Báo cáo khoa học: Function and regulation of ABCA1 – membrane meso-domain organization and reorganization pptx

Function and regulation of ABCA1 – membranemeso-domain organization and reorganization Kohjiro Nagao, Maiko Tomioka and Kazumitsu Ueda Institute for Integrated Cell–Material Sciences iCe

Function and regulation of ABCA1 – membrane

meso-domain organization and reorganization

Kohjiro Nagao, Maiko Tomioka and Kazumitsu Ueda

Institute for Integrated Cell–Material Sciences (iCeMS), Kyoto University, Japan

Introduction

The plasma membrane is critical for the life of the cell,

not only as the boundary maintaining the cytosolic

environment differently from the extracellular

environ-ment, but also as a platform for protein assembly,

which converts extracellular stimuli into intracellular

signals The skin lipid barrier prevents water loss from

our body, lipids in the myelin sheath functions as an

insulator of nerve fibers, and pulmonary surfactant

lip-ids at the air–water interface decrease alveolar surface

tension These lipids are appropriately transported in

our body and within cells, and their abnormal

deposi-tion causes cell death and various diseases (Table 1)

Several ATP-binding cassette protein A (ABCA)

sub-family proteins are involved in lipid transport between

organs and between intracellular compartments (Table 1) Furthermore, recent studies suggest that ABCA1 moves lipids not only between different mem-branes but also within the same membrane to organize and reorganize submicrometre (meso-scale) membrane domains such as lipid rafts In this review, the function and regulation of ABCA1 are summarized

Cholesterol homeostasis and ABCA1

Cholesterol is a key component of the cell membrane and is required for cell proliferation; however, excess accumulation of cholesterol is toxic to cells and its excess deposition in peripheral tissues causes

Keywords

ATP-binding cassette protein; cholesterol

homeostasis; lipid raft; membranes;

phospholipids

Correspondence

K Ueda, Institute for integrated

Cell–Material Sciences (iCeMS), Kyoto

University, Kyoto 606-8502, Japan

Fax: 81 75 753 6104

Tel: 81 75 753 6124

E-mail: uedak@kais.kyoto-u.ac.jp

(Received 17 December 2010, revised 27

February 2011, accepted 6 May 2011)

doi:10.1111/j.1742-4658.2011.08170.x

The ATP-binding cassette protein A1 (ABCA1) mediates the secretion of cellular-free cholesterol and phospholipids to an extracellular acceptor, apolipoprotein A-I, to form high-density lipoprotein Because ABCA1 is a key factor in cholesterol homeostasis, elaborate transcriptional and post-transcriptional regulations of ABCA1 have evolved to maintain cholesterol homeostasis Recent studies suggest that ABCA1 moves lipids not only between membranes but also within membranes to organize and reorganize membrane meso-domains to modulate cell proliferation and immunity

Abbreviations

ABCA1, ATP-binding cassette protein A1; apoA-I, apolipoprotein A-I; CK2, casein kinase 2; ECD, extracellular domain; HDL, high-density lipoprotein; JAK2, Janus kinase 2; LXR, liver X receptor; LXRE, liver X response element; MbCD, methyl-b-cyclodextrin; NBD, nucleotide binding domain; PC, phosphatidylcholine; PKA, protein kinase A; PKC, protein kinase C; PS, phosphatidylserine; RXR, retinoid X receptor;

SM, sphingomyelin; SREBP-2, sterol regulatory element binding protein 2.

atherosclerosis Excess cholesterol in peripheral tissues

is reversely transported as high-density lipoprotein

(HDL) to the liver Because cholesterol is not

catabo-lized in peripheral tissues, HDL formation is the only

pathway by which excess cholesterol is removed from

peripheral cells The inverse relationship between

plasma HDL levels and the risk of coronary artery

dis-ease is demonstrated [1], although genetically low

HDL per se may not predict the increased risk [2] At

least 70 mutations have been identified in the ABCA1

gene, leading to Tangier disease and familial

hypoal-phalipoproteinaemia, in which patients have a near

absence of or decrease in circulating HDL [3–8]

