CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS

CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS CHAPTER 24 – PEROXISOMAL ABC TRANSPORTERS

I NTRODUCTION

The set of proteins found in the membranes of

mammalian peroxisomes includes four half ABC

transporters (ALDP, ALDR, PMP70, P70R)

com-prising a distinct subset of the superfamily of

ABC transporters designated subfamily D Each

has an N-terminal hydrophobic transmembrane

domain with multiple transmembrane segments

(TMS) and a hydrophilic C-terminal half

con-taining a nucleotide-binding domain (NBD)

with Walker A and B motifs Limited topology

studies indicate that the C-terminal hydrophilic

halves of ALDP and PMP70 extend into the

cytosol (Contreras et al., 1996; Kamijo et al., 1990).

To provide context in what follows, we first

briefly review peroxisome function, genetic

dis-eases and biogenesis; we then focus on the

molecular, cellular and evolutionary biology of

the mammalian peroxisomal ABC transporters

Peroxisomes are typically spherical (0.1–1␮m

diameter), single-membrane-bound organelles

present in numbers ranging from a few hundred

to a few thousand in most mammalian cells

(Figure 24.1) (Gould et al., 2001; Purdue and

Lazarow, 2001; Tabak et al., 1999) The pH of the

mammalian peroxisome matrix has variously

been estimated to be more basic (Dansen et al.,

2000) or similar to (Jankowksi et al., 2001) that of

cytosol The dense, proteinaceous peroxisome matrix contains 50 or more enzymes, which par-ticipate in a variety of metabolic pathways including ␤-oxidation of straight and branched, very long (⭓C24) and long (C14–22) chain fatty acids (VLCFA and LCFA, respectively), synthe-sis of cholesterol and ether-lipids (e.g plasmalo-gens) and oxidation of polyamines, D-amino acids and, in non-primates, uric acid (Gould

et al., 2001; Sacksteder and Gould, 2000; Wanders

et al., 2001) Many of the peroxisomal oxidation

reactions liberate H2O2, which is detoxified by peroxisomal catalase The peroxisome mem-brane contains a characteristic set of peroxi-somal membrane proteins (PMPs) that, in addition to the peroxisomal ABC transporters, includes other small molecule transporters, enzymes and proteins required for import of peroxisomal matrix proteins and peroxisomal membrane biogenesis, plus many whose

func-tion is uncertain (Schäfer et al., 2001).

The rapid growth of our understanding of per-oxisome biogenesis and function over the last decade has depended in part on careful analysis

of cells from patients with inherited defects in these processes Two categories of peroxisomal genetic disorders are recognized, both with profound phenotypic consequences The first includes the peroxisomal biogenesis disorders (PBDs), a genetically heterogeneous group of

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24

P EROXISOMAL ABC

T RANSPORTERS

S HLOMO A LMASHANU AND

CHAPTER

autosomal recessive disorders characterized

by deficiency of multiple peroxisomal functions

(Gould and Valle, 2000b; Gould et al., 2001).

Zellweger syndrome (MIM#214100), a lethal

developmental and metabolic disorder, is the

phenotypic paradigm for the PBD Cell fusion

and molecular studies have identified at least

12 genes responsible for these disorders, none

of which encode peroxisomal ABC transporters

(Gould and Valle, 2000b; Gould et al., 2001)

The second category includes disorders in

which a single peroxisomal function is deficient

(Wanders et al., 2001) More than a dozen have

been recognized The exemplar is X-linked

adrenoleukodystrophy (X-ALD)(MIM#300100),

a progressive neurological disorder caused by

mutations in ABCD1, the gene that encodes

ALDP, a peroxisomal ABC transporter (see

below) (Moser et al., 2001) The principal

bio-chemical abnormality of X-ALD, accumulation

of VLCFA in plasma and tissues, together with

the existence of a VLCFA␤-oxidation pathway

in the peroxisome, has implicated ALDP in the

transport of VLCFA or VLCF acyl-CoAs into

the peroxisome Tissues and cultured cells from

X-ALD patients have a 60–80% reduction in

␤-oxidation of VLCFA

Over the last decade at least 23 genes

(designated PEX genes) have been identified

that encode proteins (peroxins) necessary for

peroxisome biogenesis (Gould and Valle, 2000b;

Gould et al., 2001; Purdue and Lazarow, 2001).

The nomenclature convention is that

ortholo-gous PEX genes in different species are indi-cated by the same number (Distel et al., 1996) Thus, Saccharomyces cerevisiae PEX1 is ortholo-gous to human PEX1.

