Peroxisome protein import: some answers, more questions

Peroxisome protein import: some answers, more questionsAlison Baker1 and Imogen Averil Sparkes2

Recent advances in the study of plant peroxisomes are

shedding new light on the importance of these organelles for

plant development, and are revealing similarities and

differences in peroxisome protein import pathways between

plants, animals and fungi. For example, the import of matrix

proteins that carry the PTS1 and PTS2 targeting signals is

coupled in plants as it is in mammals, whereas these import

pathways are separate in fungi. The expression of a human

peroxisomal ATPase partially rescues the equivalent

Arabidopsis mutant. Ubiquitination might play a role in receptor

recycling in Saccharomyces cerevisiae and exciting progress is

being made through studies of the targeting of membrane

proteins.

Addresses1 Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT,

UK2 School of Biological and Molecular Sciences, Oxford Brookes

University, Oxford OX3 0BP, UK

Corresponding author: Baker, Alison ([email protected])

Current Opinion in Plant Biology 2005, 8:640–647

This review comes from a themed issue on

Cell Biology

Edited by Patricia C Zambryski and Karl Oparka

Available online 22nd September 2005

1369-5266/$ – see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2005.09.009

IntroductionPeroxisomes perform a wide variety of metabolic pro-

cesses in eukaryotic organisms, and mutations that affect

peroxisome function have profound phenotypic conse-

quences (Tables 1 and 2). Peroxisomes have a single

membrane (Figure 1), lack DNA and import matrix

and membrane proteins posttranslationally. PEROXIN(PEX) genes encode peroxins (proteins that are required

for peroxisome biogenesis) and were first isolated through

complementation of yeast and Chinese hamster ovary

(CHO) cell mutants that are deficient in peroxisome

biogenesis. The advent of the Arabidopsis genome

sequence has allowed the in silico identification of likely

homologues [1] and their investigation through forward

and reverse genetic techniques [2,3,4��,5,6]. Of the more

than 30 PEX genes isolated in yeast, 15 putative homo-

logues have been identified in plants and just seven of

these have been partially characterised (Table 2). It

remains to be established whether the ‘missing’ peroxins


are simply too divergent to be identified on the basis of

sequence homology or if plants have novel peroxins that

are not found in other organisms. Although ‘core’ peroxi-

some protein import pathways are conserved across organ-

isms, there is already good evidence that there are

species-specific variations [7], and these could be poten-

tial targets for disease control [8].

The import of matrix proteins follows at least four defined

stages: the binding of the cargo to the receptor, cargo–

receptor complex docking at the target membrane, trans-

location of the cargo or cargo–receptor complex, and

export of the receptor (Figure 2; see [9] for detailed

review). The import of membrane proteins remains less

well understood.

Most of the available information derives from studies

using yeast and mammalian cells. In this review, we have

concentrated, where possible, on recent results in plants

that highlight the emerging number of similarities

between plant and human peroxisomal protein import.

Import of matrix proteins: receptor–cargointeraction and docking at the membranePeroxisomal matrix protein import occurs via two path-

ways. Proteins that possess the peroxisomal targeting

signal PTS1 bind the cytosolic receptor PEX5, whereas

proteins that possess PTS2 bind to the cytosolic receptor

PEX7 [10]. The receptor–cargo complex then docks with

the PEX13, PEX14 and PEX17 proteins at the mem-

brane. These proteins can be isolated from Saccharomycescerevisiae in a complex together with PEX7 and PEX5

[11]. In fungi, the import pathways for PTS1 and PTS2

are independent of one another up until the docking stage

(Figure 2). In mammals, alternative splicing of PEX5

results in short (PEX5S) and long (PEX5L) variants that

both bind PTS1-containing proteins. The long variant

also binds the PTS2–PEX7 complex, resulting in PTS2

import. The PTS1 and PTS2 import pathways also con-

verge via PEX5 in plants (Table 2; Figure 2). ArabidopsisPEX7 and PEX5 interact [12], and reduction of PEX5 and

PEX7 expression by RNA interference (RNAi) in Arabi-dopsis shows that PEX7 is required for PTS2 import,

whereas reducing PEX5 affects both PTS1 and PTS2

import (Figure 1; [4��]). Additionally, an Arabidopsis pex5-1 point mutation [3], which affects the import of PTS2

but not of PTS1 proteins [13��], is in a conserved serine.

