLCFA, very
long-chain fatty acids; X-ALD, X-linked adrenoleukodystrophy
* Corresponding author. Tel.: +49 30 2093 8860; fax: +49 30 2093
8585.
E-mail addresses: [email protected] (A. Pohl)8
[email protected] (A. Herrmann).
1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2004.12.007
pointed out that ABCB1 (MDR1 Pgp) behaved like a
bflippaseQ which would be able to transport amphiphilic
molecules (potentially also lipids) from the inner to the outer
leaflet of the plasma membrane. Since then, there have been
many indications for lipid transport mediated by ABC
proteins in cellular membranes, and a few reports on lipid
transport by purified ABC proteins in reconstituted systems.
Lipid transport by human ABC proteins has been the subject
of several review articles in the past [2–5]. In the following,
we will first give a general introduction on lipid transbilayer
movement and assays used for its determination, before
reviewing data accumulated on the involvement of eukaryotic and prokaryotic ABC proteins in lipid transport. We
will then discuss their putative role in lipid transbilayer
transport and exposure, and show models proposed for
mechanisms of lipid transbilayer transport by ABC proteins,
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A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
before relating some concluding remarks. Considering the
broad span of disciplines contributing to this topic, it
appears to be important in this article to draw a clear line
between the transport of endogenous lipids and lipid analogs
carrying a reporter group or other modifications. Similarly,
phenomena which appear to be related to the action of a
particular protein need to be distinguished from those for
which a specific transport has been proven unequivocally.
On the one hand, most attempts in reconstituted systems
have revealed only very small effects of ABC proteins on
lipid transport so far, with an influence of ATP hydrolysis
hardly significant.
In experiments on whole cells, on the other hand, the
involvement of various transport steps must be taken into
account, complicating transport rate quantification, and
hence the evaluation of their physiological significance.
lipid classes are (glycero-) phospholipids, sphingolipids,
steroids, lipopolysaccharides and triacylglycerols (for some
examples see Fig. 1). The phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), the sphingolipid
sphingomyelin (SM) and the steroid cholesterol are the
major lipids found in mammalian membranes [6,7].
The lipid transbilayer distribution in the eukaryotic
plasma membrane is asymmetrical, generally with the
majority of PC and sphingolipids in the exoplasmic leaflet,
and the aminophospholipids PE and PS mainly in the
cytoplasmic leaflet (reviewed in [8]). This asymmetry raises
several questions about the biological mechanisms by which
it is established (Fig. 2A), and about the putative biological
functions coupled to it.
2.1. Spontaneous transbilayer movement
2. Lipid transbilayer movement
Lipids form a vast group of chemically different
amphiphilic or hydrophobic substances containing a substantial portion of aliphatic or cyclic hydrocarbon. Abundant
The rate of spontaneous transbilayer movement (flipflop) in a pure lipid membrane differs for each lipid,
depending on its structure (headgroup, backbone) and its
environment (reviewed in [9]). Small, uncharged lipids
(including cholesterol) and negatively charged lipids in their
Fig. 1. Lipid substrates of ABC proteins (examples). Hydrophilic parts are indicated by blue clouds. Examples for the phospholipid moiety X are ethanolamine
(in PE), choline (in PC) or serine (in PS). Examples for the sphingolipid moiety Y are hydrogen (in ceramide), phosphorylcholine (in SM), glucose (in GlcCer),
galactose (in GalCer) or lactose (in LacCer). The fatty acid shown is oleic acid, the steroid shown is cholesterol. Lipid A structure as predominant in E. coli
[218].
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
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Fig. 2. Transbilayer lipid movement in biological membranes and models for function of lipid transporting ABC proteins (A). Lipids can move across a lipid
bilayer by passive flip-flop or mediated by proteins. Protein-mediated transbilayer movement could be energy-independent, unspecific and bidirectional (e.g.
scramblase), or energy-dependent and monodirectional (e.g. inward transport by P-type-ATPases, and outward transport by ABC proteins). The lipid specificity
varies among transporters (for details, see text). The number of lipids indicates the efficiency of lipid movement at a qualitative level. (B) Models for ABC
protein function in lipid transport and/or exposure to an acceptor A (for details, see text).
protonated form can flip across pure lipid bilayers within
seconds or minutes. In contrast, lipids with highly polar
headgroups move only slowly from one leaflet of a lipid
bilayer to the other (half-times of the order of hours to days
[10,11]). Cholesterol has been shown to decrease the
transbilayer movement of phospholipids [12,13].
2.2. Energy-independent and energy-dependent flippases
In eukaryotic cells, most lipids are synthesized asymmetrically in the membranes of the endoplasmic reticulum
(ER) and the Golgi system from which they reach, e.g. via
vesicle traffic, the plasma membrane or other organelles
(reviewed in [9]). Because lipid movement across cellular
membranes is essential for cell growth and survival, lipid
transporting proteins (flippases) are required for efficient
transbilayer lipid movement. Although some proteins have
been identified as candidate lipid transporters over the last
years (reviewed in [9,14,15]), the protein vehicles responsible for many lipid transport phenomena have not been
identified yet (Table 1).
In the mammalian ER, energy-independent flippases were
shown to mediate rapid bidirectional, rather unspecific
phospholipid flip-flop (half times of the order of minutes or
less) to ensure balanced growth of this membrane [16–18].
Similarly, rapid protein-mediated flip-flop has been demonstrated for phospholipids and glucosylceramide (GlcCer, a
precursor for complex glycosphingolipids) in the Golgi [19].
Lipid flippase activity was also found in the bacterial plasma
membrane [20,21].
Already in 1980, it was shown that the mere presence of
membrane proteins facilitates lipid flip-flop [22]. Thus, it is
not clear whether a dedicated flippase or a family of
selective flippases is involved in bidirectional lipid movement across ER and Golgi membranes [23,24].
Energy-independent, bi-directional transbilayer movement of all major phospholipids has equally been shown in
the eukaryotic plasma membrane. This flip-flop (half time of
the order of 1 min) [25,26], activated by cell stimulation and
the subsequent increase in intracellular calcium, has been
ascribed to the lipid scramblase protein. Effort has been made
to isolate and clone the potential scramblase PLSCR1 [27].
Experiments with fluorescent (nbd1) phospholipids in the
rabbit intestine brush border have indicated ATP- and Ca2+independent transbilayer movement of monoacyl phospholipids and lipids with short chains, supposedly constituting a
mechanism for the intestinal uptake of phospholipid
digestion products as lysolipids [28].
While energy-independent flippases allow lipids to
equilibrate rapidly between the two bilayer leaflets, energydependent flippases are responsible for a net transfer of
specific lipids to one leaflet of a membrane. In the plasma
membrane of eukaryotic cells, spontaneous flip-flop of
phospholipids is limited, possibly due to the high cholesterol
content, allowing the generation of a stable asymmetric lipid
1
(Typically, the abbreviation for [N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)amino] is written in capital letters (NBD). However, in order to avoid
confusion with the abbreviation for nucleotide binding domain, this style of
abbreviation was chosen).
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Table 1
ABC proteins involved in lipid transport
Organism
Family
Name
Trivial name
Involvement
Lipid analogs
Endog. lipids
Human
ABCA
ABCA1
ABC1
SL-PS
cholesterol
phospholipids
ABCA2
ABC2
ABCA3
ABCA4
ABC3
ABCR
Rim
macrophage lipid homeostasis
HDL deficiencies
Tangier disease
phagocytosis
macrophage lipid homeostasis?
neural development?
lung surfactant synthesis?
dark adaptation
Stargardt disease
macrophage lipid homeostasis?
hematopoiesis?
macrophage lipid homeostasis?
keratinocyte differentiation?
hematopoiesis?
macrophage lipid homeostasis?
macrophage lipid homeostasis?
detoxification
multidrug resistance
adrenal secretion
dendritic cell migration
ABCA6
ABCA7
ABCX
ABCA9
ABCB
ABCC
ABCD
ABCA10
ABCB1
MDR1 Pgp
ABCB4
MDR2/3 Pgp
ABCC1
MRP1
ABCC6
MRP6
ABCD1
ALDP
ABCD2
ABCG5
ALDRP
ALDL1
PMP70
PXMP1
PXMP1L
P70R
PMP69
WHITE
BCRP1
MXR1
ABCP
WHITE3
ABCG8
WHITE4
ABCD3
ABCD4
ABCG
ABCG1
ABCG2
bile formation
progessive familiar
intrahepatic cholestasis
detoxification
multidrug resistance?
LTC inflammatory response
macrophage lipid homeostasis
detoxification multidrug resistance
ABCA
SuABCA
Leishmania
ABCA
LtrABC1.1
parasite–host interaction vesicular transport
ABCB
LPgp-lp
multidrug resistance
PMP
(ABCD)
PDR/CDR
Ped3p
Yeast
Pdr5p
Cdr1p
cholesterol
phospholipids
ceramide
C6-nbd-PC
C6-nbd-PE
C6-nbd-PS
C6-nbd-SM
C6-nbd-GlcCer
C6-nbd-PC
PC
VLCFAs?
VLCFAs?
VLCFAs?
VLCFAs?
bodipy-Cer
C6-nbd-PS
C6-nbd-PC
cholesterol, PC
steroids? PS
cholesterol
steroids
cholesterol
steroids
phospholipid?
cholesterol?