Regulation

The number of peroxisomes per cell is dynamic

and varies with the metabolic state (Chang et al., 1999; Gould et al., 2001; Purdue and Lazarow,

2001; Subramani, 1998) In rodents, certain hypolipidemic drugs, plasticizers and naturally occurring lipids induce higher peroxisome numbers and the expression of genes encoding many matrix proteins and peroxisome mem-brane proteins (PMPs) including the ABC

trans-porters (Reddy et al., 1986; Zomer et al., 2000).

This coordinated induction is mediated prima-rily by activation of peroxisome proliferator-activated receptor ␣ (PPAR␣), a member of the nuclear hormone receptor superfamily (Kersten

et al., 2000; Wahli et al., 1999) Activated PPAR␣ heterodimerizes with a second nuclear hor-mone receptor, retinoid X-responsive receptor

␣, to form an active transcript factor that

recog-nizes cis-acting sequences, peroxisome

prolifer-ator responsive elements (PPRE, consensus two direct AGGA/ TCA separated by a single base pair), in the promoters of its target genes

(Juge-Aubry et al., 1997; Kersten et al., 2000)

Figure 24.1 Peroxisome morphology A, Electron micrograph of peroxisomes in human liver visualized by diaminobenzidine staining for catalase activity (image kindly supplied by M Espeel and F Roels,

University of Gent, Belgium); scale bar is 0.5 ␮m Px, peroxisome; M, mitochondrion; ER, endoplasmic

reticulum B, Immunofluorescence of human fibroblasts stained with anti-PMP70 antibody Note the

punctate appearance of normal peroxisomes C, Confocal image of S cerevisiae, grown on oleic acid to induce peroxisomes and expressing C-terminal PTS1 tagged GFP, which localizes to peroxisomes.

In normal cells, an increase in peroxisome

number results mainly from maturation and

enlargement of existing peroxisomes with

uptake of both membrane and matrix

compo-nents followed by fission into daughter

organelles (Gould and Valle, 2000a; Purdue and

Lazarow, 2001) De novo synthesis of

peroxi-somes is also possible (South and Gould, 1999)

Matrix proteins

Peroxisomal matrix proteins are synthesized

on free cytosolic ribosomes and targeted

post-translationally to the organelle by specific

cytosolic receptors that recognize cis-acting

sequences (peroxisomal targeting signals or

PTSs) in the primary peptide sequence (Gould

and Valle, 2000b; Gould et al., 2001; Purdue and

Lazarow, 2001) Most matrix proteins are

tar-geted by PTS1, a C-terminal -SKL or

conserv-ative variant thereof A few matrix proteins are

targeted by PTS2, a degenerate sequence (-R/

KX5Q/HL-) located near the N-terminus A few

matrix proteins appear to be targeted by as yet

unrecognized PTSs (Purdue and Lazarow,

2001) The PTS1 and 2 receptors have been

cloned and characterized The former, PEX5, is

a tetratricopeptide repeat protein (Dodt et al.,

1995; Fransen et al., 1995; Wiemer et al., 1995);

the latter, PEX7, is a WD40 repeat protein

(Braverman et al., 1997; Motley et al., 1997;

Purdue et al., 1997) The structure of the PEX5

tetratricopeptide repeat domain complexed with

a PTS1 peptide has been mapped in S cerevisiae

(Klein et al., 2001) and solved for human PEX5

(Gatto et al., 2000a, 2000b) Both PEX5 and

PEX7 bind their cargo proteins in the cytosol

and transport them to the peroxisome, where

they interact with specific docking proteins in

the peroxisomal membrane, release their cargo

and recycle to the cytosol (Dodt and Gould,

1996) In mammalian cells, the long isoform of

PEX5 interacts with PEX7 and is necessary for

its function (Braverman et al., 1998; Otera et al.,

1998, 2000)

Membrane proteins

Like peroxisomal matrix proteins, PMPs are

synthesized on free cytosolic ribosomes and

tar-geted to the organelle by cis-acting targeting

sequences (membrane peroxisome targeting

signals or mPTS) In contrast to the discrete,

well-defined, single targeting sequences found in

matrix proteins, the model emerging for several PMPs includes two non-overlapping targeting segments for each peptide, either of which is sufficient for localization and insertion into the peroxisome membrane In most cases these tar-geting segments are relatively long and include one or two TM segments This model derives from recent work on several integral PMPs

including: human PMP34 (Jones et al., 2001) and its fungal orthologue PMP47 (Dyer et al., 1996; Wang et al., 2001), a peroxisomal member

of the mitochondrial carrier family that proba-bly functions as an ATP/ADP transporter (van