The equivalent mutation in CHO cells similarly affects

PTS2 import and not PTS1 import because of an inability

of PEX7 to bind the mutant PEX5 [14]. The pex7-1mutant has normal PTS1 import and reduced PTS2

import [13��].

www.sciencedirect.com

Peroxisome protein import Baker and Sparkes 641

Table 1

Functions of plant peroxisomes.

Plant peroxisome type Tissue Common functions Specialized functions

Glyoxysome Seedling b-oxidation, branched-chain amino-

acid catabolism, and hydrogen

peroxide degradation

Glyoxylate cycle

Cotyledonary peroxisome Cotyledon Glycolate metabolism

Leaf peroxisome Leaf Glycolate metabolism

Root peroxisome Root Root-specific processes

Unspecialized peroxisome Stem, flower and silique ?

Table of plant peroxisome functions as determined on the basis of expression profiles of peroxisome-related genes [51]. Mutants have

been described in specific peroxisomal metabolic pathways (see [52] for recent review). In contrast to pathway-specific mutants, pex mutants

are predicted to affect all pathways because they affect the import of multiple peroxisomal proteins. Hydrogen peroxide metabolism, b-oxidation

and the degradation of branched-chain amino acids are also peroxisome functions in other eukaryotes. Unspecialized peroxisomes are so

named because no specialized functions have been ascribed to them as yet. However, the reproductive phenotypes seen in many mutants in

which peroxisome function is affected emphasize the importance of peroxisomes in these tissues.

Table 2

Phenotypes of pex mutants in Arabidopsis.

Defective

peroxin

PEX mutant Mutation Phenotype

PEX2 ted3 V275M Gain of function mutant, suppressor of det 1-1 [53].

pex2 T-DNA Embryo lethal [53].

PEX5 pex5-1 S318L Roots resistant to indole-3-butyric acid (IBA). Slight defect in seedling growth in the dark

in the absence of sucrose. PTS1 import unaltered, PTS2 import defective [3,13��].

pex5i RNAi Roots resistant to 2,4-dichlorophenoxy butyric acid (2,4-DB); seedling dependent on

sucrose for establishment; adult plants small and pale (recovered by growth in high CO2),

photorespiration defective, and PTS1 and PTS2 import defective [4��].

PEX6 pex6 R808Q Roots IBA-resistant, seedling dependent on sucrose for establishment, adult plants small

and pale, smaller siliques with reduced seed set. Fewer, large peroxisomes and reduced

level of PEX5 [23��].

PEX7 pex7-1 T-DNA at –95

(50UTR)

Roots IBA- and 2,4DB-resistant; not dependent on sucrose for seedling establishment.

PTS1 import unaltered, PTS2 import defective [13��].

pex7i RNAi Roots IBA-resistant, seedling dependent on sucrose for establishment, adult plants grow

normally in air, photorespiration unaltered. PTS1 import unaltered, PTS2 import defective

[4��].

PEX10 pex10 Ds element at

+599 (Exon 4)

Embryo lethal, homozygous mutants abort between globular and cotyledon stage [5,6].

PEX14 ped2 Q254stop Roots resistant to 2,4DB; seedling dependent on sucrose for establishment. Adult plants

compromised in photorespiration, PTS1 and PTS2 import defective [54].

PEX16 sse1 T-DNA Produce shrunken seed because of an increase in starch storage over protein and lipid

storage. PTS1 and PTS2 import are affected [39�,55].

PEX5 and

PEX7

pex5-1/pex7-1 Double mutant of

the above

Seedlings completely sucrose dependent in light and dark; cotyledon fusion sometimes

observed, adult plants have reduced stature and fertility; PTS2 import completely blocked

[13��].

Mutants in PEX genes have been isolated as a result of forward and reverse genetic screens. Forward screens have utilized the conversion of 2,4-

DB to the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) by b-oxidation. Mutants that are defective in b-oxidation remain sensitive to 2,4D.

