CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS

CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS CHAPTER 2 MEMBRANE TOPOLOGY OF THE HUMAN ABC TRANSPORTER PROTEINS

ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9

Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

2

CHAPTER

The most recent annotation* of the human

genome sequence revealed 48 genes for ABC

proteins, which were grouped into seven

subclasses, from ABCA to ABCG (see: http://

nutrigene.4t.com/humanabc.htm; see also

Chapter 3) As detailed elsewhere in this book,

ABC proteins in all organisms can be

recog-nized by their conserved motifs within the

ATP-binding domains The majority of these proteins

are membrane embedded and fulfill various

membrane transport or regulatory functions

(hence the designation ABC transporters), and

our present review deals with such human

pro-teins In contrast, the human ABCE and ABCF

subfamilies contain proteins with no known

transmembrane domains; therefore these are

outside the scope of this chapter

It is generally accepted that the minimum functional unit requirement for an ABC

trans-porter is the presence of two transmembrane

domains (TMD; IM in Chapter 1) and two

ATP-binding cassette (ABC) units These may be

present within one polypeptide chain (‘full

transporters’), or within a membrane-bound

homo- or heterodimer of ‘half transporters’

At present there are no high-resolution struc-tural data available for any mammalian ABC

transporter; therefore computer modeling

and laborious biochemical experiments are

necessary to elucidate membrane topology, i.e

the position and orientation of membrane-span-ning segments within the polypeptide chain

The generally applied experimental methods include epitope insertion, localization of glyco-sylation sites, limited proteolysis and immuno-chemical techniques Several computer-assisted empirical prediction methods are available to generate the hydrophobicity profile for a puta-tive transmembrane protein, but such an analy-sis may provide only a baanaly-sis for developing experimental strategies for the elucidation of the actual membrane topology

In the present review we summarize the available data on the membrane topology for various ABC transporters by examining the distinct subfamilies We discuss predicted topology models and their experimental re-inforcement or negation, and assess the relation-ship between phylogenetic linkages and the arrangement of membrane topology patterns

We call the reader’s attention to a recent the-matic review, also analyzing the problems of membrane topology models within the

ABC-protein kingdom (Dean et al., 2001).

In the present study, for subfamily classifica-tion, we used the Human ABC Proteins Database

* This chapter reflects the information available in June, 2001.

(http://nutrigene.4t.com/humanabc.htm, last

update May 20, 2001) We included in the

anal-ysis only those members of a given subfamily

whose full sequences were known We chose

the sequences of the longest and/or the most

common splicing variants, in cases where

alter-natively spliced cDNAs were described We

considered a membrane topology to be

‘estab-lished’ if it was supported by independent

experimental data

Protein sequences were aligned by the ClustalW server (http://www.ebi.ac.uk/

clustalw), and hydrophobicity plots were

gen-erated according to a described method (von

Heijne, 1992) For computer-assisted predictions

the HMMTOP (Hidden Markov Model for

Topology Prediction) transmembrane topology

prediction server was applied (Tusnády and

Simon, 1998, 2001), which is freely available for

noncommercial users (http://www enzim.hu/

hmmtop) This method is based on the principle

that the topology of transmembrane proteins is

determined by considering the maximal

diver-gence in the amino acid composition of defined

sequence segments

The results of the above analyses are pre-sented as hydrophobicity plots of the aligned

sequences within a given subfamily, and the

phylogenetic trees of the subfamilies are also

shown together with the plots

ABCASUBFAMILY

The Human ABC Database lists 14 members for

the ABCA subfamily, from which currently

seven are represented by full cDNA sequences

We have analyzed the membrane topology of

the following seven proteins: A1⫽ 2261 aa;

A2⫽ 2436 aa; A3 ⫽ 1704 aa; A4 ⫽ 2273 aa; A7 ⫽

2146 aa; A8⫽ 1581 aa; and A12 ⫽ 2277 aa The

ABCA subfamily, as examined here, contains

‘full transporters’, and it is noteworthy that

these proteins have the largest molecular masses

within the entire human ABC protein family

Within this subfamily the function and mem-brane topology of only two members have been

studied in detail These are ABCA1, the gene

associated with Tangier disease, and ABCA4, the

retina-specific ABC transporter whose mutations cause severe retinopathies Both of these proteins

(see Chapters 23 and 28) are located in the plasma membrane, and they share 50% identical amino acids All the suggested membrane topol-ogy models agree that both halves of these pro-teins contain one TMD and one ABC domain, and the TMDs consist of six transmembrane helices However, there are several different models for the actual distribution of the helices

In the case of ABCA1, the first model, elabo-rated by Luciani et al (1994), predicted a large

cytoplasmic domain at the extreme N-terminal part, and another large cytoplasmic domain (‘regulatory domain’) in the central portion

of the protein A hydrophobic segment with a hairpin orientation was predicted to be located within this cytoplasmic regulatory domain, thus anchoring it to the plasma membrane

After the cloning of ABCA4, a topology

model smilar to ABCA1 was first suggested

(Allikmets et al., 1997) However, Illing et al.