(Fig 1) More than 15 mutations examined show high

correlations between phospholipids, preferentially

phosphatidylcholine (PC) and cholesterol efflux [9–11],

indicating that ABCA1 influences the efflux of both

PC and free cholesterol Indeed, ABCA1, expressed in

cultured cells, mediates the secretion of both types of

lipids when lipid-free apolipoprotein A-I (apoA-I), an

Table 1 Possible substrates, localization and related diseases of ABCA subfamily proteins PE, phosphatidylethanolamine; PG, phosphatidyl-glycerol.

Possible substrates

Subcellular localization Tissue localization Related diseases ABCA1 (similarity

with ABCA1)

PS [81]

PC and cholesterol [54]

Oxysterols [105]

Cell surface, intracellular vesicles [11,31]

Macrophages, liver, small intestine, brain [5,106–108]

Tangier’s disease, atherosclerosis [5–7,13]

ABCA2 (59.2%) SM, gangliosides [109] Endosome [110] Brain [111,112] Alzheimer’s

disease [113–115]

ABCA3 (57.3%) PC, PG, PE [116–120] Intracellular vesicles,

lamellar bodies [121–123]

Lung alveolar type II cells [121,123]

Neonatal fatal surfactant deficiency and chronic interstitial lung disease [124,125] ABCA4 (66.7%) All-trans-retinal,

N-retinylidene-PE [126–128]

Intracellular disc membrane [129]

Photoreceptor cells (rod cells, cone cells) [129–131]

Stargardt muscular dystrophy, age-related macular degeneration [132–135]

endosome [136]

Brain, lung, heart and thyroid gland [136]

Dilated cardiomyopathy [136]

ABCA7 (69.3%) PC, SM,

cholesterol [77,79,137]

Plasma membrane and intracellular membranes [77,79,137,138]

Spleen, lung, adrenal, brain, liver, kidney (proximal tubule), peritoneal macrophages [79,137]

Unknown

ABCA8 (46.1%) Oestradiol-b-glucuronide,

taurocholate, LTC4, p-aminohippuric acid, ochratoxin-A [139]

ABCA12 (56.3%) Glucosylceramide [142] Lamellar bodies [143] Keratinocyte [143] Harlequin ichthyosis [144]

disorder, depression [145]

Fig 1 Putative secondary structure of human ABCA1 Five cyste-ine residues (C75, C309, C1463, C1465 and C1477) involved in the two intramolecular disulfide bonds between ECD1 and ECD2 [73] are indicated The PEST sequence [59], the 1-5-8-14, the putative calmodulin binding site [29], the PDZ binding motif [27,28] and PKA [38] and CK2 [43] phosphorylation sites are indicated (Not to scale.)

extracellular lipid acceptor in the plasma, is added to

the medium [11,12]

Transcriptional regulation of ABCA1

ABCA1-mediated cholesterol efflux is highly regulated

at the transcriptional level In peripheral cells, such as

macrophages and fibroblasts, ABCA1 gene expression is

enhanced by loading cholesterol [13] This response

is mediated by the nuclear receptors LXRa and

LXRb, whose ligands are sterol metabolites such as

22-(R)-hydroxycholesterol, 24-(S)-hydroxycholesterol,

27-hydroxycholesterol and 24-(S),25-epoxycholesterol

[14,15] LXRb is ubiquitously expressed, whereas LXRa

is restricted to the liver, adipose tissue, adrenal glands,

intestine, lungs, kidneys and cells of myeloid origin

Human LXRa expression is highly regulated and can be

autoregulated by itself, whereas human LXRb is stably

expressed even in the absence of excess cholesterol In

the basal state, LXRb and retinoid X receptor (RXR)

heterodimers are bound to liver X response elements

(LXREs) in the promoters of target genes [16] (Fig 2)