Roermund et al., 2001); human PMP22, an

abun-dant PMP of unknown function (Brosius

et al., 2002; Pause et al., 2000); and PEX13, the

docking protein for matrix protein receptors

(Jones et al., 2001) Attempts to define specific

and necessary sequence motifs in these target-ing segments have not been successful A five-residue sequence of basic five-residues implicated in initial studies of PMP47 is not a consistent fea-ture and some targeting persists when these residues are entirely replaced by alanines (Biery

and Valle, unpublished; Wang et al., 2001)

Work on PMP70 by ourselves (Almashanu and Valle, unpublished) and others (Sacksteder and Gould, 2000) indicates that the peroxisomal membrane ABC transporters also use this two targeting segment mechanism

A related question is how the newly synthe-sized, hydrophobic PMPs traverse the aqueous cytosol to acquire their proper location in the peroxisomal membrane Recent studies with cells from patients with a peroxisome biogenesis disorder (PBD) have implicated PEX19, a farne-sylated, mainly cytosolic protein, as a possible receptor for PMPs analogous to the role of PEX5 and PEX7 in the targeting of matrix proteins

(Gotte et al., 1998; James et al., 1994; Sacksteder

et al., 2000; Snyder et al., 2000) Binding studies

show that the multiple PMP targeting segments described above are recognized by PEX19

(Brosius et al., 2002; Jones et al., 2001; Sacksteder

et al., 2000) Additionally, two integral PMPs,

PEX3 and PEX16, are probably involved in this

process (Honsho et al., 1998; Muntau et al., 2000;

Snyder et al., 1999; South and Gould, 1999; South

et al., 2000) Mutations in any of these three

genes not only cause a PBD phenotype but also are associated with the distinct cellular pheno-type of absence of peroxisome membranes

(Ghaedi et al., 2000; Hettema et al., 2000;

Honsho et al., 1998; Matsuzono et al., 1999;

Muntau et al., 2000; South and Gould, 1999).

P EROXISOME ABC

The peroxisome ABC transporters are all half

ABC transporters and, as a group, are the most

thoroughly studied integral PMPs One, PMP70,

is routinely used as the standard marker

pro-tein for peroxisomal membranes (Figure 24.1).

Despite this wealth of information, much

remains to be learned about these molecules In

what follows, we review the current state of

knowledge of these interesting proteins

GENES

ALDP is encoded by ABCD1, the gene

responsi-ble for X-ALD ABCD1 spans ⬃21 kb, contains

10 exons (Sarde et al., 1994) and maps to Xq28

(Table 24.1)(Mosser et al., 1994) ABCD1

muta-tions, including frameshifting insertions and

deletions and nonsense mutations identified in

X-ALD patients, provide compelling evidence

that it is the gene responsible for this

neuro-degenerative disorder Four autosomal ABCD1

non-processed pseudogenes with 92–96% nucleotide identity located elsewhere in the genome complicate the molecular diagnosis of

X-ALD (Table 24.1) An online X-ALD database

(www.x-ald.nl) contains useful information for patients and professionals and is maintained

in a collaborative effort between the Peroxi-somal Diseases Laboratory at the Kennedy Krieger Institute, Baltimore, MD, USA and the Laboratory of Genetic Metabolic Diseases at the Academic Medical Center, Amsterdam, the Netherlands

ALDR is encoded by ABCD2 comprising 10

exons distributed over 33 kb on chromosome

12q12 (Table 24.1) The genomic organization of

ABCD2 closely resembles that of ABCD1

consist-ent with the conclusion, based on sequence similarity (64% identity), that these two mem-bers of the peroxisome ABC transporter subfam-ily diverged recently from a common ancestor

(see Figure 24.4) Moreover, this high sequence similarity suggests that the function between ALDP and ALDR may be partially redundant Thus, ALDR may have the potential to modify

the phenotype of X-ALD (Holzinger et al., 1997a) Induction of the ABCD2/Abcd2 gene in

The nomenclature is based on the guidelines for the human and the mouse ABC transporter gene nomenclature (http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html) The information on the localization, nucleotide sequence, locus and PubMed IDs was derived from the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov).