(1997) described a different model for ABCA4, with two large extracellular domains (ECDs) between the first and second transmembrane helices in each predicted TMD In this model the first TM helix within the N-terminal half was placed close to the N-terminus, while the first predicted TM helix in the C-terminal half was the very same hydrophobic segment sug-gested to form a hairpin with a membrane-associated structure in the previous models This latter model thus predicted no large intracellular regulatory domain for ABCA4

A combination of the two different models was also published (Azarian and Travis, 1997; Sun

et al., 2000), in which the N-terminal half of the

ABCA4 protein was represented by the Illing model, while the C-terminal half contained a large cytoplasmic (regulatory) domain, as

sug-gested by the model of Luciani et al (1994).

In the case of ABCA4, an elegant experimental proof for the Illing model has been recently

pub-lished (Bungert et al., 2001) Eight functional

N-glycosylation sites were mapped by mutagen-esis within the bovine ABCA4 sequence, four

in the N-terminal half, and four within the C-terminal half These results support the pres-ence of a 600 amino acid extracellular domain within the N-terminal half, and a 275 amino acid extracellular domain within the C-terminal half

of the protein The authors also presented experi-mental data suggesting that the two extracellular domains are linked by disulfide bridge(s)

Regarding ABCA1, after the initial descrip-tion of its coding region, an in-frame, upstream

methionine was discovered, and translation

from this codon results in a 60 amino acid

extension of the originally expected

polypep-tide chain (Costet et al., 2000; Pullinger et al.,

2000; Santamarina-Fojo et al., 2000) Within this

N-terminal 60 amino acid chain, sequence

anal-ysis predicted the presence of a

transmem-brane helix (between residues 22 and 44), and a

potentially cleavable signal sequence (between

amino acids 45 and 46) These predictions

trig-gered experiments to test the membrane

topol-ogy dictated by the presence of a TM helix

(TMH) in the proximity of the N-terminus,

which may serve as an anchor signal and orient

a large loop in the N-terminal half of the

protein extracellularly Fitzgerald et al (2001)

expressed various truncated and tagged

ver-sions of human ABCA1, and demonstrated that

the loop from amino acids 44 to 640 indeed

has an extracellular orientation Tanaka et al.