When cholesterol accumulates in cells, intracellular

con-centrations of oxysterols increase; subsequently, LXRb,

activated via the binding of oxysterols, stimulates the

transcription of ABCA1 [17–19] and also of LXRa

Interestingly, cholesterol feeding of mice or rats has failed to show a significant increase in hepatic ABCA1 mRNA expression [20] A promoter region, which responds to sterol regulatory element binding protein 2 (SREBP-2), was identified in the first intron of the ABCA1gene and was reported to be involved in the reg-ulation of ABCA1 expression in the liver [21] Recently, MiR-33, an intronic microRNA located within the gene encoding SREBP-2, a transcriptional regulator of cho-lesterol synthesis, was found to modulate the expression

of ABCA1 at the post-transcriptional level [22,23] An elaborate network of regulations has evolved to modu-late cholesterol synthesis and efflux to maintain choles-terol homeostasis

Post-translational regulation of ABCA1 activity

ABCA1-mediated cholesterol efflux is also highly regu-lated at the post-translational level Because cholesterol

is an essential component of cells, excessive elimination

of cholesterol can result in cell death Consequently, the ability to rapidly degrade ABCA1 in order to prevent excessive elimination is also important Indeed, ABCA1 protein turns over rapidly, with a half-life of 1–2 h [24–28] Several proteins, including a1-syntrophin,

A In the absence of excess cholesterol B When cholesterol accumulates

PC Chol

RXR

Oxysterol

LXR β

Cholesterol

Fig 2 LXR regulates ABCA1 not only in transcriptional level but also in post-translational level by direct binding In addition to its well-defined role in transcription, LXRb directly binds the C-terminal region of ABCA1 to mediate its post-translational regulation (A) In the absence of cholesterol accumulation, LXRb ⁄ RXR heterodimer binds to the C-terminal region of ABCA1 The ABCA1–LXRb ⁄ RXR complex sta-bly localizes to the plasma membrane, but is inactive in HDL formation (30,147) (B) When excess cholesterol accumulates, oxysterols bind

to LXRb leading to its dissociation from ABCA1 Because ABCA1 turns over rapidly with a half-life of 1–2 h, and because the transcription, splicing, translation and maturation of ABCA1, at more than 2000 amino acid residues, takes several hours after transcriptional activation, cells cannot cope with an acute accumulation of cholesterol for several hours This post-translational regulation allows ABCA1 to cause an immediate early response against acute cholesterol accumulation LXRb has at least two distinct roles in controlling cholesterol homeostasis (modified from [148]).

b1-syntrophin, calmodulin and apoA-I have been

reported to interact with ABCA1 and reduce the rate of

ABCA1 protein degradation [25–29]

The degradation of ABCA1 is regulated [27,28,30]

and is carried out via several pathways: (a) cell-surface

ABCA1 is endocytosed and recycled back to the

plasma membrane or delivered to the lysosomes

through early and late endosomes for degradation

[31,32]; (b) calpain protease degrades ABCA1 on the

plasma membrane [25,26] and intracellularly, especially

when apoA-I does not bind to ABCA1 [33] ABCA1 is

also degraded through the ubiquitin–proteasome

path-way [34,35] COP9 signalosome complex, which plays

an important role in the degradation of various

proteins such as IjBa, associates with ABCA1 and

controls the ubiquitinylation and deubiquitinylation of

ABCA1 [36]

Several protein kinases including protein kinase A

(PKA), protein kinase C (PKC), Janus kinase 2 (JAK2)

and casein kinase (CK2) are involved in the regulation

of ABCA1 activity and stability by apoA-I The

inter-action of apoA-I with ABCA1 increases the cellular

cAMP content and ABCA1 phosphorylation [37] This

phosphorylation is important for ABCA1 activity, as

apoA-I-dependent phospholipid efflux is decreased

sig-nificantly by the mutation of the PKA phosphorylation

site, Ser-2054, of ABCA1 [38] ApoA-I also activates

PKCa and phosphorylation of ABCA1 [39,40] This

reaction leads to the protection of ABCA1 from its

degradation by calpain On the other hand,

phosphory-lation of Thr-1286 and Thr-1305 in the PEST sequence

(rich in proline, glutamic acid, serine and threonine)