C6-nbd-PC
C6-nbd-PE
C6-nbd-PS
bodipy-PC
alkyl-lysophospholipids
beta oxidation
drug resistance
drug resistance
steroids
cholesterol
GlcCer, PS,
SM, PAF
C6-nbd-SM
C6-nbd-GlcCer
C6-nbd-PC
C6-nbd-PS
beta oxidation
peroxisome biogenesis
beta oxidation
Sea urchin
COMATOSE
CTS PXA1
PC?
N-retinylidene-PE
pseudoxanthoma elasticum
lipid transport and metabolism
beta oxidation
X-Adrenoleukodystrophy
peroxisome biogenesis
beta oxidation
bile steroid secretion
sitosterolemia
bile steroid secretion
sitosterolemia
sperm acrosome reaction?
Arabidopsis
steroids?
FA CoAs?
C6-nbd-PE
C6-nbd-PC
C6-nbd-PE
C6-nbd-PS
PE, steroids
PE
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
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Table 1 (continued)
Organism
Family
Name
Trivial name
Cdr2p
Involvement
Lipid analogs
Endog. lipids
drug resistance
C6-nbd-PC
C6-nbd-PE
C6-nbd-PS
C6-nbd-PC
C6-nbd-PE
C6-nbd-PS
phospholipid?
Cdr3p
MDR
(ABCB)
MRP/CFTR
(ABCC)
ALDp
(ABCD)
Ste6p
Yor1p
Pat1p
Pxa2p
Pxa1p
E. coli
ABCB
Pat2p
MsbA
L. lactis
ABCB
LmrA
F. novicida
?
ValA
pheromone transport
drug resistance?
drug resistance
efflux organic anions?
peroxisomal lipid transport
lyso-PC?
C6-nbd-PE
VLCFAs?
peroxisomal lipid transport
lipid A transport
drug resistance
lipid A transport?
lipid A transport?
phospholipid?
C6-nbd-PE
VLCFAs?
lipid A
phospholipids
lipid A
lipid A-linked
polysaccharide
Substrates confirmed in reconstitution experiments are in bold print. Question marks indicate functions or substrates for which an involvement has not been
proven experimentally.
distribution between the two leaflets. The depletion of
cholesterol leads to an enhanced spontaneous flip-flop of
phospholipid analogs in the red blood cell membrane [12].
PE and PS are subject to an efficient and rather rapid
energy-dependent inward transport from the outer to the
inner leaflet mediated by a protein, the aminophospholipid
translocase [29,12]. Some cell types display similar transport of PC across the plasma membrane and may contain
either a PC-specific translocase in addition to the aminophospholipid translocase, or an inward translocase of
different specificity which transports both aminophospholipids and PC (see [15]). The aminophospholipid translocase,
not yet identified on the molecular level, very likely belongs
to the novel DrS2p P-type-ATPase subfamily with over a
dozen members in eukaryotes from yeast to plant cells
[30,31,15]. Two members of this family, Dnf1p and Dnf2p,
have been shown to be essential for ATP-dependent
transport of fluorescent analogs of PS, PE, and PC from
the outer to the inner leaflet of the yeast plasma membrane
[32].
Recently, it was reported that the fluorescent analog nbd
PS is a preferred substrate of Drs2p localizing to the transGolgi-network [33].
However, it is still unclear what permits the accumulation
of the majority of PC and sphingolipids in the mammalian
outer plasma membrane leaflet. ABC proteins were suggested to be involved in the outward transport of phospholipids [34]. Alternatively, it was proposed on theoretical
grounds that the inward transport of aminophospholipids,
together with passive fluxes, would be sufficient to
accumulate choline containing lipids in the outer leaflet
[35].
Changes in the distribution of a lipid species between the
membrane leaflets can influence membrane curvature and
fusion competence, protein association and activity, as well
as various biochemical pathways (reviewed in [9]). Because
of the low compressibility of lipid monolayers, the inward
and outward transport of lipids requires a subtle balance. In
the absence of a compensatory flux, unidirectional lipid
transport by energy-coupled transporters might create an
area imbalance between the two membrane leaflets, building
up surface tension eventually relaxing by membrane
budding or invagination [36,37] (see also Section 6). This
phenomenon was suggested to be a molecular motor for the
first stage of endocytosis [38,39]. The balance between
inner and outer membrane leaflet has been generally
overlooked when the activity of potential lipid transporters
was assessed in large unilamellar vesicles (LUVs), where
surface tension generated by lipid transport could be
sufficient to eventually block the transporter itself (discussed in [37,39,40]).
2.3. Determination of lipid transbilayer distribution
The techniques used to determine the transbilayer
distribution of lipids have been described and critically
evaluated in the literature [7,41–46]. In Fig. 3, some assays
for lipid analogs as well as for endogenous lipids are shown
schematically. Since methods for the rapid quantification of
endogenous lipids are still very limited, spin-labeled (SL) or
fluorescent lipid analogs are frequently employed to
determine lipid transbilayer distribution. The relevance of
such probes has been discussed by Devaux et al. [42]:
Briefly, bulky reporter moieties and a short fatty acid chain
replacing one of the natural long chains to facilitate
membrane incorporation may affect lipid polarity and cause
perturbations, somewhat modifying the absolute values of
the transbilayer distribution of a lipid. However, compar-
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Fig. 3. Assays for the detection of lipid transbilayer distribution. (A) BSA-extraction, dithionite and ascorbate assays for fluorescent and spin-labeled lipid
analogs. The short-chain lipid analog precursor integrates into the outer membrane leaflet (1), crosses the plasma membrane (e.g. by passive flip-flop) (2) and
distributes to different intracellular membranes (e.g. by monomeric transport) (3). ER or Golgi enzymes convert part of the lipid analog precursor to the lipid
analog of interest (4), which can distribute back to the plasma membrane, where it becomes available to outward transport by transporter proteins (5). Lipid
analog is extracted from the outer plasma membrane leaflet by BSA (6), followed by the separation of cells and media. In a variant of this assay, the cells can be
directly labeled with the lipid analog of interest, if it is able to reach the inner plasma membrane leaflet by passive flip-flop or active transport, analog remaining
on the outer leaflet being removed by BSA extraction prior to outward transport incubation. Alternatively to BSA extraction, fluorescene of lipid analogs can be
quenched using dithionite, and the spin-label signal be reduced using ascorbate (7). (B) Phospholipase/sphingomyelinase assay for endogenous lipids.
Endogenous lipid is synthesized in the presence of radioactive precursors in the cell (I) and localizes to the various cellular membranes due to vesicular or
monomeric transport (II), to modification, and to the presence of transporter proteins (III). Phospholipase A2 treatment converts lipids in the outer plasma
membrane leaflet to lysolipid and fatty acid (IV). Lipid products are then analyzed by chromatography and can be compared to samples untreated with enzyme.
An analogous technique is used for SM, employing sphingomyelinase. (C) Chemical modification assays for endogenous lipids. Endogenous lipids present on
the outer plasma membrane leaflet are typically modified on the level of the headgroup. Reagents frequently used for modification are trinitrobenzene sulfonic
acid (TNBS, specific for PE) and fluorescamine. (D) Antibody, peptide or protein binding assay for endogenous lipids. Specific antibodies, respectively
peptides (e.g. Ro09-198, binding to PE [45,46]) or proteins (e.g. Annexin V, binding to PS) with affinity for a particular lipid headgroup, bind to endogenous
lipids present on the outer plasma membrane leaflet; the amount of bound antibody or binding protein is quantified.
isons between lipids with different headgroups, but with the
same fatty acid chains, can be very revealing about the
specificity of the lipid transport. Recently, various cholesterol analogs have been studied showing large variations in
the potential to mimic endogenous cholesterol [44]. Nevertheless, new assays will have to be developed to rapidly
determine the transbilayer distribution of endogenous lipids.
3. General features of the ABC protein superfamily
The ATP-binding cassette (ABC) protein superfamily
comprises a large number of transporters, channels and
regulators in pro- and eukaryotes [reviewed in [47–49]].
Their functions range from the acquisition of nutrients and
the excretion of waste products to the regulation of various
cellular processes. Generally, ABC proteins are low
capacity, but high affinity transporters, able to transport
substrates against a concentration gradient of up to 10 000
fold. Hydrolysis of ATP is required for substrate transport.
ABC proteins are mainly either import or export pumps,
bidirectional ABC proteins appear to be rare exceptions
[50]. Import pumps seem to be limited almost exclusively to
prokaryotes. Typically, ABC proteins are relatively specific
for a particular set of substrates (the multispecific ABCB1
(MDR1 Pgp) probably represents an exception in this
respect). Substrates can be amino acids, sugars, inorganic
ions, peptides, proteins, lipids and various organic and
inorganic compounds. ABC proteins consist of nucleotide
binding domains (NBDs), and of transmembrane (TM)
domains (TMDs) of usually six alpha-helices. NBDs and
TMDs can occur as separate proteins (frequently found in
prokaryotes) or fused together. The transmembrane domains
vary considerably between different ABC proteins, whereas
the nucleotide binding domains are highly conserved (e.g.