(2001) obtained similar results, and they also

predicted a large extracellular loop within the

C-terminal half of the protein (between amino

acids 1368 and 1655) However, the two

pub-lications contain contradicting data

concern-ing whether cleavage occurs at position 45

According to the results of Tanaka et al (2001),

the polypeptide chain is cleaved at this

posi-tion during maturaposi-tion, and they suggest that

ABCA1 is present in the plasma membrane

with a large N-terminal segment extending to

the extracellular space Fitzgerald et al (2001),

using a bulkier N-terminal GFP-tag, found no

cleavage of ABCA1 at this position

In summary, the experimental data obtained for ABCA1 and ABCA4 support a similar

membrane topology for the two proteins, with

a domain arrangement of

TMH1-ECD1-

TMH(2–6)-ABC1-TMH7-ECD2-TMH(8–12)-ABC2, where H indicates helix and ECD, an

extracellular domain The primary sequence

alignment and hydropathy analysis of the

aligned ABCA sequences (as presented in

Figure 2.1) are in line with this assumption In

the projection of Figure 2.1, the location of TM

helices within ABCA1, as predicted by Tanaka

et al., is used as a guide to label similarities

within the hydrophobicity patterns of the

mem-bers in this subfamily Indeed, a similar domain

arrangement and membrane topology can be

predicted for all the currently known members

of the ABCA subfamily However, the

align-ment also reveals that ABCA3 and ABCA8 have

relatively short ECDs within their N-terminal and C-terminal halves

ABCB SUBFAMILY

Three members of this subfamily are ‘full trans-porters’, with ABCB1 (MDR1 or Pgp), ABCB4 (MDR3) and ABCB11 (sisterPgp or BSEP), all localized in the plasma membrane, in polar-ized cells in the apical membrane All the other family members are ‘half transporters’, includ-ing ABCB2 and ABCB3 (TAP1 and TAP2) resid-ing in the endoplasmic reticulum ABCB6, ABCB7, ABCB8 and ABCB10 are mitochondrial membrane proteins, while ABCB9 has a puta-tive lysosomal localization It is well estab-lished that ABCB2/TAP1 and ABCB3/TAP2 form a noncovalent heterodimer, and actively translocate peptides into the endoplasmic reticulum (see Chapter 26) Dimer formation for the mitochondrial half transporters has not been experimentally shown, but the presence

of mitochondrial targeting signals within the

N-terminal regions of both ABCB7 (Csere et al., 1998), and ABCB8 (Hogue et al., 1999) has been

demonstrated

For the first recognized mammalian ABC transporter, the original topology model of ABCB1 (MDR1-Pgp) predicted six TM helices

in both TMDs of the protein, each followed

by an ABC domain (Chen et al., 1986) This

membrane topology has been fully supported

by later epitope insertion experiments (Kast

et al., 1995, 1996) ABCB4 and ABCB11 are close

relatives of ABCB1, and similar membrane topology arrangements can be predicted for

both of these proteins (Figure 2.2).

Hydrophobicity plots of the aligned sequences reveal that the positioning of predicted helices in the ABCB family of half transporters is more closely related to the C-terminal halves (TMD2) of the full trans-porters than to the N-terminal halves, as

pre-sented in Figure 2.2 The hydrophobicity plots

of the half transporters support the six TM helix model in their TMDs, although the N-terminal regions are clearly extended, and contain hydro-phobic regions which may correspond to TM helices Indeed, in the case of TAP1 and TAP2, additional four and three TM helices, respec-tively, were predicted (Abele and Tampe, 1999)

It is worth mentioning that currently no reliable algorithms are available for such predictions, and visual inspection and heuristic adjustments

of the sequences can be most helpful for

predict-ing the probable location of some TM helices,

such as TM2 in ABCB6, or TM6 in ABCB8

ABCC SUBFAMILY

The ABCC subfamily consists of 11 members

in the human genome and most of these

(ABCC1–6 or MRP1–6) have been identified

as active membrane transporters for various

organic anions, several of which are discussed

in other chapters in this volume Although the

majority of the characterized ABC proteins are

active pumps, the cystic fibrosis transmembrane

conductance regulator, ABCC7 (CFTR), is a

chloride channel which may also regulate other

channel proteins The sulfonylurea receptors, ABCC8 (SUR1) and ABCC9 (SUR2), are best described as intracellular ATP sensors, regulat-ing the permeability of specific K⫹ channels (with which they form transmembrane com-plexes) Nothing is currently known about the function of ABCC10 and ABCC11

The membrane topology of human CFTR/ ABCC7 was originally predicted based on the MDR1 model, and supported experimentally

by glycosylation site insertion mutagenesis

(Chang et al., 1994) This study strongly

sup-ported the original model, with a TMD1-ABC1-R-TMD2-ABC2 domain arrangement Each TMD is predicted to consist of six TM helices, and a regulatory domain (R) is present between

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Figure 2.1 Relative similarity dendrogram and the hydrophobicity plots of the aligned sequences of the ABCA subfamily transporters The shaded areas show transmembrane helices with locations supported by experimental results from studies of ABCA1 and ABCA4 (see text).

the two halves of the protein, which seems to

be unique for CFTR

The membrane topology models for human ABCC1 have been independently formulated

by two research groups (Bakos et al., 1996;

Stride et al., 1996) They found that when the

CFTR/ABCC7 and MRP1/ABCC1 sequences

were aligned, the hydrophobicity analysis of the

aligned sequences yielded a close matching of

putative transmembrane segments, thus also

suggesting a six plus six transmembrane helix

topology for MRP1/ABCC1 However, ABCC1

contains an additional N-terminal segment of

about 230 amino acids, which has no counter-part in CFTR/ABCC7 On the basis of the hydropathy profiles and limited proteolysis experiments, the hydrophobic N-terminal segment of ABCC1 was suggested to be mem-brane embedded, with four to six

transmem-brane helices (Bakos et al., 1996; Stride et al.,

1996)