(Fig 1) within the cytoplasmic domain of ABCA1

pro-motes calpain degradation and is reversed by apoA-I

[41] Unsaturated fatty acids destabilize ABCA1 by

phosphorylation through a PKCd pathway [42]

Phos-phorylation of Thr-1242, Thr-1243 and Ser-1255 by

CK2 decreases the ABCA1 flippase activity, apoA-I

binding and lipid efflux [43] Calmodulin interacts with

ABCA1 at a close or overlapping position to the CK2

phosphorylation site and this interaction regulates

calpain-mediated ABCA1 degradation [29] The

inter-action of apoA-I with ABCA1 for only minutes

stimu-lates autophosphorylation of JAK2, which in turn

activates ABCA1-dependent lipid efflux [44]

Interest-ingly, the apoA-I-mediated activation of JAK2 also

activates STAT3, which is independent of the lipid

efflux activity of ABCA1 The apoA-I⁄ ABCA1

path-way in macrophages is proposed to function as an

anti-inflammatory receptor through activation of JAK2⁄

STAT3 [45] Cyclosporine A and FK506 were reported

to abolish ABCA1-dependent lipid efflux by inhibiting

the Ca2+-dependent calcineurin⁄ JAK2 pathway [46]

CDC42, a member of the Rho GTPase family, is reported to interact with ABCA1 and the interaction enhances apoA-I-mediated cholesterol efflux [47] Palm-itoylation of ABCA1 is also involved in the trafficking and function of ABCA1 [48]

We have reported [30] that the LXRb⁄ RXR complex binds to ABCA1 when the intracellular concentration

of oxysterols is low, and the ABCA1–LXRb⁄ RXR complex is distributed on the plasma membrane but is inert in terms of cholesterol efflux (Fig 2) When cho-lesterol accumulates and the intracellular concentration

of oxysterols increases, oxysterols bind to LXRb and the LXRb⁄ RXR complex dissociates from ABCA1 Once free from LXRb⁄ RXR, ABCA1 is active in the formation of HDL and decreases the local cholesterol concentration immediately Upon binding to oxysterols, LXRb⁄ RXR activates the transcription of ABCA1 and other genes Consequently, LXRb can cause a post-translational response, as well as a transcriptional response, to maintain cholesterol homeostasis This novel mechanism is an immediate early response to cope with rapid increases in intracellular cholesterol, such as when macrophages consume apoptotic cells

Effects of Tangier mutations on ABCA1

From patients with Tangier disease and familial hypo-alphalipiproteinaemia, more than 70 mutations have been identified in the ABCA1 gene Most mutations reside in extracellular domains (ECDs) (putative

apoA-I binding site) and nucleotide binding domains (NBDs) (driving-force-supplying site) of ABCA1 [3] (Fig 1) Mutations can be categorized into three groups (Table 2) The first is the ‘maturation defect mutant’ and the majority of mutations belong to this group Wild-type ABCA1, modified with complex oligosaccha-rides, is mainly localized to the plasma membrane and sometimes in the intracellular compartments [11,31]; however, maturation defect mutants of ABCA1, modi-fied with high mannose type oligosaccharides, have impaired trafficking and are localized inappropriately

to the endoplasmic reticulum [10,11,49] Because apoA-I interacts with ABCA1 on the cell surface, mutants which do not reach the plasma membrane are unable to mediate apoA-I-dependent lipid efflux The second group is the ‘apoA-I binding defect mutant’ This group is represented by the C1477R mutant of the second ECD (Table 2) Although C1477R mutant is expressed in the plasma membrane like wild-type ABCA1, apoA-I binding is abolished [9,10,49,50] The third group is the ‘lipid translocation defect mutant’, represented by the W590S mutation in the first ECD of ABCA1 (Table 2) The subcellular distribution of