Such mutants result either as a consequence of mutation in one of the genes coding enzymes of the b-oxidation pathway or as a result of a defect

in peroxisome biogenesis that results in b-oxidation deficiency caused by a failure to import the enzymes. Thus, these mutants have a normal

‘short root’ phenotype when grown on 2,4-D but a long root phenotype when grown on 2,4-DB [2]. Similarly, b-oxidation mutants have a long

root phenotype when grown on indole butyric acid (IBA) because of their inability to convert IBA to indole acetic acid (IAA) but remain sensitive

to IAA [3]. As might be expected, pex mutants are pleiotropic, the severity of the lesion depending on the particular allele (e.g. complete or partial loss

of function) and the particular process affected. For example, the pex5-1 mutant has a mild phenotype caused by a compromised PTS2

import pathway. pex7-1 and pex7i are more severe and the pex7-1/pex5-1 mutant is most severe because of a complete block in PTS2 import.

pex5i and ped2 are affected in both import pathways and display deficiency in photorespiration that is not seen in the PTS2-deficient mutants.

This is because the photorespiratory enzymes are all PTS1-targeted proteins. The most severe phenotypes are seen in the null mutants pex2

and pex10, which are embryo lethal, demonstrating an essential role for peroxisomes in plant development.

www.sciencedirect.com Current Opinion in Plant Biology 2005, 8:640–647

642 Cell biology

Figure 1

Plant peroxisomes. (a) Electron micrograph of a peroxisome from Arabidopsis cell culture. The large catalase crystal and single membrane

surrounding the peroxisome are clearly visible. Image courtesy of Maıte Vicre. Scale bar represents 100 nm. (b) Nicotiana tabacum epidermal

cells transiently expressing a matrix marker, CFP–SKL (green), or a membrane marker, eYFP–PEX10 (red). The membrane marker labels the rim

of the peroxisome whereas the matrix marker fills the peroxisomes in the adjacent cell. Both markers are not expressed in the same cell.

Scale bar represents 2 mm. (c) Arabidopsis roots from a control plant expressing PTS2–GFP marker (bottom panel) and a transgenic plant

expressing a PEX5 RNAi construct together with either PTS1–GFP (top panel) or PTS2–GFP (middle panel). Lower levels of PEX5 in the

transgenic plant affect the import of both PTS1 and PTS2 proteins, which are no longer imported into the punctate peroxisome structures

and are mislocalised to the cytosol. Scale bar represents 30 mm. Reproduced from [4��] with the permission of American Society for Biochemistry

and Molecular Biology. Copyright 2005 by American Society for Biochemistry and Molecular Biology.

Import and export of matrix proteinreceptorsPEX5 and PEX7 follow the extended shuttle route in

which import of the receptor–cargo complex into the

peroxisome is followed by complex dissociation and

export of the receptor back into the cytosol for further

rounds of import (Figure 2). PEX5 becomes accessible to

a peroxisomal matrix protease during the import cycle

[15], but there is still some debate as to whether the

complex remains associated with the peroxisome mem-

brane or completely translocates into the matrix [16].

Cargo-loaded PEX5 is imported, and re-export of the

receptor is ATP-dependent [17]. Amino acids 1–17 of

PEX5 are required for re-export [18]. The PEX5 cycle is

discussed in detail by Azevedo et al. [19]. PEX5 is thus a

complex protein that performs multiple interactions with

different binding partners at different phases of the


import cycle (Figure 2; [19]). Likewise, PEX7 has

recently been shown to cycle between the peroxisome

lumen and the cytosol [20��].

How is cargo unloaded? PEX8, which bridges the docking

and PEX2/PEX10/PEX12 complexes on the trans side of

the membrane [11], interacts with PEX5 and has been

proposed to act as an unloading factor. As the peroxisome

lumen of both mammalian [21] and S. cerevisiae [22]

peroxisomes has been reported to be alkaline, changes

in protein conformation that are mediated by differences

in pH might play a role.