Subsequent investigations of the membrane topology of human ABCC1 by epitope inser-tion (Kast and Gros, 1997, 1998) and by

muta-tion of glycosylamuta-tion sites (Hipfner et al., 1997)

fully supported the above topology and

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MDR1 MDR3 sPgp

B1 B4 B11 B6 B7 B2/TAP1 B3/TAP2 B9 B8 B10

Figure 2.2 Relative similarity dendrogram and the hydrophobicity plots of the aligned sequences of the

ABCB subfamily transporters The shaded areas show transmembrane helices, predicted with locations as

projected from experimental results based on ABCB1 (see text).

revealed the presence of five TM helices in

the N-terminal segment These results indicated

a domain arrangement of

TMD0-L0-TMD1-ABC1-L-TMD2-ABC2, in which TMD0

repre-sents the N-terminal five TM helix extension,

while L0 and L represent intracellular linker

sequences In fact, by aligning the linear

sequences and the hydrophobicity plots for

all full-size ABC transporters present in the

sequence database in 1997, we concluded that

a common membrane topology and domain

arrangement distinguishes a subfamily (MRP

or ABCC subfamily) within the ABC kingdom

(Tusnády et al., 1997).

In the present review the amino acid sequences of 11 members of the ABCC

sub-family (those with full cDNA sequences) were

aligned, and the hydrophobicity plots of the

aligned protein sequences were determined

(Figure 2.3) This comparison indicates that

seven proteins in the family (ABCC1–3, ABCC6,

and ABCC8–10) form a subcluster within which

each member possesses the N-terminal TMD0

domain, first described for ABCC1 Thus this

subgroup is characterized by the

TMD0-L0-TMD1-ABC1-L-TMD2-ABC2 arrangement The

N-terminal TMD0 domain is absent from

ABCC4, ABCC5 and CFTR/ABCC7

Recent studies revealed that the TMD0 domain of ABCC1 does not play a crucial role

either in the transport activity of the protein,

or in the proper routing of the protein into the

basolateral membrane compartment However,

the presence of the L0 region (together with the

TMD1-ABC1-L-TMD2-ABC2 core) is necessary

for the ABCC1 GS-conjugate transport activity,

and for the proper intracellular routing of the

protein (Bakos et al., 1998) Thus, the ABCC1 L0

polypeptide was found to be membrane

associ-ated, and a 10 amino acid deletion within

this region, encompassing a putative

amphi-pathic helix, abolished the L0–membrane

inter-action and eliminated transport function, while

not affecting membrane routing (Bakos et al.,

2000) We have concluded from these studies

that the L0 region forms a distinct structural and

functional domain, which interacts with both the

membrane and the core region of the transporter

In harmony with the above conclusions, the cytoplasmic amino-terminal of CFTR/ABCC7

(which corresponds to the L0 domain) was

found to have a major role in the control of CFTR

channel gating, via physical interaction with the

regulatory (R) domain (Naren et al., 1999) Of

course, the actual sites of interactions still need to

be explored in these different proteins

ABCD SUBFAMILY

Four half transporters with TMD-ABC arrangements, ABCD1/ALDP, ABCD2/ALDR, ABCD3/PMP70 and ABCD4/PMP70R, are the members of this family They are localized to the peroxisomal membrane and their mutant forms are involved in different inherited per-oxisomal disorders It has been proposed that peroxisomal transporters need to dimerize in order to exert their function Co-immuno-precipitation experiments demonstrated the homodimerization of ABCD1, while a hetero-dimerization of ABCD1 with ABCD3 or ABCD2, and heterodimerization of ABCD2

with ABCD3 were found (Liu et al., 1999).

The presence of six TM helices in the TMDs

of the ABCD half transporters is generally pre-dicted, but the experimental verification of this prediction has yet to be approached

ABCG SUBFAMILY

The members of this subfamily are also half transporters, with a unique domain arrange-ment of ABC-TMD, i.e the ABC domain located at the N-terminus Four proteins have been described with full sequences in this subgroup The best-characterized transporter

is ABCG2 (MXR/BCRP), whose overexpression confers multidrug resistance Interestingly, this protein performs an active drug extrusion from the cells and is N-glycosylated in its mature form These data suggest that ABCG2 is local-ized in the plasma membrane, while several other half ABC transporters in the ABCB and ABCD subfamilies have been suggested to localize in membranes of various intracellular organelles, e.g the TAP proteins are located in the ER There is genetic evidence that ABCG5 and ABCG8 form heterodimers, as it was found that mutations of either of these genes cause the recessive genetic disease sitosterolemia

(Berge et al., 2000; Lee et al., 2001) On the other

hand, overexpression of the human ABCG2 multidrug resistance protein in a heterologous insect cell system generated an active, drug-stimulated ATPase activity, strongly suggest-ing that this protein can act as a homodimer

(Özvegy et al., 2001).