Walker motifs). The substrate specificity is believed to be
determined by the transmembrane domains, including the
loops connecting the individual helices [47].
4. Eukaryotic ABC proteins
Structurally, eukaryotic ABC proteins typically possess
two nucleotide binding domains and two TM domains,
probably representing the minimal functional unit (full-size
protein). In some ABC proteins, deviating organizations of
the domains can prevail [49] (Fig. 4), e.g. half-size proteins
with one NBD and one TM domain each, thought to require
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
35
Fig. 4. Domain organization of human ABC proteins involved in lipid transport. Transmembrane domains (TMDs) are shown as membrane-spanning helices,
nucleotide binding domains are marked NBD. Intracellular domains (ICDs) and posttranslational modifications (e.g. glycosylations) are not shown.
dimerization in order to be functional. ABC proteins with
nucleotide binding domains only (families E, F) are likely
not directly involved in membrane transport, and are
thought to have regulatory functions.
4.1. Human ABC proteins
The 49 human ABC proteins currently known can be
classified into 7 families (A–G) according to sequence
similarity [49,51]. An overview can be found on Michael
Mqller’s website (http://nutrigene.4t.com/humanabc.htm).
Several human ABC proteins found to be mutated in lipidlinked diseases (families A, B, C, D, and G) were suggested to
be involved in lipid transport. Although diseases due to
complete loss of function of single ABC proteins do not
appear to have a very high incidence in the population,
polymorphisms in ABC genes are likely to play a role in
medically relevant phenomena. At present, direct transport of
lipid substrates has only been shown for a small number of
human ABC proteins. The ABC proteins identified in
mammals so far (e.g. in mouse, rat, pig) are highly similar
to members of the human ABC protein families, and will
therefore not be discussed separately in this work.
4.1.1. Human ABCA (ABC1) family
Out of the 12 ABCA family members, all of which are
full-size proteins, 8 are assumed to transport lipophilic
substrates. For a number of these, an involvement in lipid
transport was deduced from lipid dependent expression, and
homology to ABCA1, but has not yet been confirmed
experimentally.
ABCA1 (ABC1), studied intensely with regards to lipid
transport, is expressed in numerous tissues such as the
trachea, lung, adrenal gland, spleen and uterus [52], in some
of which its expression is steroid-dependent [53]. ABCA1GFP chimeras localize to the plasma membrane and to
intracellular vesicles in transfected HeLa cells [54]. ABCA1
has been implicated with the transport of cholesterol and
phospholipids (see Section 6).
In mammals, the majority of cholesterol is synthesized de
novo in the liver, and delivered to peripheral cells by
lipoproteins. Since peripheral cells are unable to degrade
cholesterol, any surplus of cholesterol must either be stored
in the cytosol in the form of esters, or released from the cell.
ABCA1 mutations can be responsible for some cases of
familial high density lipoprotein (HDL) deficiency, e.g.
Tangier disease [55–57], characterized by impaired efflux
of cholesterol and phospholipids from peripheral cells onto
apolipoproteins such as apoA-1. Cholesterol accumulation in
macrophages and apolipoprotein degradation lead to tissue
deposition of cholesterol esters and increase the risk of
arteriosclerosis in the patients. While Tangier cells typically
fail to bind nascent apoA-I [58,59], the expression of ABCA1
in cultured cells has been found to enhance the binding of
apoA-I to the plasma membrane [60], and to increase the
efflux of cellular phospholipid and cholesterol to this
apolipoprotein [61]. Both sequential and parallel mechanisms
have been proposed for the transport of phospholipids and
cholesterol by ABCA1 (see also Section 6).
ABCA1 has been suggested to directly transport the
aminophospholipid PS (typically restricted to the cytoplasmic leaflet of mammalian plasma membranes) to the
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exoplasmic leaflet [61], where the presence of PS facilitates
apoA-1 binding, leading thus indirectly to cholesterol efflux
[61–63]. Indeed, the exposure of endogenous PS (detected
via prothrombinase activity and binding of Annexin V)
upon Ca2+ induced stimulation was found to be low in red
blood cells, resp. thymocytes, derived from mice lacking
ABCA1, and could be partially restored upon ABCA1
transfection [61]. A recent study on apoptotic murine cells
confirmed elevated levels of exposed PS to cause a strong
increase in apoA-I binding, although not sufficient to trigger
phospholipid and cholesterol efflux to apoA-I [64].
In contrast, Wang et al. [65] recently proposed direct
transbilayer transport of both cholesterol and phospholipids
via ABCA1, following experiments in which phospholipid/
apoA-I particles made by ABCA1 were unable to stimulate
passive cholesterol efflux when added to a second set of cells.
In addition to a role in lipid loading of apolipoproteins,
ABCA1 has been implicated with PS exposure on the outer
plasma membrane leaflet of apoptotic cells and phagocytizing macrophages [66]. In the absence of apoliposomes, Ca2+
induced externalization of spin-labeled (SL) analogs of PS,
but not of PC, was reduced in mice lacking ABCA1 [61].
The large ABC protein ABCA2, found in high levels in
human brain (possibly in oligodendrocytes), colocalizes
with a lysosomal/endosomal marker [67]. Vulevic et al.
[67] have associated ABCA2, which contains a signature
motif for lipocalins (protein family binding small hydrophobic molecules), with the transport of steroids and lipids
due to colocalization with an analog of the steroid
estramustine, and increased estramustine resistance upon
ABCA2 gene overexpression.
Kaminski et al. suggested a role for ABCA2 in macrophage lipid metabolism and neural development, after
having found an induction of ABCA2 mRNA during steroid
loading [68]. Based on these observations and the unique
expression profile, Schmitz and Kaminski [69] inferred a
role of ABCA2 in transbilayer lipid transport of neural cells.
ABCA3 is expressed exclusively in type II epithelial lung
cells expressing the gene surfactant protein A. It is
hypothesized to play a role in the formation of pulmonary
surfactant [70], a mixture containing PC and various
surfactant proteins, which reduce surface tension on the
surface of the alveoli. Mutations in ABCA3 were found in
several cases of infants with fatal surfactant deficiencies
[71]. ABCA3 is localized in the plasma membrane and in
the limiting membrane of lamellar bodies, in which
surfactant is stored prior to exocytotic delivery into the
alveolar space. Mulugeta et al. have therefore speculated
that ABCA3 might transport PC into or other lipids out of
lamellar bodies, which are highly enriched in PC [72].
ABCA4 (ABCR or Rim protein) [73] is localized in the
photoreceptor outer segment disc membranes of the retina
[74,75]. Reconstitution studies, in which its ATPase activity
was stimulated by retinal, lead to the hypothesis that
ABCA4 may function as an active retinoid transporter
[76]. ABCA4 appears to be highly substrate-specific, being
involved in dark-adaptation through the transport of the
lipid product all-trans-N-retinylidene-PE across the disc
membrane following the photobleaching of rhodopsin. This
transport allows all-trans-retinal to be reduced to all-transretinol on the surface of the disc membrane. Thus, ABCA4
mediated transport is an important step in the recycling of
all-trans-retinal to 11-cis-retinal for the regeneration of
rhodopsin. Studies on the ATPase activity of reconstituted
ABCA4 strongly suggested PE to be required to couple the
binding of retinoids to ABCA4 ATPase activity [76].
Defective ABCA4 can cause the degeneration of the macula
lutea and consequent deterioration of vision (Stargardt
disease), presumably through the accumulation of the
lipofuscin fluorophore N-retinylidene-N-retinyl ethanolamine (A2E) [77,78].
ABCA6 shows expression in the lung, heart, brain, liver
and ovaries. As ABCA6 expression is suppressed by
cholesterol loading [79], the protein was attributed a
potential role in macrophage lipid homeostasis.
ABCA7 is expressed mainly in myelo-lymphatic tissues.
The protein is localized either in the plasma membrane or
the endoplasmic reticulum [80], and was implicated with the
specification of hematopoietic cell lineages during development [81]. As the loading of macrophages with steroids
increases, and unloading decreases the expression of the
ABCA7 gene, Kaminski et al. proposed ABCA7 to be
involved in macrophage transbilayer lipid transport [82].
ABCA7 overexpression in HeLa cells increased the
exposure of endogenous ceramide (Cer) on the outer plasma
membrane leaflet, and raised levels of endogenous PS [83].
As ABCA7 is upregulated during keratinocyte differentiation, this led to speculations about a regulator role for
ABCA7 in lipid transport during terminal keratinocyte
differentiation. Very recently, apolipoprotein-mediated
release of cellular cholesterol and phospholipids was
reported from HEK293 cells transiently or stably expressing
ABCA7 [80,84]. Furthermore, the transfection of GFPtagged ABCA7 of L929 cells triggered apolipoproteinmediated assembly of cholesterol-containing HDL. In
contrast, Wang et al. [65] reported ABCA7-mediated efflux
of phospholipid and SM, but in contrast to ABCA1 not of
cholesterol, from HEK293 cells.
ABCA9, highly homologous to ABCA6, shows ubiquitous expression, the highest levels being found in the heart,
brain and fetal tissues. ABCA9 is induced during macrophage differentiation. Different from ABCA7, the expression of ABCA9 in macrophages decreases upon steroid
loading of the cells. Piehler et al. suggested that ABCA9
might act on monocyte differentiation and macrophage lipid
homeostasis [85].
