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Biochimica et Biophysica A

Review

Function of prokaryotic and eukaryotic ABC proteins in lipid transport

Antje Pohla,b, Philippe F. Devauxb, Andreas Herrmanna,*

aHumboldt-University Berlin, Institute of Biology, Invalidenstr. 42, D-10115 Berlin, GermanybInstitut 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]

1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbalip.2004.12.007

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-oxa-

1,3-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).

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 eukary-

otic 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,

cta 1733 (2005) 29–52

A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–5230

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.

2. Lipid transbilayer movement

Lipids form a vast group of chemically different

amphiphilic or hydrophobic substances containing a sub-

stantial portion of aliphatic or cyclic hydrocarbon. Abundant

Fig. 1. Lipid substrates of ABC proteins (examples). Hydrophilic parts are indicate

(in PE), choline (in PC) or serine (in PS). Examples for the sphingolipid moiety Y a

galactose (in GalCer) or lactose (in LacCer). The fatty acid shown is oleic acid, th

[218].

lipid classes are (glycero-) phospholipids, sphingolipids,

steroids, lipopolysaccharides and triacylglycerols (for some

examples see Fig. 1). The phospholipids phosphatidylcho-

line (PC), phosphatidylethanolamine (PE), phosphatidylser-

ine (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

The rate of spontaneous transbilayer movement (flip-

flop) 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

d by blue clouds. Examples for the phospholipid moiety X are ethanolamine

re hydrogen (in ceramide), phosphorylcholine (in SM), glucose (in GlcCer),

e steroid shown is cholesterol. Lipid A structure as predominant in E. coli

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).

1 (Typically, the abbreviation for [N-(7-nitrobenz-2-oxa-1,3-diazol-4

yl)amino] is written in capital letters (NBD). However, in order to avoid

confusion with the abbreviation for nucleotide binding domain, this style o

abbreviation was chosen).

A. Pohl et al. / Biochimica et Biophysica Acta 1733 (2005) 29–52 31

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 asym-

metrically 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 respon-

sible 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 demon-

strated 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 move-

ment across ER and Golgi membranes [23,24].

Energy-independent, bi-directional transbilayer move-

ment 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 phospho-

lipids 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, energy-

dependent 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

-

f

Table 1

ABC proteins involved in lipid transport

Organism Family Name Trivial name Involvement Lipid analogs Endog. lipids

Human ABCA ABCA1 ABC1 macrophage lipid homeostasis SL-PS cholesterol

phospholipidsHDL deficiencies

Tangier disease

phagocytosis

ABCA2 ABC2 macrophage lipid homeostasis? steroids?

neural development?

ABCA3 ABC3 lung surfactant synthesis? PC?

ABCA4 ABCR

Rim

dark adaptation N-retinylidene-PE

Stargardt disease

ABCA6 macrophage lipid homeostasis?

ABCA7 ABCX hematopoiesis? cholesterol

phospholipids

ceramide

macrophage lipid homeostasis?

keratinocyte differentiation?

ABCA9 hematopoiesis?

macrophage lipid homeostasis?

ABCA10 macrophage lipid homeostasis?

ABCB ABCB1 MDR1 Pgp detoxification C6-nbd-PC

C6-nbd-PE

C6-nbd-PS

C6-nbd-SM

C6-nbd-GlcCer

steroids

cholesterol

GlcCer, PS,

SM, PAF

multidrug resistance

adrenal secretion

dendritic cell migration

ABCB4 MDR2/3 Pgp bile formation C6-nbd-PC PC

progessive familiar

intrahepatic cholestasis

ABCC ABCC1 MRP1 detoxification C6-nbd-SM

C6-nbd-GlcCer

C6-nbd-PC

C6-nbd-PS

multidrug resistance?

LTC inflammatory response

ABCC6 MRP6 pseudoxanthoma elasticum

lipid transport and metabolism

ABCD ABCD1 ALDP beta oxidation VLCFAs?

X-Adrenoleukodystrophy

peroxisome biogenesis

ABCD2 ALDRP

ALDL1

beta oxidation VLCFAs?

ABCD3 PMP70

PXMP1


peroxisome biogenesis

ABCD4 PXMP1L

P70R

PMP69


ABCG ABCG1 WHITE macrophage lipid homeostasis cholesterol, PC

ABCG2 BCRP1

MXR1

ABCP

detoxification multidrug resistance bodipy-Cer

C6-nbd-PS

C6-nbd-PC

steroids? PS

ABCG5 WHITE3 bile steroid secretion cholesterol

steroidssitosterolemia

ABCG8 WHITE4 bile steroid secretion cholesterol

steroidssitosterolemia

Sea urchin ABCA SuABCA sperm acrosome reaction? phospholipid?

cholesterol?

Leishmania ABCA LtrABC1.1 parasite–host interaction vesicular transport C6-nbd-PC

C6-nbd-PE

C6-nbd-PS

ABCB LPgp-lp multidrug resistance bodipy-PC

alkyl-lyso-

phospholipids

Arabidopsis PMP

(ABCD)

Ped3p COMATOSE

CTS PXA1

beta oxidation FA CoAs?

Yeast PDR/CDR Pdr5p drug resistance C6-nbd-PE PE, steroids

Cdr1p drug resistance C6-nbd-PC

C6-nbd-PE

C6-nbd-PS

PE


Organism Family Name Trivial name Involvement Lipid analogs Endog. lipids

Cdr2p drug resistance C6-nbd-PC

C6-nbd-PE

C6-nbd-PS

phospholipid?