Symbol Previous Alias Map Number Genomic mRNA Locus OMIM PMID

symbol, location of exons NT NM ID ABC #

OMIM, Online Mendelian Inheritance in Man PMID, Pubmed entry or Pubmed indexed for MEDLINE.

cells from X-ALD patients or in the Abcd1

knockout mice by exposure to 4-phenylbutyrate

temporarily corrected the deficiency of VLCFA

␤-oxidation (Kemp et al., 1998) If this response

could be maintained, it would be a promising

therapeutic strategy for X-ALD Expression of

ABCD2 is induced by fibrates and other

xenobi-otics as well as certain endogenous lipids in a

response that depends on PPAR␣ (Fourcade

et al., 2001) Survey of 2 kb of 5⬘ flanking

sequence of rat Abcd2 identified several

candi-date PPREs (see above) but none of these were

functional in transient transfection assays with

chimeric promoter/reporter constructs Thus,

either the responsible PPRE is located more

remotely or the PPAR␣-dependent regulation

of the Abcd2 involves a different mechanism

(Fourcade et al., 2001) An earlier study showed

that the human and murine ABCD2 genes share

more than 500 bp of conserved 5⬘ flanking

sequence with potential Sp1- and AP-2-binding

sites but no TATAA box Moreover, in transient

transfection assays with chimeric promoter/

reporter constructs, 1.3 kb of the 5⬘-flanking

region of human and murine ABCD2 genes

was shown to be necessary for upregulation by

9-cis-retinoic acid and forskolin, while no effect

of PPAR␣ could be detected (Pujol et al., 2000)

PMP70, encoded by ABCD3, is abundantly and widely expressed and, like ABCD2, is

induced in mammalian liver following the

administration of fibrates (Kamijo et al., 1990)

A rat Abcd3 cDNA was initially identified by

screening an expression library (Kamijo et al.,

1990) and the sequence of the rat cDNA was

used, in turn, to clone the human PMP70 cDNA

and structural gene (Gärtner et al., 1992, 1998).

Human ABCD3 maps to chromosome 1p21–p22

(Gärtner et al., 1993) and comprises 23 exons

dis-tributed over 65 kb of genomic DNA (Table 24.1).

The proximal promoter region of human and

murine ABCD3 genes have a high GC content

and multiple consensus Sp1-binding sites,

fea-tures consistent with its broad tissue expression

(Gärtner et al., 1998) Several PPRE-like sequences

with the correct spacing are present in the

5⬘ flanking sequence of ABCD3 (Berger et al.,

1999; Fourcade et al., 2001) The organization of

ABCD3 differs from ABCD1 and ABCD2 genes

with only 2 of 22 introns falling in positions

cor-responding to ABCD1 introns This observation

plus the fact that the first exon of ABCD1 is

exceptionally large (⭓1286 bp) lead to the

specu-lation that the modern ABCD1 gene may have

arisen from an ancient retrotransposition of a

cDNA derived from a ABCD3-like gene followed

by intron acquisition in the 3⬘ half of the gene

(Gärtner et al., 1998).

P70R, also known as PMP69, is encoded

by ABCD4 This gene maps to chromosome

14q24.3, covers 16 kb and has 19 exons

(Holzinger et al., 1998; Shani et al., 1997) The position of several ABCD4 introns corresponds

to those in ABCD3, consistent with the

sugges-tion, based on sequence similarity, that these two genes are more closely related to each

other than to ABCD1 and 2 Also, as in ABCD3,

the 5⬘ flanking sequence of ABCD4 has a high

GC content, contains several consensus Sp1-binding sites and lacks a TATAA box No PPREs were identified in the 5⬘ 1.8 kb Variant ABCD4 transcripts result from the use of alternative polyadenylation sites and alternative exon splicing events including one that confers an

alternative C-terminus (Holzinger et al., 1997b, 1998; Shani et al., 1997).

Northern blot studies of RNA harvested from adult mice have shown different expression patterns for the four peroxisomal ABC

trans-porters (Albet et al., 1997; Berger et al., 1999).

Abcd1 is mainly expressed in heart, lung, intes-tine and spleen; Abcd2 in skeletal muscle and brain; Abcd3 in all tissues studied with greatest abundance in liver and kidney; while Abcd4

mRNA is 10-fold more abundant in kidney than in other tissues analyzed

The expression of the four peroxisomal ABC transporter genes is also regulated

differen-tially during mouse brain development Abcd1

mRNA is most abundant in embryonic brain and gradually decreases during maturation;

Abcd2 and Abcd4 mRNA accumulates in the early postnatal period; and Abcd3 transcripts

increase during the second and third postnatal

weeks (Albet et al., 1997; Berger et al., 1999).