ABCA10, as ABCA9 highly homologous to ABCA6, is
ubiquitously expressed, with high gene expression levels in
the heart, brain, and the gastrointestinal tract. As its gene
expression in macrophages is suppressed by cholesterol
loading, Wenzel et al. hypothesized on the involvement of
ABCA7 in macrophage lipid homeostasis [86].
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
4.1.2. Human ABCB (MDR/TAP) family
The 4 full-size and 7 half-size proteins of the ABCB
family show highly varied specificities (e.g. amphiphilic
compounds, peptides, iron, phospholipids, bile salts) [51].
The proteins associated with antigen processing ABCB2, 3
(TAP1, 2), and the bile salt export pump ABCB11 (BSEP)
are ABCB protein family members.
ABCB1 (Multidrug Resistance 1 P-glycoprotein, MDR1
Pgp) [87] is a full-size protein with two transmembrane
domains and two nucleotide binding domains, which
appears to be functional as a monomer [88]. It occurs in
the apical membrane domain [89] of epithelia with secretory
functions (e.g. adrenal gland, kidney), at the pharmacological borders of the body (intestine, blood–brain barrier, fetomaternal barrier) [90], and in many tumor tissues [91].
ABCB1 has a surprisingly broad spectrum of (mainly
cationic or electrically neutral) amphiphilic substrates [92].
One of its important physiological roles appears to be the
protection of the organism against toxins by exporting these
into the bile, urine, or gut. In tumors, the overexpression of
ABCB1 is one of the principal factors responsible for
multidrug resistance (MDR) against a variety of structurally
unrelated drugs. ABCB1 is involved in other phenomena as
well, in which, interestingly, lipid transport often seems to
be implicated: It was found to mediate the secretion of the
steroid aldosterone by the adrenals [93], and its inhibition
blocked the migration of dendritic immune cells [94],
possibly related to the outward transport of the lipid platelet
activating factor (PAF, see below). Ueda et al. reported
ABCB1 mediated transport of the steroids cortisol and
dexamethasone, but not of progesterone in ABCB1 transfected cells [95]. Inhibition studies have also led to
speculations about the transport of cholesterol by ABCB1
[96] (see also Section 6). Short-chain and long-chain (nbd
and radiolabeled) analogs of PC, PE, PS, SM, and GlcCer
were found to be expelled from ABCB1 overexpressing
cells [97–99]. Among the endogenous lipids, the short-chain
PC PAF [100] is an ABCB1 substrate (ABCB1 antisense
oligonucleotides blocking PAF secretion in human mesangial cells). Other potential substrates are GlcCer (rescued
from cytosolic hydrolysis in the presence ABCB1, and
strongly reduced in the absence of ABCB1) [101] and PS
(as found by Annexin V binding experiments in ABCB1
overexpressing human gastric carcinoma cells) [99]. Interestingly, the exposure of endogenous SM to the outer
plasma membrane leaflet of human myeloblastic cells was
reduced upon ABCB1 inhibition, a possible hint for ABCB1
mediated transport of this lipid involved in signalling [102].
While the inability of the ABCB1 mouse homologs Mdr1a/
1b to restore the transport of PC into the bile of mice lacking
Mdr2 (homologous to human ABCB4) [103] could suggest
that natural long-chain PC is not an ABCB1 substrate, it is
also conceivable that Mdr1a/1b activity was too low in this
system. Multispecific transport of diverse endogenous lipids
via ABCB1 could affect the transbilayer distribution of
lipids, in particular of species normally predominant on the
37
inner plasma membrane leaflet, such as PS and PE.
Reconstitution experiments with ABCB1 have thus far
yielded ambiguous results: While Romsicki and Sharom
[104] found a low ATP dependent increase in reoriented
short-chain nbd analogs of PC, PE, PS and SM, Rothnie et
al. [40] observed low reorientation of short-chain nbd
analogs of PC, PE, Cer and of short-chain SL analogs of
PC, PE, GlcCer, and SM which was ATP independent.
Interestingly, the activity of ABCB1 was dependent on
cholesterol. Due to the small size of the vesicles containing
the reconstituted protein, lateral pressure (surface tension)
might have prevented substantial transport of lipids via
ABCB1 [105] (see Section 2). The structure and potential
mechanisms of ABCB1 will be discussed in Section 7.
ABCB4 (MDR2/3 Pgp), a full-size protein, is a close
relative of ABCB1 (MDR1 Pgp), sharing 75% of its amino
acid sequence [49]. Unlike ABCB1, ABCB4 is highly
substrate specific, exclusively transporting (short-chain nbd
analogs of) PC, as observed in ABCB4 transfected porcine
cells [97]. The secretion of PC into the bile appears to be the
physiological function of ABCB4 [103] (see also Section 6),
the protein being present in high amounts in the canalicular
membranes of hepatocytes. In mice lacking ABCB4, PC
secretion into the bile is abolished [103], and the transbilayer movement of radioactively labeled endogenous PC
appears to be slightly enhanced in fibroblasts from mice
overexpressing the ABCB4 gene [106]. In some cases of
progressive familiar intrahepatic cholestasis (type III) in
humans, ABCB4 has indeed been found to be defective
[107].
4.1.3. Human ABCC (CFTR/MRP) family
All 13 ABCC family members are full-size proteins.
However, they differ in the number of transmembrane
domains (two for ABCC4, ABCC5, ABCC11 and
ABCC12, while all others possess a third TM domain,
TMD0 (Fig. 4)). The major functions of the ABCC proteins
are, among others, the protection against toxic compounds
and the secretion of organic anions [108]. Examples for
ABCC family members are the cystic fibrosis transmembrane conductance regulator ABCC7 (CFTR), defective in
mucoviscidosis, and the sulfonylurea receptors ABCC8,9
(SUR 1, 2), regulating associated potassium channels.
ABCC8 is defective in patients with familial persistent
hyperinsulinemic hypoglycemia of infancy. The ABCC
family members differ in substrate specificity, tissue and
organelle distribution [109].
ABCC1 (MRP1) [110] is located in the basolateral domain
of polarized cells [111], and displays wide tissue distribution
[109]. ABCC1 transports a large variety of toxins across the
plasma membrane, either unconjugated or conjugated with
glutathione, sulfate or glucuronate [112]. While it is unclear
whether ABCC1 contributes significantly to multidrug
resistance in tumor cells [109], it protects particularly
sensitive organs by expelling toxins into the blood (the
internal environment [113], in contrast to the apically located
38
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
ABCB1 (MDR1 Pgp) which exports toxins into the external
environment). Additionally, ABCC1 mediates the leukotriene C (LTC) dependent inflammatory response by the
transport of the arachidonic acid derivative LTC4 [114].
ABCC1 transfected pig kidney epithelial cells showed
increased outward transport of short-chain nbd analogs of
SM, and GlcCer [115], and the transport of short-chain (PS,
PC) and long-chain PC nbd analogs in erythrocytes was
equally attributed to ABCC1 in an inhibition study [116].
Upon reconstitution, ABCC1 transported an nbd PC analog
[117]. However, thus far, no endogenous lipids have been
found to be ABCC1 substrates.
Despite similarities in the substrate spectrum of ABCC1
and several other ABCC proteins, Raggers et al. did not
observe translocation of short-chain nbd lipid analogs by
other ABCC proteins tested besides ABCC1 [101].
ABCC6 (MRP6) is expressed exclusively in the liver and
kidney, where the gene product is localized on the
basolateral domain of the plasma membrane [118]. ABCC6
defects result in pseudoxanthoma elasticum (PXE)
[119,120], a disorder characterized by a calcification of
the elastic fibers of the eye, skin, and vasculature, leading to
decreased visual acuity, characteristic skin lesions, and
peripheral vascular disease. Additionally, frequently found
high plasma triglyceride levels and low plasma HDL
cholesterol in PXE patients suggest an involvement of
ABCC6 in lipid transport and metabolism [121].
The molecular basis of this disease is not solved, and
PXE might be a primary metabolic disorder with secondary
involvement of elastic fibers [122].
The transport of glutathione conjugates upon ABCC6
gene expression in Sf9 insect cells point to a role for ABCC6
in the transport of organic anions [123], thought to confer
low levels of resistance to certain anticancer agents [124].
4.1.4. Human ABCD (ALD) family
All four known members of the ABCD family are half-size
proteins found in peroxisomes, single-membrane organelles
involved in beta-oxidation of long and very long chain fatty
acids, synthesis of bile acids, cholesterol plasmalogens and
metabolism of amino acids and purines. ABCD proteins have
been implicated with the transport of fatty acids (FA),
coenzyme A (CoA), or FA-CoA, although ATP independent
transport of very long chain fatty acids (VLCFA) into
peroxisomes [125] might argue against direct VLCFA transport via ABCD proteins. ABCD half-size proteins can form
not only homodimers (ABCD1–ABCD1), but also heterodimers (ABCD1–ABCD2, ABCD1–ABCD3, ABCD2–
ABCD3) [126]. Although the heterodimers might be functional, the differing tissue gene expression of ABCD
members argues against obligatory heterodimerization [127].