W590S is indistinguishable from that of wild-type

ABCA1 Furthermore, W590S mutation does not affect

apoA-I binding [9,10,49,50]; however, W590S mutation

reduced phosphatidylserine (PS) flopping activity of

ABCA1, detected with the annexin V binding assay

[49], and impaired sodium taurocholate-dependent

cho-lesterol and phospholipid efflux by ABCA1 [51] These

results suggest that W590S mutation affects the lipid

translocation activity of ABCA1 and that the two

activities of ABCA1 (apoA-I binding and lipid

translo-cation) can be separable (Fig 3A) Additionally,

W590S mutation retarded the dissociation of apoA-I

from ABCA1 [51] Lipid translocation by ABCA1 is

supposed to facilitate the dissociation of apoA-I from

ABCA1 ApoA-I is reported to undergo a

conforma-tional transition in response to lipid [52], and lipidated

apoA-I does not interact with ABCA1 [53,54] The

con-formational transition of apoA-I caused by lipid

load-ing durload-ing bindload-ing to ABCA1 may facilitate the

dissociation of apoA-I from ABCA1 [55] (Fig 3A)

Subcellular localization and function of

ABCA1

ABCA1 localizes mainly to the plasma membrane but

sometimes also localizes in the intracellular

compart-ments Two distinct mechanisms have been proposed

for ABCA1-mediated HDL formation One is that

ABCA1 mediates the complex formation of apoA-I

with phospholipids and cholesterol on the cell surface

(cell-surface model), and the other is that apoA-I binds

to ABCA1 on the cell surface and ABCA1⁄ apoA-I

complexes are subsequently internalized ApoA-I⁄ lipid

complexes are formed (probably via ABCA1 activity)

in late endosomes and re-secreted by exocytosis

(retro-endocytosis model) Takahashi and Smith [56] first

showed that, following internalization, apoA-I is

recycled back to the cell surface to be re-secreted The

internalized ABCA1 and apoA-I were reported to

co-localize within late endosomes, and ABCA1 rapidly

shuttled between intracellular compartments and the plasma membrane [31,57] Trapping ABCA1 on the plasma membrane by cyclosporine A treatment reduces apoA-I-mediated cholesterol efflux [58] Deletion of the PEST sequence blocks its endocytosis and decreases apoA-I-mediated efflux of cholesterol after loading the late endosome⁄ lysosome pool of cholesterol by

Fig 3 Membrane meso-domain organization and reorganization by ABCA1 (A) Two proposed mechanisms for HDL formation (i) Membrane phospholipids and cholesterol are translocated by ABCA1 and (ii) are loaded to apoA-I directly bound to the ECDs of ABCA1 to generate nascent HDL particles [55] (iii) Membrane phospholipid translocation via ABCA1 induces bending of the mem-brane bilayer to create high curvature sites, to which apoA-I binds and solubilizes membrane phospholipid and cholesterol to create nascent HDL particles [95] (B) Regulation of cell signalling by ABCA1 (i) ABCA1 translocates membrane phospholipids and cho-lesterol and (iv) changes membrane meso-damain organization, such as lipid rafts, that lead to suppressed receptor-mediated sig-nalling events [99].

Table 2 Effects of mutations on the functions of ABCA1.