Similarly, in situ hybridization studies in rat

brain showed different spatial and temporal

expression patterns of Abcd1 and Abcd3 during postnatal development (Pollard et al., 1995).

Abcd3 expression was low at birth and

increased to a peak between the second and third week in hippocampus and cerebellum By

contrast, Abcd1 expression was maximal at

birth in all areas of the brain and decreased

thereafter (Pollard et al., 1995) Administration

of fenofibrate strongly increased the expression

of the Abcd2 and Abcd3 in rat intestine and

liver, respectively, but not the expression of

Abcd1 (Albet et al., 1997) These observations

suggest that transcriptional regulation is an

important variable in the expression of the

per-oxisomal half ABC transporters and must be

taken into account when considering possible

in vivo heterodimerization partners.

All known peroxisomal ABC transporters are

half transporters that must dimerize to be

func-tional The partially overlapping patterns of

developmental and tissue expression suggest

that both hetero- and homodimerization are

possible Using a yeast two-hybrid system, Liu

et al found hetero- and homodimerization of

the C-terminal halves of ALDP, ALDR and

PMP70 (Liu et al., 1999) P70R was not tested.

Two mutations in ALDP (P484R and R591Q)

known to cause X-ALD impaired both

hetero-and homodimerization These results were

supported by co-immunoprecipitation

experi-ments showing homodimerization of ALDP

and heterodimerization of ALDP with either

ALDR or PMP70 ALDR also heterodimerized

with PMP70 Formation of ALDP homodimers

and ALDP/PMP70 heterodimers was also

demonstrated by co-immunoprecipitation of

in vitro synthesized proteins (Smith et al., 1999).

Considering the protein interaction and

expres-sion studies, it seems likely that both

homo-dimerization and heterohomo-dimerization of certain

peroxisomal half ABC transporters takes place

and that this may vary from tissue to tissue The

functional consequences of the choice of

part-ners are not known

MEMBRANE

As discussed above, our understanding of how

PMPs achieve their proper and specific location

in the peroxisomal membrane is not well

under-stood Nevertheless, recent work suggests that

PEX19, a farnesylated protein located primarily

in the cytosol, appears to have a major role

in PMP import PEX19 interacts in vitro with

numerous PMPs including ALDP, PMP70 and

ALDR (Gloeckner et al., 2000; Sacksteder et al.,

2000; Snyder et al., 2000) More specifically, a

region of ALDP located towards the N-terminus

(between amino acids 67–186) has been shown to

be important for proper peroxisomal targeting

and an overlapping fragment (residues 1–203)

interacts in a two-hybrid system with PEX19

(Gloeckner et al., 2000) Similarly, expressing a

series of N-terminal and C-terminal deletions of PMP70 tagged with a C-terminal green fluores-cent protein (GFP) in Chinese hamster ovary (CHO) cells, we localized a peroxisomal mem-brane targeting signal to the N-terminal 80 amino acids of PMP70 as well as a second site influencing targeting in the C-terminal 100 amino acids (Almashanu and Valle, unpublished observations) Additionally, we made a chimeric protein with the 183 N-terminal residues of PMP70 followed by the 428 C-terminal residues

of its Escherichia coli homologue (YDDA) tagged

with GFP When expressed in CHO cells, the chimeric PMP70/YDDA-GFP localized to perox-isomes while YDDA-GFP alone showed a non-peroxisomal pattern Mutagenesis studies of conserved residues in the N-terminal 80 amino acids of PMP70 failed to detect a discrete target-ing sequence This suggests that recognition of PMP70 by the targeting apparatus depends on more general secondary structure features rather than on specific residues

By analogy to other ABC transporters, the N-terminal hydrophobic half of the peroxisomal ABC transporters is expected to have six TM segments However, the location and number

of these hydrophobic segments has been diffi-cult to establish with certainty (see also Chapter 2) Analyses with multiple protein motif prediction algorithms failed to identify six unequivocal TM segments (TMS) in any of

the peroxisomal half ABC transporters (Figure

24.2) Moreover, there was some variation in the

location of the predicted TMS In Figure 24.2,

we present the TMSs predicted by several algo-rithms for 11 PMP70 homologues, from bacte-ria to mammals The location of five TMSs is clear; the position of the sixth is less certain The initial description of rat PMP70 by