ABCD1 (ALDP) mRNA is highly expressed in human
liver, heart, skeletal muscle, lung and intestine [128].
Defects in ABCD1 result in the inherited neurometabolic
disorder X-linked adrenoleukodystrophy (X-ALD) [129],
characterized by elevated levels of VLCFA in nervous
system white matter and the adrenal cortex [130], leading to
neuron demyelinization, renal insufficiency and testicular
dysfunction. Increased VLCFA levels have been attributed
to impaired peroxisomal beta-oxidation, and transfection
with ABCD1 cDNA was shown to restore beta-oxidation in
X-ALD fibroblasts [131] (see also yeast homolog Pat1p;
Section 4.5). Recently, mitochondrial beta-oxidation was
reported to affect peroxisomal beta-oxidation, and a role of
ABCD1 in the interaction of peroxisomes with mitochondria was suggested [132]. ABCD1 has been implicated with
peroxisome biogenesis, restored upon ABCD1 overexpression in Zellweger cells (having a defect in the peroxisomal
protein Pex2p) [133].
ABCD2 (ALDRP) is closely related to ABCD1 (63%
amino acid identity). Its mRNA is highly expressed in human
brain and heart [128]. Similar to ABCD1, transfection with
ABCD2 cDNA restored beta-oxidation in X-ALD (ABCD1
defect) fibroblasts [134], and peroxisome proliferation could
be induced upon pharmacologically increased ABCD2
expression in X-ALD cells [135]. In X-ALD fibroblasts,
ABCD2 induction by steroid depletion was found to reduce
the accumulation of VLCFA [136]. While defects in ABCD2
as a cause for X-ALD were considered to be unlikely,
ABCD2 could be a heterodimeric partner for ABCD1 in some
tissues, acting as a modifier gene accounting for the high
phenotypic variability of X-ALD [127].
ABCD3 (PMP70, PXMP1) shows 36% amino acid
identity with ABCD1. The mRNA of its mouse homolog
is highly expressed in the liver, kidney, heart, lung and
intestine [128]. The overexpression of ABCD3 was found to
restore beta-oxidation in X-ALD fibroblasts and to normalize peroxisome biogenesis in Zellweger cells [133] (see also
yeast homolog Pat2p, Section 4.5).
ABCD4 (PXMP1L, P70R, PMP69) shares 25% of
ABCD1 amino acid sequence. ABCD4 mRNA is highly
expressed in human kidney, spleen and testis [128]. As
ABCD2 and 3, it has been suggested to act as a modifier
gene contributing to X-ALD phenotypic variability, possibly
heterodimerizing with the other members of this protein
family [137].
4.1.5. Human ABCG (WHITE) family
The five characterized ABCG proteins are half-size
proteins. In contrast to other proteins, the ABC-domain is
N-terminal, followed by the transmembrane domain (Fig. 4)
[138]. A number of ABCG members are thought to be
involved in the transport of steroids (ABCG1, 5, 8),
additionally, some appear to transport phospholipids
(ABCG1) and toxins (ABCG2).
ABCG1 (WHITE) [139] is thought to be active either as a
homo- or a heterodimer (possibly with ABCG2) [140]. It
shows an ubiquitous gene expression pattern [138] and
localizes primarily to ER and Golgi [140]. ABCG1 derives
its trivial name from its homology to the Drosophila white
protein, which transports guanine and tryptophane as
precursors for eye pigments [141]. ABCG1 itself appears
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
to serve a different function, presumably in the transport of
phospholipids and steroids out of macrophages, as its gene
expression in macrophages was found to be induced during
cholesterol influx, and suppressed by lipid efflux via HDL.
The inhibition of ABCG1 expression resulted in reduced
HDL-dependent efflux of cholesterol and PC from these
cells [140].
ABCG2 (BCRP, MXR, ABCP) [142] is found in the
placenta, intestinal epithelium, liver canaliculi, breast ducts
and lobules, as well as in veinous and capillary endothelium
[143]. Recent studies suggest the protein to be active as a
homotetramer [144] in the plasma membrane [145]. It is
assumed to prevent the tissue uptake of xenobiotics [143],
transporting various drugs across the plasma membrane in
an ATP dependent process [146]. Transfection studies
proved the overexpression of ABCG2 to induce multidrug
resistance in a previously drug sensitive cell line [147]. In
addition to drug transport, the reduced accumulation of a
short-chain Bodipy analog of ceramide in ABCG2 overexpressing cells has given hints for a transport of lipid
analogs [146]. Recently, we have found increased outward
movement of short-chain nbd analogs of PS and PC, but not
of PE, and increased exposure of endogenous PS in a human
gastric carcinoma cell line overexpressing ABCG2 [148]. In
Lactococcus lactis transfected with human ABCG2,
ABCG2-associated ATPase activity was significantly stimulated by cholesterol and estradiol, pointing to a possible role
of ABCG2 in the transport of steroids [149].
ABCG5 (WHITE3) and ABCG8 (WHITE4) are encoded
by neighbouring genes [150]. Their mouse homologues are
expressed in the liver and intestine [151]. Very recently,
ABCG5 and ABCG8 have been demonstrated to function as
an obligate heterodimer to promote steroid secretion into the
bile [152–154]. Mutations in either ABCG5 or ABCG8
result in an identical clinical phenotype; and the expression
of both genes is required for either protein to be transported
to the plasma membrane. Defects in ABCG5 and 8 can
result in sitosterolemia, a disorder characterized by
increased uptake of steroids (among them the plant steroid
beta-sitosterol) in the intestine, combined with decreased
biliary excretion, resulting in cholesterol deposits in skin
and tendons, and in premature coronary artery disease
[150,155,156].
The overexpression of human ABCG5 and 8 in mice
promoted bilary cholesterol secretion, reduced cholesterol
absorption, and increased hepatic cholesterol synthesis [157].
Furthermore, no significant sitosterol transport by
ABCB1 (MDR1 Pgp), ABCC1 (MRP1), and ABCG2
(BCRP) was found [158].Taken together, these data
demonstrate a central role of ABCG5 and ABCG8 in in
vivo cholesterol excretion [159] (see also Section 6).
4.2. Sea urchin ABC proteins
SuABCA is a full-size ABC protein found abundantly in
the sea urchin sperm plasma membrane, and is possibly
39
implicated in the sperm acrosome reaction during fertilization. Due to its close homology to human ABCA3,
Mengerink and Vacquier suggested SuABCA to be involved
in phospholipid or cholesterol transport [160].
4.3. Eukaryotic parasite ABC proteins
ABC proteins found in parasites are of particular clinical
relevance due to the occurrence of multidrug resistant
strains. Specific attention will be given here to ABC
proteins found in unicellular eukaryotes of the Trypanosomatidae family, causing Leishmaniasis and Trypanosomiasis, major and globally widespread parasitic diseases.
In Leishmania spp., three different families of ABC
proteins are known (reviewed in [161,162]), homologous to
the mammalian ABC families ABCA, ABCB, and ABCC,
respectively.
4.3.1. Leishmania ABCA family
LtrABC1.1 [161], a full-size protein containing two
transmembrane domains and two nucleotide binding
domains, is found mainly in the plasma membrane and
flagellar pocket of Leishmania [163]. LtrABC1.1 expression appears to be uncorrelated with multidrug resistance. In
a Leishmania tropica cell line overexpressing LtrABC1.1,
the accumulation of short-chain nbd analogs of PC, PE, and
PS was found to be reduced [163], regardless of the nature
of the phospholipid head group. Furthermore, LtrABC1.1
overexpression reduced vesicular transport. Parodi-Talice et
al. have suggested a role for LtrABC1.1 in lipid movement
across the plasma membrane and in vesicle trafficking.
Finally, Legare et al. noted the potential importance of
members of the Leishmania ABCA family in the interaction
of the parasite with its host, an engulfing macrophage cell
[162].
4.3.2. Leishmania ABCB family
Leishmania Pgp-like proteins (LPgp-lp), about 37%
identical to mammalian ABCB1s, are full-size ABC
proteins [161]. They have been shown to transfer multidrug
resistance upon transfection [164]. In Leishmania enriettii,
Leishmania Pgp-like protein is located mainly in different
intracellular vesicles [165]. Upon the overexpression of the
LPgp-lp gene in L. tropica, the parasite cells accumulated
lower amounts of a short-chain Bodipy analog of PC and the
antiproliferative alkyl-lysophospholipids miltefosine and
edelfosine than did the controls [166].
4.4. Plant ABC proteins
Ped3p (peroxisome defective protein 3, also designated
COMATOSE, CTS, PXA1), predicted to be a full-size protein
[167,168], is found in arabidopsis glyoxysomes, specialized
peroxisomes occurring in cells of storage organs (endosperms, cotyledons) and senescent organs [168]. Both halves
of Ped3p show significant sequence homology to the human
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A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
half-size protein ABCD1 (ALDP) [169]. In Ped3p defective
plants, fatty acid beta-oxidation is impaired [168,169],
causing a defect in gluconeogenesis that severely inhibits
seedling germination in the absence of sucrose [170]. As
Ped3p mutants accumulate fatty acyl CoAs, Footitt et al.
proposed the protein to be a transporter of fatty acyl CoAs
with little specificity concerning chain length [169].