Cdr3p C6-nbd-PC

C6-nbd-PE

C6-nbd-PS

phospholipid?

MDR

(ABCB)

Ste6p pheromone transport lyso-PC?

drug resistance?

MRP/CFTR

(ABCC)

Yor1p drug resistance C6-nbd-PE

efflux organic anions?

ALDp

(ABCD)

Pat1p Pxa2p peroxisomal lipid transport VLCFAs?

Pat2p Pxa1p peroxisomal lipid transport VLCFAs?

E. coli ABCB MsbA lipid A transport lipid A

phospholipids

L. lactis ABCB LmrA drug resistance C6-nbd-PE lipid A

lipid A transport?

F. novicida ? ValA lipid A transport? 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.

Table 1 (continued)


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 trans-

port of PC across the plasma membrane and may contain

either a PC-specific translocase in addition to the amino-

phospholipid translocase, or an inward translocase of

different specificity which transports both aminophospholi-

pids 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 trans-

Golgi-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 sug-

gested to be involved in the outward transport of phospho-

lipids [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 (dis-

cussed 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-

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 choles-

terol analogs have been studied showing large variations in

the potential to mimic endogenous cholesterol [44]. Never-

theless, 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

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 lipid-

linked diseases (families A, B, C, D, andG)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]. ABCA1-

GFP 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 cytoplas-

mic leaflet of mammalian plasma membranes) to the

http://nutrigene.4t.com/humanabc.htm


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 phagocytiz-

ing 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 hydro-

phobic 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 macro-

phage 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-trans-

retinol 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 ethanol-

amine (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 develop-

ment [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 differentia-

tion, 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 GFP-

tagged ABCA7 of L929 cells triggered apolipoprotein-

mediated 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 ubiqui-

tous expression, the highest levels being found in the heart,

brain and fetal tissues. ABCA9 is induced during macro-

phage differentiation. Different from ABCA7, the expres-

sion 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].


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 pharmacolog-

ical borders of the body (intestine, blood–brain barrier, feto-

maternal 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 trans-

fected 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 mesan-

gial 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]. Inter-

estingly, 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

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 trans-

bilayer 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 transmem-

brane 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


ABCB1 (MDR1 Pgp) which exports toxins into the external

environment). Additionally, ABCC1 mediates the leuko-

triene 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 knownmembers 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 trans-

port via ABCD proteins. ABCD half-size proteins can form

not only homodimers (ABCD1–ABCD1), but also hetero-

dimers (ABCD1–ABCD2, ABCD1–ABCD3, ABCD2–

ABCD3) [126]. Although the heterodimers might be func-

tional, 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 mitochon-

dria was suggested [132]. ABCD1 has been implicated with

peroxisome biogenesis, restored upon ABCD1 overexpres-

sion 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 normal-

ize 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


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 over-

expressing 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 stimu-

lated 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

implicated in the sperm acrosome reaction during fertiliza-

tion. 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 Trypanoso-

matidae family, causing Leishmaniasis and Trypanosomia-

sis, 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 expres-

sion 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 (endo-

sperms, cotyledons) and senescent organs [168]. Both halves

of Ped3p show significant sequence homology to the human


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. 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 compar-

ison and phylogenetic classification. Both half-size and full-

size 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 pro-

cesses 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 excep-

tional, 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].

4.5.3. 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].

4.5.4. ALDp family (ABCD homolog)

Pat1p (also designated Pxa2p) and Pat2p (also desig-

nated 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 Gram-

negative 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 glucos-

amine unique to Gram-negative bacteria, is a major

component of the outer membrane, representing the hydro-

phobic 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.


The structure and potential mechanisms of MsbA will be

discussed in Section 7.

LmrA, a half-size protein in L. lactis forming homo-

dimers, 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 ATP-

dependent transport of fluorescent short-chain nbd PE [197].

However, short-chain nbd PC was not recognized, suggest-

ing headgroup-specificity, different from the low substrate

specificity of ABCB1 despite the high sequence conserva-

tion. 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 physiolog-

ical 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 mem-

brane 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

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 outward-

directed 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 outward-

directed 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 move-

ment. 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 mem-

brane with the polar OH group facing the aqueous phase and

the alkyl chain oriented toward the bilayer center, choles-

terol 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 canal-

icular membrane, but rather facilitation of lumenal choles-

terol uptake (e.g. by mixed bile salt and PC micelles),

possibly by pushing it partly into the aqueous phase, as

suggested recently [205].


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 conforma-

tional 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. Conforma-

tional 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 bind-

ing 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 half-

size 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 to308), 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

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 2�6 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

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 conforma-

tional 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 compar-

ison 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 specu-

lated on a common mechanism in im- and exporters,

considering the binding protein as the substrate in the case

of importer proteins [212].


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 2resolution) [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 substan-

tial 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.

By rotation of the E. coli MsbA NBDs by 1508 relativeto 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 cytoplas-

mic 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 multi-

specificity [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 mole-

cules can be sufficient to help increase or break down this

asymmetry, having for example potential effects on signal-

ling. 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].


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, knock-

out 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 Sphingo-

lipids: Sphingolipid Synthesis and Organisation, and Lipid

flippases—Protein-mediated lipid translocation: Regulation

and physiological significance of transbilayer lipid distribu-

tion, and the COST action D22 Lipid–Protein Interaction.

Note added in proof

A detailed discussion of the mechanism of ABC protein-

mediated 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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