The export of PEX5 (and presumably of PEX7) is an

ATP-dependent process. PEX1 and PEX6 belong to the

AAA family of ATPases. These peroxins interact with

each other, are anchored to the peroxisome membrane by



Figure 2

Import of peroxisomal matrix proteins. Receptor–cargo complexes composed of cargo proteins containing PTS1 (blue star) or PTS2 (red star)

bind to receptors PEX5 or PEX7, respectively. The PEX7–cargo complex requires accessory factors for import; in plants and mammals it binds

PEX5, in S. cerevisiae it binds PEX18 and PEX21, and in Neurospora crassa it binds PEX20 [56]. PEX18 is able to functionally replace the amino

terminus of PEX5 for receptor docking [57]. The receptor (purple circle)–cargo (purple star) complex in yeast and mammals docks at the

membrane. The protein components that are involved in docking, PEX13, PEX14 and PEX 17, are coloured green. It is unclear whether the

receptor translocates fully into the matrix or remains associated with the translocation complex. PEX8 in S. cerevisiae has been shown to

bridge two complexes that are involved in docking (green; PEX13, PEX14 and PEX 17) or translocation (yellow; PEX2, PEX10 and PEX12) in

yeast and mammals. Additional protein components that are involved in steps downstream of receptor–cargo translocation are shown (blue;

PEX4, PEX22, PEX26, PEX15, PEX1 and PEX6) where PEX6 binds PEX15 in S. cerevisiae or PEX26 in humans. At this stage, it is unclear how

receptors are recycled back to the cytosol for further rounds of import. In plants, much less is understood about all of the steps involved in

the import of peroxisomal matrix proteins.

binding to PEX15 (in S. cerevisiae) or PEX26 (in humans)

and are required for peroxisome biogenesis.

An Arabidopsis PEX6 mutant has been isolated (Table 2)

and had reduced numbers of enlarged peroxisomes and

significantly reduced levels of the PTS1 receptor PEX5

[23��]. Human fibroblast lines that are defective in PEX6

also have fewer enlarged peroxisomes and reduced levels

of PEX5. The growth defects of the Atpex6mutant can be

partially restored by overexpression of human PEX6 or

Arabidopsis PEX5 [23��]. These results are consistent with

several other studies that have proposed a role for PEX1

and PEX6 in receptor recycling [17,24,25�].


Export of peroxisomal matrix proteinreceptors: role of ubiquitination?Ubiquitination is an important posttranslational modifi-

cation that is involved in signalling (monoubiquitination)

and in targeting proteins for degradation (polyubiquitina-

tion). PEX5 and PEX18 in S. cerevisiae are ubiquitinated

[25�–27�,28]. In wildtype S. cerevisiae cells, PEX5 is

monoubiquitinated at the peroxisomal membrane. Simi-

lar levels of monoubiquitinated PEX5 are present in

wildtype cells and in the cells of mutants that are defec-

tive in the ubiquitin-conjugating (UBC) E2 enzymes,

UBC4, UBC5 and UBC1 [25�]. Hence, monoubiquitina-

tion was proposed to occur via a fourth E2 enzyme PEX4.


644 Cell biology

PEX4 interacts with PEX10 [29], a protein that has a

RING-finger domain. This domain is also found in

some E3 enzymes, providing substrate specificity for

the E2s. Although there is no evidence that PEX10 acts

in this way, PEX5 ubiquitination is dependent on PEX10

[25�,26�]. A model has been proposed in which PEX5

is monoubiquitinated by PEX4 in a RING-complex-

dependent manner and then extracted from the mem-

brane by the PEX1/PEX6/PEX15 complex [25�]. An

analogous function in removal of proteins from the endo-

plasmic reticulum (ER) membrane for degradation is

performed by the AAA protein p97/VCP/CDC48. The

crystal structure of the amino-terminal domain of

PEX1 shows similarity to the amino-terminal domain

of VCP [30]. Thus, Kragt et al. [25�] propose that mono-

ubiquitination is a signal for receptor recycling. In the

absence of any of the peroxins involved downstream of

receptor–cargo docking/translocation (PEX4, PEX22,

PEX1, PEX6 and PEX15) PEX5 is polyubiquitinated

by UBC4, UBC5 and UBC1 and degraded [25�–27�].This is a very testable model and future studies should

determine if it is correct and applicable to higher eukar-

yotes.