4.5.3. MRP/CFTR family (ABCC homolog)
Yor1p is a plasma membrane located [182] full-size
protein in S. cerevisiae. It is related to the human ABCC
family [183]. Besides conferring drug resistance, Yor1p has
been suggested to be involved in the efflux of organic
anions. The deletion of Yor1p in S. cerevisiae resulted in
increased accumulation of a short-chain nbd analog of PE
[174].
4.5. Yeast ABC Proteins
In yeast, the 31 ABC proteins identified so far have been
classified into 6 clusters, including 10 subclusters, according
to predicted topology, binary amino acid sequence comparison and phylogenetic classification. Both half-size and fullsize proteins exist in yeast. Besides I.1 and VI, all
subclusters seem to have human homologues [171].
4.5.1. PDR/CDR family
Pdr5p is a full-size protein located in the plasma
membrane [172] of Saccharomyces cerevisiae, mediating
multidrug resistance. It confers resistance to progesterone
and deoxycorticosterone, which are also inhibitors of Pdr5p
drug transport, suggesting these steroids to be direct
transport substrates of Pdr5p [173]. Pdr5p deletion in S.
cerevisiae leads to an increased accumulation of short-chain
nbd PE [174]. Notably, the depletion of Yor1p and Pdr5p
causes reduced surface exposure of endogenous PE [32].
Cdr1p and Cdr2p, candida drug resistance proteins of
the human pathogenic yeast C. albicans, are full-size
proteins composed of two homologous halves, each
comprising a TM domain and a nucleotide binding domain
[175]. Cdr3p, highly homologous to Cdr1p and Cdr2p,
shows the same domain organization, but appears not to be
involved in drug resistance [176]. In C. albicans strains with
a disrupted CDR1 gene, the (already low) exposure of
endogenous PE on the outer plasma membrane leaflet,
revealed via TNBS labeling, was reported to be further
reduced [177]. However, taking into account the limitations
of the TNBS labeling approach and the very low percentage
of labeled PE, the results have to be taken with caution.
Furthermore, gene disruption may also affect other processes involved in PE exposure, for example, intracellular
lipid trafficking. In transfected S. cerevisiae cells, Cdr1p
and Cdr2p elicited in-to-out transport of short-chain nbd
analogs of PE, PC and PS. In contrast and rather exceptional, Cdr3p mediated the out-to-in transport of these
analogs [178].
4.5.2. MDR family (ABCB homolog)
Ste6p, a S. cerevisiae full-size ABC protein transporting
the farnesylated dodecapeptide mating pheromone a-factor
[179], was reported to confer resistance against the lyso-PC
analog ET-18-OCH3 (Edelfosine) in transfected yeast cells,
presumably by outward transport of the drug [180].
However, failure to reproduce these results led to retraction
of the original article [181].
4.5.4. ALDp family (ABCD homolog)
Pat1p (also designated Pxa2p) and Pat2p (also designated Pxa1p) [184,185] are S. cerevisiae half-size proteins
related to human ABCD3 (PMP70) and ABCD1 (ALDP),
respectively. Pat1 and Pat2 are thought to heterodimerize
[185] in the peroxisomal membrane, and are required for the
import of long-chain fatty acids into peroxisomes, Pat1 and
Pat2 deletion causing a partial deficiency in long-chain fatty
acid beta-oxidation [184]. These data also support the
hypothesis that ABCD1 and ABCD3 are involved in
VLCFA transport (see Section 4.1).
5. Prokaryotic ABC proteins
ABC proteins exist in both Gram-positive and Gramnegative bacteria (reviewed in [48]). Most of them are
concerned with the import of small solutes, depending on
specific binding proteins, but exporters exist as well. In
prokaryotes, ABC proteins can possess either separate NBD
and TM domains, or fused domains.
5.1. Prokaryotic ABCB family
MsbA is a half-size ABC protein [186,187] active as a
homodimer [188] in the Escherichia coli inner membrane. It
is a close bacterial homolog of ABCB1 (MDR1 Pgp) as was
deduced from protein sequence homology [189,190]. It is an
essential ABC protein in prokaryotes, conserved in all
bacteria, with more than 30 orthologs identified today [191].
MsbA plays an important role in the transport of lipid A
from the inner to the outer membrane of Gram-negative
bacteria. Lipid A, a hexa-acylated disaccharide of glucosamine unique to Gram-negative bacteria, is a major
component of the outer membrane, representing the hydrophobic anchor of lipopolysaccharides on the outside of the
outer membrane. MsbA defects cause the accumulation of
lipid A and phospholipids in the inner membrane, lethal to
E. coli [187,192]. The stimulation of the ATPase activity of
MsbA reconstituted into liposomes by lipid A [193], but not
by short-chain nbd phosphatidylglycerol [23], provides
further indications for specific transport of lipid A by MsbA
[23]. Very recently, newly synthesized lipid A, and possibly
PE, has been shown to accumulate on the cytoplasmic half
of the E. coli inner membrane upon the inactivation of
MsbA in a temperature-sensitive mutant [194] arguing for
acceleration of transbilayer movement by MsbA.
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
The structure and potential mechanisms of MsbA will be
discussed in Section 7.
LmrA, a half-size protein in L. lactis forming homodimers, extrudes various drugs [195]. Like MsbA, LmrA is
homologous to both halves of human ABCB1 (MDR1 Pgp)
[196,189], which it can complement functionally in human
lung fibroblasts, causing multidrug resistance [189].
Recently, Reuter et al. could show functional substitution
of temperature-sensitive mutant MsbA in E. coli, suggesting
transport of lipid A by LmrA [190].
Reconstituted LmrA has been found to mediate ATPdependent transport of fluorescent short-chain nbd PE [197].
However, short-chain nbd PC was not recognized, suggesting headgroup-specificity, different from the low substrate
specificity of ABCB1 despite the high sequence conservation. It is not known whether LmrA also transports
endogenous phospholipids of L. lactis. In the light of the
rapid, ATP-independent lipid flip-flop mediated by proteins
in the inner membrane of bacteria [20,21,198–200] with
half-times of the order of one minute, it remains open
whether lipid transport by LmrA is of physiological
relevance for L. lactis.
5.2. Val A
ValA is an ABC protein in Francisella novicida with
high homology to E. coli MsbA, able to rescue MsbA
defective E. coli [201]. Due to this finding and decreased
cell surface exposure of a lipopolysaccharide epitope in the
absence of functional ValA, McDonald et al. suggested
ValA to be required for the transport of lipid A molecules
linked to core polysaccharide across the inner membrane.
6. Putative functions in lipid transbilayer transport and
exposure
The studies summarized above document the physiological importance of ABC proteins in lipid transport, their
malfunction being able to cause severe diseases. This raises
the question of the specific function of ABC proteins in lipid
transport (Fig. 2B).
Protein-mediated lipid transport can serve essentially two
functions, which may not exclude each other:
(i)
Lipid transbilayer transport, establishing, preserving or
perturbing a distinct asymmetric transbilayer lipid
distribution (asymmetric distribution of lipid species
between the two leaflets, or asymmetry in the number
of lipid molecules per leaflet, resulting in an area
difference between the two leaflets)
(ii) Exposure of lipids to acceptors: Besides being membrane constituents, some phospholipids are constituents
of bile and of pulmonary surfactant, and various
steroids act as hormones. In addition to movement
from one membrane leaflet to the other, some lipids
41
require therefore transport to other membranes, onto
lipoproteins, or into the extracellular lumen.
So far, there is no indication for an involvement of ABC
proteins in the generation of an asymmetric transbilayer
distribution of abundant lipids such as phospholipids or
cholesterol. All available quantitative data indicate that the
activity of ABC transporters in cellular membranes cannot
compete with the inward-directed activity of the flippases
described in Section 2 to affect transbilayer distribution on a
qualitative level. However, the activity of ABC proteins
may not be negligible. Several studies indicate that outwarddirected lipid transport mediated by ABC proteins can
modulate the transbilayer distribution even of abundant
phospholipids inward transported by energy-dependent
flippases in the mammalian plasma membrane. An example
is the enhanced exposure of aminophospholipids on the
outer plasma membrane leaflet of ABCB1 (MDR1 Pgp),
Yor1p, Pdr5p and ABCA1 expressing cells. ABC proteins
may thus affect physiological functions associated with
asymmetric transbilayer lipid distribution. Their outwarddirected lipid transporting activity may also counteract
vesicle budding driven by energy-coupled flippases (see
Section 2.2): While the overexpression of ABC genes in
yeast can lead to endocytosis defects [202,174], the loss of
ABCA1 function in Tangier fibroblasts is associated with
enhanced endocytosis [203], supporting a functional link
between lipid transport and vesicle biogenesis.
Furthermore, ABC proteins may play a role in the
transbilayer distribution of marginal, but physiologically
relevant lipids exhibiting slow passive transbilayer movement. In Section 7, models for lipid transbilayer transport by
ABC proteins will be discussed.