Class

Q597R a–c

DL693b,c N935Sb

A1046D b M1091T b

S1506Lb N1800Hb,d R2081Wb

W590S a–e

a [11] b [10] c [49] d N1800 is predicted to reside in the loop between TM11 and TM12 e [9] f [146].

acetylated low-density lipoprotein treatment [59] These

results together support the idea that nascent

lipopro-tein particles are formed in intracellular compartments

and subsequently secreted from the cell

There are also many reports that ABCA1-mediated

cholesterol efflux to apoA-I mainly occurs on the

cell surface and that the retroendocytosis pathway

does not contribute significantly to HDL formation

[33,60,61] The majority of internalized apoA-I is

directly transported to late endosomes and lysosomes

for degradation, and blocking endocytosis does not

decrease apoA-I-dependent cholesterol efflux

Although apoA-I is specifically taken up by

macro-phages, only a small fraction of apoA-I is re-secreted

from these cells Furthermore, the majority of

re-secreted apoA-I is degraded in the medium,

suggest-ing that the mass of retroendocytosed apoA-I is not

sufficient to account for HDL formation; however,

these studies were performed using macrophages and

other cells without cholesterol loading

As pointed out by Oram [62], the mechanism of

HDL formation is probably different whether excess

cholesterol has accumulated within cells or not

ABCA1 and apoA-I are endocytosed via a

clathrin-and Rab5-mediated pathway clathrin-and recycled rapidly back

to the cell surface, at least in part via a Rab4-mediated

route; approximately 30% of the endocytosed ABCA1

is recycled back to the cell surface [32] When

clathrin-mediated endocytosis is inhibited, the level of ABCA1

at the cell surface increases and apoA-I internalization

is blocked Under these conditions, apoA-I-mediated

cholesterol efflux from cells that have accumulated

lipoprotein-derived cholesterol is decreased, whereas

efflux from cells without excess cholesterol is increased

[32] These results suggest that the retroendocytosis

pathway of ABCA1⁄ apoA-I contributes to HDL

for-mation when excess lipoprotein-derived cholesterol has

accumulated in cells This study is also in agreement

with previous studies that blocking retroendocytosis of

ABCA1 does not affect cholesterol efflux from cells in

the absence of excess cholesterol [33,60,61] ABCA1

probably follows the same constitutive recycling

path-way as the LDL receptor [63]

Lipid acceptor for ABCA1

Because cholesterol and phospholipids are very

hydro-phobic, lipid acceptors which solubilize them in

aque-ous solutions are required for lipid secretion Under

physiological conditions, apoA-I and apoE, containing

amphipathic helices, function as acceptors of lipids

secreted into serum by ABCA1 Although other

amphipathic-helical-peptide-containing proteins (e.g

apoA-II, apoC-I, C-II, C-III, PLTP and serum amy-loid A) also function as lipid acceptors for ABCA1-dependent cholesterol efflux, their physiological contri-butions remain to be clarified [64–67] Synthetic amphipathic helical peptide (37pA) also promotes cho-lesterol and phospholipid efflux from ABCA1-express-ing cells [68,69] Furthermore, the 37pA peptide synthesized with d amino acids is as effective as that with l amino acids From these results, the amphi-pathic helix is considered to be a key structural motif for peptide-mediated lipid efflux from ABCA1 [68]

We reported that sodium taurocholate can also serve

as an acceptor for cholesterol and phospholipids trans-located by ABCA1 [51]; therefore, the detergent-like property of the amphipathic helix might be important

as a lipid acceptor

It is proposed that apoA-I directly binds to ABCA1

in the process of HDL formation, shown by several groups via crosslinking experiments [54,65,70–72] Because the 3-A˚ crosslinker can crosslink apoA-I with ABCA1, the pair of reactive amino acids of ABCA1 and apoA-I are within a distance of £ 3 A˚ [70] Hozoji

et al found that two intramolecular disulfide bonds are formed between ECD1 and ECD2 of ABCA1, and these two disulfide bonds are necessary for apoA-I binding and HDL formation [73] (Fig 1) It is reported that apoA-I is not crosslinked with the ATPase-deficient mutant form of ABCA1 [74] Fur-thermore, fluorescent-labelled apoA-I does not bind to cells expressing ATPase-deficient mutant [51,75] These results suggest that the large ECD of ABCA1 is the direct binding site for apoA-I and its ATP-dependent conformational change is required for apoA-I binding But, it is also proposed that apoA-I interacts with a special domain on the plasma membrane apart from ABCA1 and solubilizes membrane lipids