Import of peroxisomal membrane proteinsPeroxisome membrane proteins (PMPs) have targeting

signals (mPTS) and import machinery that are comple-

tely different to those of matrix proteins (Figure 3). Basic

residues that are adjacent to one or more transmembrane

domains are important components of mPTSs, and some

PMPs have multiple mPTSs that might be functionally

redundant or might work co-operatively to bring about

efficient targeting [31,32]. In S. cerevisiae different mPTSs

might function to bring about targeting to distinct classes

of peroxisome [33�], and there is an intriguing possibility

Figure 3

Import of peroxisomal membrane proteins. Domain mapping of human PEX

1–51 in yellow) is involved in binding PEX3, the central domain (amino acids

acids 124–299 in green) is required for binding mPTS of certain PMPs [41,4

PEX19 acting as a chaperone/receptor and binding PMPs. This complex su

PEX16 is also involved in this process in mammals [38]. It is unclear how PM

(PEX3) appear to insert independently of PEX19 and PEX3, hinting at a seco


that a similar mechanism could operate in plants that have

multiple peroxisome types (Table 1).

The possible involvement of the ER in PMP trafficking

remains controversial [34] but a recent report has pro-

vided definitive evidence that PEX3 in S. cerevisiae trafficsfrom the ER to peroxisomes [35��]. It is possible that not

all PMPs take this route and the pathways involved

remain unknown. Arabidopsis PEX2 and PEX10 are tar-

geted to peroxisomes independently of known ER trans-

port routes [36].

Two peroxins, PEX3 and PEX19, are implicated in PMP

import in mammals and fungi (Figure 3; reviewed in [37])

and homologues of these peroxins exist in the Arabidopsisgenome [1,32]. PEX16 in mammals is also required for

PMP import [38] and the Arabidopsis sse1 ( pex16) mutant

has defective peroxisomes [39�], although whether this is

due to a defect in the import of PMPs remains to be

established. Recently, evidence has accumulated that

supports the notions that PEX19 functions as a cycling

receptor/chaperone for PMPs [40] and that PEX3 func-

tions as the membrane-bound receptor for PEX19 [41].

Recombinant PEX19 can interact simultaneously with

PEX3 and cargo [42].

What features of PMPs does PEX19recognize?Functionally equivalent PEX19 binding sites have been

identified in PEX13 and PEX11 from S. cerevisiae [43��].These are short linear, probably helical, peptides that

contain both basic and hydrophobic residues. In vivoexperiments showed that the PEX19-binding site of

PEX13 is necessary for targeting, but is only sufficient

in combination with at least one transmembrane domain

19 isolated three domains: the amino-terminal region (amino acids

60–91 in red) binds PEX14, and the carboxy-terminal domain (amino

8]. PMP import in yeast and mammals is proposed to occur through

bsequently docks at the peroxisome membrane by binding PEX3.

Ps are inserted into the membrane. Certain PMPs in mammals

nd PMP targeting pathway [40].



[43��]. These results allowed the development of a pre-

diction matrix for PEX19-binding sites. When applied to

mammalian PMPs, this matrix correctly identified the

mPTS of Adrenoleukodystrophy protein (ALDP), a per-

oxisomal ATP-binding cassette (ABC) transporter. This

binding site overlaps with the previously identified

ALDP targeting signal [44] and is capable of binding

both HsPEX19 and ScPEX19, thereby showing the evo-

lutionary conservation of mPTS recognition between

yeast and humans [45��].

Is there a non-PEX19-dependent PMPimport pathway?Despite strong evidence in support of the role of PEX19

as a cycling chaperone/receptor that is recruited to the

peroxisomemembrane by PEX3, there are results that are

not easily explained by this model. A mutant PEX13 that

failed to bind PEX19 could be imported into peroxisomes

and integrated into the peroxisome membrane [46]. The

defect in PMP import inHansenula or Saccharomyces pex19mutants could be partially overcome by overexpressing

either full-length PEX3 or a green fluorescent protein

(GFP)-fusion protein comprising only the first 50 amino

acids of PEX3 [47], which are insufficient to bind PEX19

[41]. The insertion of PEX3 into the peroxisome mem-

brane is not dependent on PEX19 in mammalian cells

[40] although PEX19 was required for PEX3 exit from the

ER in S. cerevisiae [35��]. These results might hint at as-

yet-undescribed PMP targeting pathways (Figure 3).

Is PEX19 a multifunctional protein?A role for PEX19 in the assembly of the PTS1 import

complex has been proposed on the basis of two findings.