Most examples of ABC proteins implicated in lipid
transport indicate a role not primarily in lipid asymmetry,
but rather in the exposure of specific lipids on the
exoplasmic leaflet, allowing their uptake by acceptors and
further transport to specific sites: The lipopolysaccharide
precursor lipid A has to be delivered from the outer leaflet
of the inner membrane to the outer membrane of bacterial
cells, and PC transported across the canalicular membrane
by ABCB 4 (MDR3 Pgp) is taken up by bile salts in the bile
canalicular lumen. The necessity of appropriate exposure of
membrane-bound lipid to a cognate acceptor can maybe best
be illustrated for cholesterol: Deeply buried in the membrane with the polar OH group facing the aqueous phase and
the alkyl chain oriented toward the bilayer center, cholesterol has recently been shown to experience rapid flip-flop
in PC vesicles, and membranes of erythrocytes and
presumably most other cells (reviewed in [204]). The
primary function of ABCG5/8 may therefore very likely
not be cholesterol transbilayer transport across the canalicular membrane, but rather facilitation of lumenal cholesterol uptake (e.g. by mixed bile salt and PC micelles),
possibly by pushing it partly into the aqueous phase, as
suggested recently [205].
42
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
A similar mechanism may be relevant for ABCA1 in the
delivery of cholesterol to apoA-1, although this seems to be
more complicated: Here, PC is involved in the release of
cholesterol from the respective membranes, which has also
been suggested for ABCG5/8 [205].
Taking into account the opening of the central pore of
ABCB1 (MDR1 Pgp) to the lipid phase in the presence of
nucleotide (see Section 7), one could even ask whether ABC
proteins may take up substrates from the same leaflet from
which they are delivered to an acceptor.
While being hypothetical at present, different modes of
lipid exposure by ABC proteins could be imagined, which
may or may not include the transport of these lipids from the
opposite leaflet of the membrane to the side of acceptor
localization (Fig. 2B). In principle, transport and exposure
of the lipid to the acceptor could be a two-step process or be
combined in one step as proposed in the vacuum cleaner
model [1]. In the absence of an acceptor, the lipid might be
released to the exoplasmic leaflet (modifying transbilayer
lipid distribution as a side effect), while the activity of the
ABC protein is likely underestimated in this case.
7. Models for lipid transbilayer transport by ABC
proteins
ABC proteins display variable transmembrane domains
(TMDs), while the nucleotide binding domains (NBDs) are
highly conserved in ABC proteins of diverse origins. It is
therefore assumed that all ABC proteins bind and hydrolyze
ATP in a similar fashion and use a common mechanism on
the NBD level to power the translocation of substrate via the
TMDs [206]. In addition to biochemical data, structures
obtained by X-ray and electron cryo crystallography have
been used to propose models for the mechanism of substrate
transport by ABC proteins.
The main models currently discussed are the tilting
model and the rotating helix model [207]: In the tilting
model (Fig. 5), the TMDs are considered as rigid entities.
The protein possesses a chamber open towards the
cytoplasmic face. The substrate enters the chamber (e.g.
from the cytoplasmic aqueous phase or the inner leaflet of
the bilayer). ATP binding and hydrolysis cause conformational changes (tilting) of the TMDs, leading to the release
of the substrate into the exoplasmic or periplasmic aqueous
phase now accessible from the chamber.
In the rotating helix model (Fig. 6), the individual
movement of TM helices is taken into account. Conformational changes lead to the rotation of the TM helices, such as
to reorient the substrate binding site from the inner leaflet of
the lipid bilayer to the aqueous chamber, from where the
substrate is released either into the exoplasmic/periplasmic
aqueous phase or to the membrane outer leaflet. As both
models focus on different aspects, they may not be mutually
exclusive. The order of substrate binding, nucleotide binding and hydrolysis, and conformational changes, as well as
the interactions between the protein domains are subject of a
current discussion.
In the following, we will summarize information on
substrate transbilayer transport on the basis of structures of
prokaryotic export (MsbA) and import (BtuCD) ABC
proteins, and of the eukaryotic export protein ABCB1
(MDR1 Pgp) in the presence or absence of nucleotide.
7.1. Information derived from MsbA
The X-ray crystal structures of a homodimer of the halfsize ABC protein MsbA (see Section 5.1) from E. coli (4.5
2 resolution) and Vibrio cholera (3.8 2 resolution) obtained
by Chang and Roth [188] and Chang [208] in the absence of
nucleotide have been interpreted to reflect two different
conformational states of MsbA involved in transport of lipid
A, revealing an open (E. coli) and a closed conformation (V.
cholera) [191]. In E. coli MsbA, the TM helices are tilted
by 308 to 408 relative to the normal of the membrane, being
in intermolecular contact on the outer membrane leaflet side.
They form a cone shaped structure with large openings (25
2) towards the inner membrane leaflet at each of the dimer
interfaces. The openings lead into a cone-shaped chamber in
the interior of the TMDs. The cytoplasmic side of the
chamber contains positively charged residues, while the
periplasmic side is essentially hydrophobic. The base of the
chamber facing the cytoplasm is up to 45 2 wide.
The NBDs do not have intermolecular contact and are
separated by about 50 2. An intracellular domain (ICD) is
situated between the TMDs and the NBDs. However,
whether the tertiary structure proposed corresponds to a
native conformation has been questioned by several authors
[207,209,210] (see below).
In V. cholera MsbA, the TM helices are less tilted (108 to
308), and the openings towards the inner membrane leaflet
are more narrow (12 2) than in the E. coli structure.
However, the chamber volume remains large enough to
accommodate a lipid A molecule. TMDs and NBDs make
intermolecular contact. Compared to E. coli MsbA, the
alpha-domain of the NBD is rotated about 1208 along the
dimer axis and contacts the opposing NBD.
Chang [191] proposed the following transport model
(Fig. 5): In the open conformation, the NBDs are not
firmly attached to the ICD region connecting TMDs and
NBDs, and can rotate freely relative to the TMD. Substrate
recognition, perhaps by charge–charge interactions, at the
cytoplasmic chamber opening induces nucleotide binding,
changing the conformation of the NBDs and promoting
their dimerization. Dimerization drives trapping of the
substrate in the chamber (closed conformation), from
where it spontaneously flips to the periplasmic side due
to energetically unfavourable interaction of its hydrophobic
chains with the polar part of the chamber. The NBD dimer
cooperatively hydrolyzes ATP, leading to conformational
changes in the NBDs. Relayed through the ICD, the
conformational changes cause the TMDs to open towards
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
43
Fig. 5. (A) Backbone structure of E. coli and V. cholera MsbA. Structures of E. coli MsbA (Eco) (Chang and Roth [188]) (left) and V. cholera (VC) (Chang
[208]) MsbA (right) were obtained from the Protein Data Bank, 1JSQ and 1PD4, respectively. The transmembrane domains (TMDs) and nucleotide binding
domains (NBDs) are shown in yellow and green, respectively. The six positively charged residues in the chamber are in blue. (B) Model proposed for lipid
transport via MsbA (tilting model). The protein is in its open conformation (1). Substrate recognition (2) induces nucleotide binding, which promotes NBD
dimerization and trapping of the substrate that flips to the opposite side (closed conformation) (3). ATP hydrolysis causes opening of the TMDs to the outer
membrane side, releasing the substrate (4). ADP and Pi dissociation reset the protein into its initial state. See Section 7.1 for details, and also a model published
by Chang [191].
the periplasmic side, releasing the substrate. Upon the
dissociation of ADP and Pi, the NBDs separate from each
other and the TMDs are reset to the resting state. While
the two MsbA structures favor a tilting mechanism, the
data could also be consistent with the rotation of certain
helices during the catalytic cycle [191]. The subsequent
transfer of lipid A across the periplasmic space and to the
outer leaflet of the outer membrane is not fully understood
[193].
7.2. Information derived from BtuCD
The ABC protein BtuCD mediates the import of vitamin
B12 in E. coli. Although BtuCD has not been implicated
with lipid transport, its structure (resolved in the absence of
nucleotide by X-ray crystallography with a resolution of
3.2 2 [206]) provides important information on the
mechanism of substrate transport. The BtuCD complex is
a heterotetramer consisting of two copies each of the BtuC
(TMD) and BtuD (NBD) subunits. The overall structure
has been described to resemble an inverted portal. The two
TMDs consist of 10 helices each, unlike the 26 helices
predicted for a number of other ABC proteins. At the
interface of the TMDs, a cavity opens to the periplasmic
space and spans 2/3 of the predicted lipid membrane. The
cavity is closed to the cytoplasm by the so-called gate. The
cytoplasmic loop between the TM helices 6 and 7 (L loop)
provides most of the interface with the NBD. Due to the
lack of an ICD between the TMDs and the NBDs, the
NBDs are located just below the membrane surface. The
two TMDs, as the two NBDs, are in close contact to each
other.
The following transport mechanism has been proposed
[206]: The substrate (on the periplasmic side) interacts
44
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
Fig. 6. ABCB1 (MDR1 Pgp) structure and model proposed for lipid transport (rotating helix model). The protein is in its barrel conformation (left: structure;
right: model) (1). Substrate binding at the inner membrane leaflet induces nucleotide binding, leading to helix rearrangements which open a gap between two
TMDs, enabling the substrate to enter the central pore (2). The substrate moves from the central pore into the outer membrane leaflet or the extracellular milieu
(3). ATP hydrolysis resets the transporter into its initial state (4). See Section 7.3 for details. Electron micrographs of ABCB1 were taken from Rosenberg et al.