Kamijo et al included a protease sensitivity

study indicating that the C-terminal

hydropho-bic half of PMP70 faces the cytosol (Kamijo et al.,

1990) Similarly, immunohistochemical studies with antibodies directed at specific segments

of ALDP indicated that the C-terminal half of the protein projects into the cytosol (Contreras

et al., 1996) Additionally, protease treatment

followed by immunoblot analysis showed that the C-terminal segment of ALDP could be released from the surface of intact rat liver

peroxisomes and retain its ability to bind ATP

in vitro (Contreras et al., 1996) We are unaware

of any topology studies of ALDR or P70R

THE PEROXISOMAL MEMBRANE

Homologues from several non-mammalian

species have been identified (Smith et al.,

1999); those from S cerevisiae have been well

characterized

PXA1/PAL1/PAT2 (Hettema et al., 1996;

Shani et al., 1995; Swartzman et al., 1996) and

PXA2/PAT1 (Hettema et al., 1996; Shani and

Valle, 1996) are the two yeast homologues of the mammalian half ABC transporters Taking advantage of the induction of PMPs and peroxi-somal matrix proteins by growth on oleic acid as

a sole carbon source, Shani et al used degenerate

PCR primers corresponding to the conserved

Walker A and Walker B sequences of ABCD1 and ABCD3 to clone PXA1 The conceptual

pro-tein product Pxa1p has 758 amino acids, a pre-dicted molecular mass of 87 kDa and is slightly more similar to ALDP than to PMP70 (Shani

et al., 1995) Swartzman et al used an alternate strategy to clone a cDNA identical with PXA1

Figure 24.2 Alignment of the human peroxisomal ABC transporters Amino acid sequences were

aligned using the MegAlign program (DNASTAR Inc.) Identical residues in two or more polypeptides

are boxed in black The indicated transmembrane segments were predicted using the transmembrane

region detection programs available at http://www.us.expasy.org The labeled solid overlines designate

the EAA-like motif and the NPDQ motif (see text) The heavy solid overline designates an N-terminal helical

hydrophobic region of unknown function present in ALDP, ALDR and PMP70.

over the 758 C-terminal amino acids but with an

additional 112 N-terminus codons with a

pre-dicted protein mass of 100 kDa (Swartzman

et al., 1996) The shorter Pxa1p is clearly

func-tional in that it rescues growth on oleic acid in

mutant yeast lacking PXA1 (Shani et al., 1995).

Functional studies with Pal1p were not reported

and the N-terminal extension of Pal1p is not

homologous with the peroxisomal ABC

trans-porters of other species Thus, the significance of

this N-terminal extension is uncertain

PXA2/PAT1, originally identified as YKL741,

encodes a half ABC transporter with highest

similarity to mammalian PMP70 and ALDP

Shani et al showed that the combined

disrup-tion of PXA1 and PXA2 gave a growth

pheno-type identical to that of either single disruption

(Shani and Valle, 1996) and that the stability of

Pxa1p was reduced in yeast with a PXA2

dele-tion Finally, in co-immunoprecipitation studies

with either Pxa1p or Pxa2p, they showed direct

physical evidence for the heterodimerization of

the two proteins to assemble a full peroxisomal

half ABC transporter Thus, the functional

trans-porter in yeast is a heterodimer of Pxa1p/Pxa2p

Availability of sequence information of

peroxiso-mal ABC transporters from yeast and mamperoxiso-mals

provides the opportunity to look for conserved

sequence motifs of possible functional

signifi-cance In addition to the TM segments and the

Walker A and B and C segments of the NBD, at

least two additional conserved motifs can be

identified in the peroxisomal ABC transporters

The first, the EAA-like motif, is a 15 amino acid

sequence (- N S E E I A F Y X G X K X E X

where, in a comparison of Pxa1p, Pxa2p, PMP70

and ALDP, the designated residues are present

in three out of four and the underlined residues

are present in all four proteins) The EAA-like

motif is present between TMS 4 and 5 and

resembles the central core of a 30-residue

sequence (the EAA motif) found in a similar

location in many prokaryotic ABC transporters

(Saurin et al., 1994; Shani et al., 1996b)

Muta-genesis studies in Pxa1 showed that

conserv-ative missense mutations at E294 and G301

reduce the function of Pxa1p but do not alter

stability or targeting (Shani et al., 1996a).

Recent mutagenesis and crosslinking studies

of the prokaryotic EAA motif suggest that this

sequence may interact with certain regions of

the ATP-binding cassette (Hunke et al., 2000;

Mourez et al., 1997) Photoaffinity labeling

and mass spectrometry study of the human P-glycoprotein (Pgp, ABCB1) showed that its EAA-like motif (cytoplasmic loop 2) was one of nine tryptic peptides that bind to

photoaffinity-labeled substrate analogues (Ecker et al., 2002).