No experimental data are available as yet

on the exact membrane topology of the ABCG transporters However, here we combined some experimental data with empirical predictions to localize the transmembrane helices We used the

HMMTOP transmembrane topology prediction

server for this purpose in ABCG2, with the

following restrictions: the N-terminal 290 amino

acid chain, which harbors the ABC domain,

should be intracellular, and – as the protein

was shown to be N-glycosylated (Özvegy

et al., 2001) – the predicted extracellular loops

should contain consensus N-glycosylation site(s)

The model predicted six TM helices with the following localization in the linear sequence of

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C1/MRP1 C3 C2 C6 C8/SURI C9/SUR2 C10 C5 C11 C4

C7/CFTR

Figure 2.3 Relative similarity dendrogram and the hydrophobicity plots of the aligned sequences of the

ABCC subfamily transporters The shaded areas show transmembrane helices, predicted with locations as

projected from experimental results based on ABCC1 (see text).

ABCG2: 394–416, 427–449, 474–497, 506–530,

539–563 and 632–651 Two of the potential

N-glycosylation sites (at positions 418 and at

596) were predicted within extracellular loops

1 and 3, respectively Figure 2.4 shows the

hydrophobicity plots of the aligned ABCG

sequences, with the HMMTOP predicted TMDs

superimposed This analysis suggests a similar

location of the six TM helices in the TMDs for all

ABCG proteins

Membrane topology models without

experi-mental studies are just ‘educated

hallucina-tions’ (to quote J Riordan), but even at this

stage they have an important stimulatory role

in searching for structure–function

relation-ships As demonstrated in the case of CFTR

or Pgp-MDR1, biochemical experiments may

efficiently validate such models, while in other

cases, e.g in the ABCA subfamily,

experiment-based models still remain contradictory The

various membrane topology predictions in the

ABCC-MRP family have led to numerous

experi-mental and theoretical studies, yielding

impor-tant information regarding functional domains

and those involved in membrane routing/ targeting

A membrane topology is still difficult to define for any given ABC transporter, but a cor-rect prediction for domain arrangements may provide a major help in devising useful anti-bodies, site-directed mutants and even specific functional modulators or inhibitors We can hardly wait for the detailed crystal-based struc-tures of these large human membrane proteins, but until then there is still much fun to be had

in working out more and more sophisticated and accurate models

After completing this chapter, the structure of

a bacterial ABC transporter, MsbA of Escherichia coli, determined by X-ray crystallography to a

resolution of 4.5 Å (Chang and Roth, 2001, see Chapter 7)was published MsbA is a half trans-porter with a TMD-ABC domain arrangement, and the functional protein is a homodimer The published structure reveals that each MsbA subunit contains a TMD with six transmem-brane helices, an ABC domain, and an ‘intracel-lular domain’ which is composed of three intracellular loops, connecting TMH2 to TMH3, TMH4 to TMH5, and TMH6 to the ABC domain A very important result of the pub-lished MsbA structure is the first demonstration that the membrane-spanning segments of this ABC transporter are indeed ␣-helices Helix organization and interactions similar to those in the MsbA protein most probably will be charac-teristic for many other ABC transporters

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Hutchinson, A., Chidambaram, A., et al.

ATP-binding transporter gene (ABCR) is mutated

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topology and glycosylation of the human

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G5 G8 G2/BCRP

G1

Figure 2.4 Relative similarity dendrogram and

the hydrophobicity plots of the aligned sequences

of the ABCG subfamily transporters The shaded

areas show transmembrane helices, predicted with

locations as projected from the HMMTOP server

data and experimental results, based on ABCG2

(see text).

multidrug resistance-associated protein

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Bakos, É., Evers, R., Szakács, G., Tusnády, G.E.,

multidrug resistance protein (MRP1) lack-ing the N-terminal transmembrane domain

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Accumulation of dietary cholesterol in sitos-terolemia caused by mutations in adjacent ABC transporters Science 290, 1771–1775

Bungert, S., Molday, L.L and Molday, R.S.

bind-ing cassette transporter ABCR and its relation-ship to ABC1 and related ABCA transporters

Identification of N-linked glycosylation sites

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Chang, X.B., Hou, Y.X., Jensen, T.J and

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