Transport substrates

Substrates transported directly by ABCA1 are still controversial [55] (Table 1) Several models have been proposed for the mechanism of ABCA1-mediated HDL formation: (a) a two-step process model in which ABCA1 first mediates PC efflux to apoA-I, and this apoA-I–PC complex accepts cholesterol in an ABCA1-independent manner; (b) a concurrent process model in which PC and cholesterol efflux by ABCA1 to apoA-I are coupled to each other; and (c) a third model in which ABCA1 generates a specific apoA-I binding site

on the plasma membrane with subsequent translocation

of PC and cholesterol to apoA-I When it was proposed [74,76], the two-step model looked the most plausible, because photoactive PC could be crosslinked with

ABCA1 whereas direct binding of photoactive

choles-terol to ABCA1 could not be detected [74]; however,

analysis of the functions of ABCA7 raised questions

about this model Human ABCA7, which has the

high-est homology (69.3%) to ABCA1, mediates the

apoA-I-dependent efflux of PC and cholesterol, similar to

ABCA1 [77]; however, human ABCA7 mediates

choles-terol release much less efficiently than ABCA1 [78], and

PC but not cholesterol are loaded onto apoA-I by

mouse ABCA7 [79] These results cannot be explained

by the two-step process model Instead, they suggest

that ABCA1 has higher affinity for cholesterol

trans-port than ABCA7, and are consistent with the

concur-rent process model [80] The third model was originally

that ABCA1 mediates the translocation of PS to the

outer leaflet to form a special membrane domain, where

apoA-I binds and solubilizes membrane lipids [81]

Membrane meso-domain organization

and reorganization

On the plasma membrane, various meso-scale (10–

100 nm) domains, such as lipid rafts [82], are supposed

to be dynamically organized and reorganized and

involved in various cellular functions, such as signal

transduction ABCA1 is involved in membrane

meso-domain reorganization, as ABCA1 expression results

in a significant redistribution of cholesterol and

sphin-gomyelin (SM) from Triton X-100-resistant membrane

domains [83] Caveolin also redistributes from

punctu-ate caveolae-like structures to the general area of the

plasma membrane [83] Macrophages from

ABCA1-deficient mice exhibited increased lipid rafts on the cell

surface [84] and ABCA1 made cells more sensitive to

methyl-b-cyclodextrin (MbCD) treatment [80] and

generated cholesterol-oxidase-accessible membrane

domains [85]

Importantly, membrane meso-domain reorganization

by ABCA1 is independent of apoA-I [83,85] It is

possi-ble that active lipid translocation via ABCA1, the flop

of phospholipids and cholesterol from the inner to

outer leaflet, leads to membrane destabilization Since

ABCA1 is not associated with Triton X-100-resistant

membrane domains [86,87], the domains are

destabi-lized via lipid translocation by ABCA1 located outside

the domains It is reported that ABCA1 is associated

with Luburol WX-resistant membranes in

cholesterol-loaded monocyte-derived macrophages [87], that

ABCA1 and flotillin-1 are co-localized in these

deter-gent-resistant membranes and can be co-precipitated

[88], and that expression of caveolin-1 enhances

apoA-I-dependent cholesterol efflux in hepatic cells [89]

ABCA1 may localize just outside Triton X-100-resistant

membrane domains But ABCA1 is not recovered from either Triton X-100- or Luburol WX-resistant mem-branes in fibroblasts [87] It is not clear how lipid trans-location by ABCA1 in non-raft regions reorganizes other domains of the plasma membrane