These results suggest that the EAA-like motif participates in substrate binding/translocation

or in the interaction of these processes with ATP binding/hydrolysis

The second conserved motif, which we

designate the NPDQ motif (see Figure 24.3 for

sequence consensus),is located between TMS 2 and 3 Preliminary studies replacing one, two

or four of the residues in the conserved NPDQ core with alanines did not affect targeting (Almashanu and Valle, unpublished) We could not identify this motif in other categories of ABC transporters including other mammalian half ABC transporters like the TAP proteins Therefore, we considered that it might be spe-cific for the peroxisomal ABC transporters and used it to search for the subset of ABC trans-porters with homology to the known peroxi-some members of the superfamily Interestingly, our results suggest that the NPDQ motif is a specific signature for peroxisomal transporters among eukaryotes but that it is also present in a small subset of prokaryotic transporters (see section on evolution, below)

The spectrum of physiological ligands and the direction of transport is not known with cer-tainty for any of the mammalian peroxisomal half ABC transporters A variety of studies sug-gest, however, that one or more of the peroxi-somal ABC transporters transport straight or branched LCFA or VLCFA or their acyl-CoA derivatives into the peroxisome Two impor-tant variables in the transport of fatty acids across membranes are the chain length and the site of activation of the fatty acid to its acyl-CoA derivative The latter is accomplished by acyl-CoA synthetases that differ in chain length specificity and subcellular location In general, the longer the chain length, the more difficult it becomes for fatty acids to move across the per-oxisomal membrane; medium-chain fatty acids

do not require transporters while LCFAs do Activation of fatty acids makes them more polar and impairs movement across the peroxi-somal membrane (Hettema and Tabak, 2000)

The most detailed studies of the functions of the peroxisomal ABC transporters have utilized

S cerevisiae as a model system In this regard,

yeast offer several advantages over mammalian

cells for the study of peroxisome biogenesis

and function (Kunau et al., 1993) In contrast to

mammalian peroxisomes, the peroxisomes of

yeast are more easily isolated and their

compo-nents more easily induced Moreover, fatty acid

␤-oxidation in yeast is limited to peroxisomes,

while in mammalian cells, ␤-oxidation of fatty

acids occurs in both peroxisomes and

mito-chondria In S cerevisiae, Pxa1p and Pxa2p

het-erodimerize to form the functional peroxisome

ABC transporter that is essential for growth

on LCFAs, especially oleic acid, C18:1, as a sole

carbon source (Hettema et al., 1996; Shani and

Valle, 1996) ␤-Oxidation of LCFA in intact cells

deleted for either the PXA1 or PXA2 gene is

reduced to approximately 20% of the wild-type

level In detergent lysates of these same mutant

yeasts, ␤-oxidation of LCFA is unaffected,

indi-cating that the peroxisome membrane is a

bar-rier in the intact mutant yeast (Hettema and

Tabak, 2000; Hettema et al., 1996) Using

proto-plasts in which the plasma membrane has been

selectively permeabilized by digitonin, it was

shown that C18:1-CoA, but not C8:0-CoA, enters

peroxisomes in a Pxa1p/Pxa2p and

ATP-dependent process (Hettema and Tabak, 2000;

Verleur et al., 1997) This result is consistent with

the observation that the acyl-CoA synthetase

with activity towards long-chain fats is

extraper-oxisomal in yeast (Hettema et al., 1996) Thus,

the available evidence indicates that the yeast

PXA transporter is necessary for the transport

of LCF acyl-CoAs into the peroxisome

Our understanding of peroxisomal ABC transporter function in mammalian cells derives

largely from observations made in cells or

tis-sues with mutations in one or more of the

trans-porters Mutations in ABCD1 cause X-ALD,

a neurodegenerative disorder with a highly

variable clinical phenotype (Moser et al.,

2001) Biochemically, X-ALD patients

accumu-late VLCFA in plasma and tissues and exhibit

deficient VLCFA ␤-oxidation with decreased

activity of the peroxisomal VLCFA acyl-CoA

synthetase (VLCS) that activates VLCFAs to

their CoA thioesters (Moser et al., 2001) The

lat-ter is thought to be localized on the matrix side

of the peroxisome membrane (Lazo et al., 1990;