[209] (see Figs. 3a and d of the publication) with permission.
through the TMD via the connecting L loop with the
nucleotide binding site (this step might be particular for
importers, in which nucleotide binding site and substrate are
located on opposite sides of the membrane). The binding and
hydrolysis of ATP at the nucleotide binding site induce close
contact of the two NBDs, leading to long-range conformational changes, which force the TMDs to spread and to open
the gate of the translocation pathway at the interface of the
two NBDs. The substrate passes through the pathway open to
the cytoplasmic space either by diffusion or due to forces
applied by the TMDs. The release of ADP and Pi causes the
ABC protein to return into its resting state, including
reorientation of the NBDs. In contrast to the mechanism
proposed for MsbA, NBDs and TMDs remain in contact
throughout the transport cycle. BtuCD structure and comparison to the ATP-bound form of Rad50, a DNA double-strand
break repair enzyme with an ABC-type ATPase domain
[211], suggests a tilting mechanism, with the particularity of
describing substrate import. Locher and Borths have speculated on a common mechanism in im- and exporters,
considering the binding protein as the substrate in the case
of importer proteins [212].
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
7.3. Information derived from ABCB1 (MDR1 Pgp)
The structure of the full-size ABCB1 (see Section 4.1.2)
monomer was determined in the presence and absence of
nucleotide using electron cryo crystallography (20 2
resolution) [209]. In the absence of nucleotide, the TMDs
are approximately parallel and form a barrel surrounding a
central pore appearing to be open at the extracellular face of
the membrane, and closed at the intracellular face. Small
densities protrude into the pore. The binding of nucleotide
leads to substantial reorganization of the TMDs involving
the repacking and rotation of the TM helices within the
membrane: In the presence of nucleotide, the TMDs consist
of three clearly segregated domains, enclosing a central pore
less obviously closed towards the intracellular face, and
additionally open to the lipid phase along one side with a
gap appearing between two domains. This gap, equivalent
to most of the depth of the bilayer, could give substrates
from the lipid bilayer access to the central pore. Due to the
technique employed, data concerning the NBDs are
insufficient for exhaustive characterization.
The following transport model was proposed (Fig. 6)
[209,213]: Substrate binds to the protein from the inner
membrane leaflet. Subsequently, ATP binding leads to
conformational changes. The TMDs part in order to enable
the substrate to enter the central pore, giving it access to the
exoplasmic milieu. ATP hydrolysis resets the transporter
into its initial state as also suggested for BtuCD [212]. This
is in agreement with the finding that the binding and
hydrolysis of ATP can be independent events associated
with distinct conformational states of the transporter
[214,215]. Data from the two structures including substantial TM helices repacking upon nucleotide binding is
consistent with the helix rotation model, while not being
easily reconcilable with the tilting model.
When comparing the different structures and proposed
models of transport, important differences are obvious for
the ABC proteins discussed here. As it is unclear whether
the TMDs of different subfamilies are related to each other
either evolutionarily or structurally [209], TMDs might be
custom-made for the respective substrate. Obviously, the
TMDs for an importer of a hydrophilic molecule (e.g.
BtuCD) must differ from that for an exporter of amphiphilic
substrates (e.g. MsbA, ABCB1), whereas the mechanism of
transport could be a common one. While BtuCD has
sequence similarity to ABCB1 only on the level of the
NBDs, the ABCB family members ABCB1 and MsbA
share sequence similarity in both TMDs and NBDs [210].
However, the lack of a TMD:TMD interface and the large
distance between NBDs in the proposed E. coli MsbA
structure, incompatible with disulfide cross-linking studies
and structural data for ABCB1, lead Stenham et al. [210] to
question the validity of the proposed tertiary structure of E.
coli MsbA. In contrast, the tertiary structure of BtuCD
contains a parallel TMD:TMD interface and an NBD:NBD
interface as found in isolated prokaryotic NBDs.
45
By rotation of the E. coli MsbA NBDs by 1508 relative
to the cognate TMDs, Stenham et al. [210] have obtained a
structural model for ABCB1 with a consensus NBD:NBD
interface and a parallel TMD:TMD interface, consistent
with crosslinking data and electron crystallography studies.
According to this model, the TMDs surround a chamber
open at the exoplasmic surface and closed at the cytoplasmic surface. The rotating helix model appears to be
particularly suited for lipid transport, as the substrate can
access the transport pathway from the membrane, as
suggested to be necessary for ABCB1 mediated transport,
which might also partially explain ABCB1 substrate multispecificity [1]. Meanwhile, the various structures discussed
here reflect conformational snapshots of ABC proteins
during the transport cycle, and may not provide a sufficient
basis to unravel the transport mechanism and all involved
conformational intermediates.
It also remains to be established whether MsbA and
ABCB1 can serve as models for other lipid-transporting
ABC proteins not belonging to the ABCB family. Further
structural data from other ABC proteins is required to reveal
whether the TMDs of lipid-transporting ABC proteins are
structurally similar, and function according to the same
principle.
8. Conclusions
A long way from being solely a subject for basic
research, lipid transport reaches far into the clinical domain.
The transport of lipids across a membrane can have a net
secretory function for a cell or an organelle, or concern
mostly the transbilayer distribution of lipids in a membrane.
Cholesterol transport by ABCA1 out of macrophages, PC
transport by ABCB4 (MDR3 Pgp), the transport of fatty
acids into peroxisomes by members of the ABCD (ALD)
family, and steroid secretion by ABCG5 and 8 seem to be
examples for secretion events, where rather high quantities
of lipids must be efficiently transported to other membranes,
onto lipoproteins, or into the extracellular lumen. Here, the
exposure to appropriate acceptors appears to be an essential
step. In a membrane with an asymmetric distribution of the
lipid species across the two leaflets, on the other hand, a
comparatively slow transport of relatively few lipid molecules can be sufficient to help increase or break down this
asymmetry, having for example potential effects on signalling. This could be the case for the transport of PS by
ABCA1 in apoptotic cells, serving as a signal for
phagocytosis. Meanwhile, other proteins existing in the
same membrane may transport lipids in the same or the
opposite direction as ABC proteins. Knock-out studies in
mammals on ABC proteins have yielded unexpected results,
likely due to functional compensation by other ABC
proteins [216]. Similarly, transfection with one ABC gene
has been shown to influence the expression of another ABC
gene [217].
46
A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52
In addition, ABC proteins have been proposed to be
transporters, channels and regulators, meaning they can
potentially affect transbilayer transport of lipids directly, or
indirectly through regulation of other proteins.
This makes clear why experimental setups are needed
which quantify lipid transport in a comparable way, verify
its attribution to a particular protein in the absence of a
transporter background, determine its specificity and permit
the distinction between transport events against or with a
concentration gradient, requiring or not the hydrolysis of
ATP, and the presence of acceptor molecules.
It should be noted that ABC proteins implicated in the
transport of lipids can be localized in the plasma membrane,
as well as in intracellular membranes, complicating direct
measurements on cells. Now that all ABC proteins in
humans, and many in other organisms, have been identified,
reconstitution into vesicles large enough to allow lipid
redistribution (e.g. giant unilamellar vesicles (GUVs))
appears to be a promising technique to answer many current
questions. As the number of unequivocally identified lipid
transporter proteins is small, putative lipid bulk transporters
will be of particular interest as models.
However, the identification of lipid transport remains
difficult. Short-chain lipid analogs offer the advantage of
easy integration into and extraction from membranes, but
only reach a certain level of accordance with endogenous
lipids for these same reasons.
Endogenous lipids can be bound by certain antibodies or
lipid binding peptides or proteins such as Annexin V, and
they can be chemically modified (e.g. by TNBS), or
extracted from membranes via lipid transfer proteins. At
the same time, reliable techniques for the quantification of
endogenous lipids on a specific leaflet exist for a few lipid
species only. The combination of results obtained in model
systems with data from cells overexpressing ABC genes
upon stimulation, selection or transfection, as well as from
cells lacking functional ABC proteins upon mutation, knockout or inhibition will therefore be indispensable to obtain a
clear picture of the role of these proteins in lipid transport.
At the present state, the ABC protein superfamily can
already be considered to be of great importance in the
transport of lipids in prokaryotic as well as in eukaryotic
organisms, while the map of proteins responsible for lipid
transport, far from complete today, continues to be drawn.
Acknowledgements
The authors thank Thomas Pomorski, Erwin Schneider
(Humboldt University Berlin) and Francisco Gamarro
(Instituto de Parasitologia y Biomedicina, Granada), as well
as David Stroebel and Cecile Breyton (IBPC, Paris) for
critical reading of the manuscript. This work was supported
by grants from the EC research training networks Sphingolipids: Sphingolipid Synthesis and Organisation, and Lipid
flippases—Protein-mediated lipid translocation: Regulation
and physiological significance of transbilayer lipid distribution, and the COST action D22 Lipid–Protein Interaction.
Note added in proof
A detailed discussion of the mechanism of ABC proteinmediated transport is presented in the recent view of
Higgins and Linton [219]. First biochemical evidence for
the binding of N-retinylidene-PE to ABCA4 and for
dissociation of this lipids from ABCA4 upon binding and
hydrolysis of ATP has been shown recently [220].
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