First, PEX19 can compete with PEX5 and PEX13 for

binding to PEX14 at overlapping but non-identical bind-

ing sites [46]. Second, PEX19 is detected in complexes in

the peroxisome membrane and cytosol [47]. A domain-

mapping study of HsPEX19 concluded that there are

three domains in PEX19, the amino-terminal region that

binds PEX3 [41,48], a central region that binds PEX14

and the carboxy-terminal region that binds cargo PMPs

([48]; Figure 3).

As well as having potentially distinct roles in PMP import

and membrane complex assembly, PEX19 has been

reported to be involved in the internalisation of the

plasma membrane type IIa sodium-dependent phosphate

co-transporter and in the regulation of a tumour suppres-

sor pathway in mice [49,50]. Whether these observations

are in any way connected to PEX19’s role in peroxisome

assembly or whether PEX19 is a truly multifunctional

protein remains to be seen.

ConclusionsRecent results suggest that the import of peroxisome

matrix proteins in mammals might be more similar to

that in plants than to that in yeast. This observation


prompted the claim that this ‘establishes Arabidopsis asan excellent model for human peroxisome biogenesis

disorders’ [13��]. Further characterisation of the import

system could therefore result in plant systems being

studied not only in terms of agricultural benefits but also

because parallels could also lend insight into mammalian

peroxisome biogenesis disorders.

Note added in proofThe reader is referred to two publications that appeared

too late for inclusion in this review [58,59].

AcknowledgementsWe thank Dr Maıte Vicre and Dr Makoto Hayashi for the images usedin Figure 1a and c respectively, and we acknowledge the support ofBiotechnology and Biological Sciences Research Council (BBSRC)grants (C19029 and C19030) for our current work.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest�� of outstanding interest

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23.��

Zolman BK, Bartel B: An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPaseimplicated in peroxisomal function. Proc Natl Acad Sci USA2004, 101:1786-1791.

ArabidopsisPEX6was isolated from a forward geneticsscreen and found tobe rescued by human PEX6 cDNA. At the molecular level, the Arabidopsismutantdisplaysseveralcharacteristics thataresimilar to thoseof thehumanpex6 mutant: fewer peroxisomes, enlarged peroxisomes and decreasedlevels of PEX5 that can be compensated by overexpression of PEX5. Thisindicates that PEX6 might be involved in receptor recycling in plants.

24. Collins CS, Kalish JE, Morrell JC, McCaffery JM, Gould SJ:The peroxisome biogenesis factors Pex4p, Pex22p, Pex1p,


and Pex6p act in the terminal steps of peroxisomal matrixprotein import. Mol Cell Biol 2000, 20:7516-7526.

25.�

Kragt A, Brouwer TV, van den Berg M, Distel B: TheSaccharomyces cerevisiae peroxisomal import receptorPex5p is monoubiquitinated in wild type cells. J Biol Chem2005, 280:7867-7874.

Like Kiel et al. [27�], the authors propose that the polyubiquitinated form ofPEX5 in S. cerevisiae pex mutants might be subject to targeted degrada-tion. They show that PEX5 is monoubiquitinated in wildtype cells andpropose this ubiquitination state is required for receptor recycling.

26.�

Platta H, Girzalsky W, Erdmann R: Ubiquitination of theperoxisomal import receptor Pex5p. Biochem J 2004,384:37-45.

First published report that PEX5 in S. cerevisiae is ubiquitinated.

27.�

Kiel J, Emmrich K, Meyer HE, Kunau WH: Ubiquitination ofthe peroxisomal targeting signal type 1 receptor, Pex5p,suggests the presence of a quality control mechanismduring peroxisomal matrix protein import. J Biol Chem 2005,280:1921-1930.

S. cerevisae PEX5 is ubiquitinated in pex mutants and has an implied rolein receptor recycling. In wildtype cells, the proteasome is involved in PEX5turnover. The authors propose that ubiquitination might act as a qualitycontrol mechanism.

28. Purdue PE, Lazarow PB: Pex18p is constitutively degradedduring peroxisome biogenesis. J Biol Chem 2001,276:47684-47689.

29. Eckert JH, Johnsson N: Pex10p links the ubiquitin conjugatingenzyme Pex4p to the protein import machinery of theperoxisome. J Cell Sci 2003, 116:3623-3634.