Steinberg et al., 1999b) Functional interaction

between ALDP and peroxisomal VLCS is also

implied by the observation of a synergistic effect

on VLCFA ␤-oxidation when both VLCS and

ALDP were overexpressed in humans and

mouse fibroblasts (Steinberg et al., 1999a;

Yamada et al., 1999) Several hypotheses

regard-ing the functional relationship between VLCS and ALDP and their role in the pathogenesis of X-ALD have been proposed but the mechanism remains obscure A relationship between fatty acid ␤-oxidation and PMP70 has also been

sug-gested Imanaka et al (1999) demonstrated a

two- to threefold increase in ␤-oxidation of palmitic acid (C16:0) in CHO cell line overex-pressing PMP70, whereas the oxidation of ligno-ceric acid (C24:0) decreased about 30–40% In summary, current data suggest that ALDP and PMP70 are involved in the transport of LCFA and VLCFA or their CoA derivatives across the peroxisomal membrane

Creation of mouse models, each lacking one

of the four peroxisomal half ABC transporters, also has the promise of providing functional insight Targeted knockouts for the genes encoding ALDP, ALDR and PMP70 have been

produced (Forss-Petter et al., 1997; Lu et al., 1997; Yamada et al., 2000) The X-ALD mouse

model has some of the human biochemical fea-tures including high levels of VLCFA in brain and adrenal gland However, the mice appear

to lack the neurological phenotype of humans

as they do not develop symptoms or evidence

of cerebral or spinal cord demyelination by up

to 2 years of age The phenotypes of both the ALDR and the PMP70 knockout mice do not correspond to a recognizable human disease

Mice lacking PMP70 have impaired metabolism

of very long branched-chain fatty acids includ-ing pristanic acid, phytanic acid and bile acid

precursors (Jimenez-Sanchez et al., 2000).

Biochemical studies of purified peroxisomal ABC transporters in reconstituted lipid vesicles should help to clarify our understanding of their function but have not yet been described

The superfamily of ABC proteins is large and diverse The availability of whole genome sequences from an increasing number of orga-nisms has led to the identification of many new members and to a better understanding of the set of ABC transporters characteristic of each species Taking advantage of this sequence information, we searched for a specific signa-ture sequence for the ABCD subfamily that would identify all known peroxisomal ABC

transporters in different species We used a short

segment of a highly conserved motif located at

the second predicted loop of the mammalian

peroxisomal half ABC transporters (the NPDQ

motif; Figure 24.3) and used it to perform

BLAST searches of different NCBI databases

This specifically identified all the known

peroxi-somal half ABC transporters as well as their

apparent orthologues whose subcellular

local-ization is yet to be determined in other

eukary-otes Additionally this search identified a

subset of prokaryotic half ABC transporters

Interestingly, these prokaryotic transporters are

all half ABC transporters, each is encoded by a

single gene without subdivision into an operon

We used more than 30 of the hits (Table 24.2) to

run the ClustalX algorithm, producing a

multi-ple sequence alignment, and we analyzed this

comparison to generate a phylogenetic

relation-ship by maximum parsimony (Figure 24.4) This

analysis indicates that among the four

mam-malian peroxisomal half ABC proteins P70R

rep-resents the ancestral gene, more closely related

to the bacterial and the plant homologues

Pxa1p and Pxa2p, the two S cerevisiae

homo-logues, are more divergent from the ancestral gene and closer to the PMP70/ALDP branch

Interestingly, two of the Caenorhabiditis elegans

homologues are closely related to P70R, two to PMP70 and only one to ALDP/ALDR This sup-ports the hypothesis that genes encoding these two transporters diverged relatively recently, a hypothesis that is also supported by the genomic organization of these two genes (see section on genes, above) In addition, the genomic

organi-zation of ABCD3 gene, when compared to that of ABCD1 and ABCD2, suggests that the modern ABCD1 gene may have arisen from an ancient

retrotransposition event followed by intron

acquisition (Gärtner et al., 1998) The prokaryotic

homologues identified in this search number

one per species but Haemophilus influenzae Rd and possibly E coli have two.

A separate and additional aspect of the

evolu-tionary history of the human ABCD1 gene is the presence of four autosomal ABCD1 pseudogenes

Figure 24.3 Sequence alignment of the NPDQ motif from 30 homologues of human ALDP We used

ClustalX 1.81 to align the protein sequences listed in Table 24.2 Color code for residues: G, brown;

P, yellow; conserved K, R, red; conserved D, E, purple; conserved neutrals, green, blue, teal

Residue number is indicated on the right.