We analysed the effects of the cellular SM level on the function of ABCA1 using a CHO-K1 mutant cell line, LY-A [90], which has a missense mutation in the ceramide transfer protein CERT, and reported that the decrease in SM content in the plasma membrane stim-ulates apoA-I-dependent cholesterol efflux by ABCA1 [91] The amount of cholesterol available to cold MbCD extraction is increased when SM content is reduced However, apoA-I-dependent cholesterol efflux

is not observed without ABCA1 expression even when

SM content is reduced, suggesting that the cholesterol available to cold MbCD cannot be loaded onto

apoA-I spontaneously When ABCA1 is expressed, the cho-lesterol available to cold MbCD is increased by 40% This effect of ABCA1 is independent of apoA-I These results suggest that ABCA1 translocates membrane lipids in detergent-soluble domains and makes (acti-vates [92] or projects [55]) cholesterol available to cold MbCD The effect of the reduction of plasma mem-brane SM content on the function of ABCA1 is quite different from that on the function of ABCG1, since ABCG1-mediated cholesterol efflux decreases when cellular SM content is reduced [93] This is consistent with previous reports that lipids loaded onto lipid-poor apoA-I by ABCA1 are provided by Triton X-100-sen-sitive membrane domains, whereas lipids loaded onto HDL, the lipid acceptor for ABCG1, are derived from Triton X-100-resistant membrane domains [86,87], and that treating rat fibroblasts with SMase increased apoA-I- or apo-E-dependent cholesterol efflux [94] Several studies suggest that ABCA1 generates spe-cial membrane meso-domains Membrane phospho-lipid translocation via ABCA1 induces bending of the membrane bilayer to create high curvature sites, such

as is created in 20-nm-diameter small unilamellar vesi-cles, to which apoA-I binds and solubilizes membrane phospholipid and cholesterol to create nascent HDL particles [95] (Fig 3A) In agreement with this concept, plasma membrane protrusions [54] and apoA-I binding

to protruding plasma membrane domains [96] have been observed in cells expressing ABCA1 Such apoA-I binding could be enhanced by PS molecules translocated

to the exofacial leaflet by ABCA1 [81], because the presence of PS enhances vesicle curvature [97] How-ever, there are still clear differences in the dependence

on apoA-I concentration between ABCA1-mediated lipid efflux and in vitro solubilization of phospholipid vesicles [95] It has been calculated that only a portion

of the apoA-I associated with the cell surface binds

directly to ABCA1 [72,98] One plausible explanation

may be that direct interaction with ABCA1 causes

conformational changes in apoA-I to make it open

and receptive to lipids Activated apoA-I may interact

with the special meso-domains created by ABCA1

Recently it was reported that LXR signalling at a

metabolic checkpoint modulates T cell proliferation

and immunity [99] T cell activation is accompanied by

the downregulation of LXR target genes, ABCA1 and

ABCG1 [100] Loss of LXR expression confers a

pro-liferative advantage to lymphocytes, resulting in

enhanced homeostatic and antigen-driven responses

Conversely, ligand activation of LXR inhibits

mitogen-driven T cell expansion Cellular cholesterol is required

for increased membrane synthesis during cell

prolifera-tion Additional mechanisms may also be involved,

such as changes in membrane meso-domain

organiza-tion, e.g lipid rafts, that lead to enhanced

receptor-mediated signalling events Functional deficiencies of

ABCA1 and ABCG1 cause enhanced inflammatory

responses of macrophages, especially after treatment

with lipopolysaccharide or other toll-like receptor

ligands [84,101–103] ABCA1 and ABCG1 may

pro-mote membrane lipid redistribution [83,104], which

modulates raft-dependent cell signalling (Fig 3B)

Acknowledgements

This study was supported by a Grant-in-aid for

Scien-tific Research (S) from the Ministry of Education,

Cul-ture, Sports, Science and Technology of Japan, the

Bio-oriented Technology Research Advancement

Insti-tution, and the World Premier International Research

Center Initiative, MEXT, Japan

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