30. Shiozawa K, Maita N, Tomii K, Seto A, Goda N, Akiyama Y,Shimizu T, Shirakawa M, Hiroaki H: Structure of the N-terminaldomain of PEX1 AAA-ATPase — characterization of a putativeadaptor-binding domain. J Biol Chem 2004, 279:50060-50068.

31. Murphy MA, Phillipson BA, Baker A, Mullen RT: Characterizationof the targeting signal of the Arabidopsis 22-kD integralperoxisomal membrane protein. Plant Physiol 2003,133:813-828.

32. Hunt JE, Trelease RN: Sorting pathway and molecular targetingsignals for the Arabidopsis peroxin 3. Biochem Biophys ResCommun 2004, 314:586-596.

33.�

Wang X, McMahon MA, Shelton SN, Nampaisansuk M, Ballard JL,Goodman JM: Multiple targeting modules on peroxisomalproteins are not redundant: discrete functions of targetingsignals within PmP47 and Pex8p. Mol Biol Cell 2004,15:1702-1710.

Characterisation of PMP47 and PEX8 targeting signals under variousgrowth conditions indicate that the contribution of different targetingsignals within a single protein to efficient targeting depends upon themetabolic state of the cell.

34. Heiland I, Erdmann R: Biogenesis of peroxisomes topogenesisof the peroxisomal membrane and matrix proteins.FEBS J 2005, 272:2362-2372.

35.��

Hoepfner D, Schildknegt D, Braakman I, Philippsen P, Tabak HF:Contribution of the endoplasmic reticulum to peroxisomeformation. Cell 2005, 122:85-95.

S. cerevisiae pex3 mutants lack detectable peroxisomes. Real-timeimaging of newly synthesised YFP tagged PEX3 and PEX19 in S. cere-visiae pex3 mutants and wildtype cells revealed that PEX3 is found first inthe ER and subsequently co-localises with a peroxisome matrix marker.PEX3 trafficking and peroxisome synthesis is dependent on PEX19. Thisis compelling evidence that peroxisomes are derived from the ER.

36. Sparkes IA, Hawes C, Baker A: AtPEX2 and AtPEX10 aretargeted to peroxisomes independently of known ERtrafficking routes. Plant Physiol 2005, 139:in press.

37. Schliebs W, Kunau WH: Peroxisome membrane biogenesis:the stage is set. Curr Biol 2004, 14:R397-R399.

38. South ST, Gould SJ: Peroxisome synthesis in the absence ofpreexisting peroxisomes. J Cell Biol 1999, 144:255-266.

39.�

Lin Y, Cluette-Brown JE, Goodman HM: The peroxisomedeficient Arabidopsis mutant sse1 exhibits impaired fatty acidsynthesis. Plant Physiol 2004, 135:814-827.



SHRUNKEN SEED 1 (SSE1) was previously predicted to be a plant PEX16homologue. Here, the authors show that SSE1 is involved in peroxisomebiogenesis; GFP–SSE1 fusion is targeted to peroxisomes, sse1 mutantslack normal peroxisomes.

40. Jones JM, Morrell JC, Gould SJ: PEX19 is a predominantlycytosolic chaperone and import receptor for class 1peroxisomal membrane proteins. J Cell Biol 2004,164:57-67.

41. Fang Y, Morrell JC, Jones JM, Gould SJ: PEX3 functions as aPEX19 docking factor in the import of class I peroxisomalmembrane proteins. J Cell Biol 2004, 164:863-875.

42. Shibata H, Kashiwayama Y, Imanaka T, Kato H: Domainarchitecture and activity of human Pex19p, a chaperone-likeprotein for intracellular trafficking of peroxisomal membraneproteins. J Biol Chem 2004, 279:38486-38494.

43.��

Rottensteiner H, Kramer A, Lorenzen S, Stein K, Christiane LF,Volkmer-Engert R, Erdmann R: Peroxisomal membraneproteins contain common Pex19p-binding sites that are anintegral part of their targeting signals. Mol Biol Cell 2004,15:3406-3417.

This study provides definitive evidence for the function of PEX19 as aPMP import receptor. The authors used yeast two-hybrid and peptide-scanning techniques to identify PEX19-binding sites in PEX13 andPEX11. These sites, which were short linear peptides composed ofpredominantly basic and hydrophobic residues, were functionally inter-changeable and necessary for PMP targeting in vivo. In combination witha transmembrane domain, they were sufficient for correct targeting. Theauthors used their results to develop and test an algorithm for definingPMP targeting signals in yeast by assessing PEX19-binding sites.

44. Landgraf P, Mayerhofer P, Polanetz R, Roscher A, Holzinger A:Targeting of the human adrenoleukodystrophy protein to theperoxisomal membrane by an internal region containing ahighly conserved motif. Eur J Cell Biol 2003, 82:401-410.

45.��

Halbach A, Lorenzen S, Landgraf C, Volkmer-Engert R,Erdmann R, Rottensteiner H: Function of the PEX19-bindingsite of human Adrenoleukodystrophy protein as targetingmotif in man and yeast. J Biol Chem 2005, 280:21176-21182.

This study demonstrates the conservation of PMP-targeting sitesbetween yeast and mammals. The authors show that the applicationof an algorithm that was developed by Rottensteiner et al. [43��] for yeastPMPs to known mammalian PMPs defined both sites shown previously tobe required for targeting to the peroxisome membrane and novel sites. APEX19-binding site in the human Adrenoleukodystrophy Protein (ALDP)that was identified by the algorithm was shown to bind both human andyeast PEX19 in vitro and to be responsible for ALDP targeting in vivo. ThePEX19-binding site in ALDP could functionally substitute for that of yeastPEX13.

46. Fransen M, Vastiau I, Brees C, Brys V, Mannaerts GP,Van Veldhoven PP: Potential role for Pex19p in assemblyof PTS-receptor docking complexes. J Biol Chem 2004,279:12615-12624.


47. Otzen M, Perband U, Wang DY, Baerends RJS, Kunau WH,Veenhuis M, Van der Klei IJ: Hansenula polymorpha Pex19p isessential for the formation of functional peroxisomalmembranes. J Biol Chem 2004, 279:19181-19190.

48. Fransen M, Vastiau I, Brees C, Brys V, Mannaerts GP,Van Veldhoven PP: Analysis of human Pex19p’s domainstructure by pentapeptide scanning mutagenesis.J Mol Biol 2005, 346:1275-1286.

49. Ito M, Iidawa S, Izuka M, Haito S, Segawa H, Kuwahata M,Ohkido I, Ohno H, Miyamoto K: Interaction of a farnesylatedprotein with renal type IIa Na/Pi co-transporter in response toparathyroid hormone and dietary phosphate. Biochem J 2004,377:607-616.

50. Wadhwa R, Sugihara T, Hasan K, Taira K, Reddel RR, Kaul SC:A major functional difference between the mouse andhuman ARF tumour suppressor proteins. J Biol Chem 2002,277:36665-36670.

51. Kamada T, Nito K, Hayashi H, Mano S, Hayashi M, Nishimura M:Functional differentiation of peroxisomes revealed byexpression profiles of peroxisomal genes in Arabidopsisthaliana. Plant Cell Physiol 2003, 44:1275-1289.

52. Hayashi M, Nishimura M: Entering a new era of research onplant peroxisomes. Curr Opin Plant Biol 2003, 6:577-582.

53. Hu J, Aguirre M, Peto C, Alonso J, Ecker J, Chory J: A role forperoxisomes in photomorphogenesis and development ofArabidopsis. Science 2002, 297:405-409.

54. Hayashi M, Nito K, Toriyama-Kato K, Kondo M, Yamaya T,Nishimura M: AtPex14p maintains peroxisomal functions bydetermining protein targeting to three kinds of plantperoxisomes. EMBO J 2000, 19:5701-5710.

55. Lin Y, Sun L, Nguyen LV, Rachubinski RA, Goodman HM: Thepex16p homolog SSE1 and storage organelle formation inArabidopsis seeds. Science 1999, 284:328-330.

56. Sichting M, Schell-Steven A, Prokisch H, Erdmann R,Rottensteiner H: Pex7p and Pex20p of Neurospora crassafunction together in PTS2-dependent protein import intoperoxisomes. Mol Biol Cell 2003, 14:810-821.

57. Schafer A, Kerssen D, Veenhuis M, Kunau WH, Schliebs W:Functional similarity between the peroxisomal PTS2 receptorbinding protein Pex18p and the N-terminal half of the PTS1receptor Pex5p. Mol Cell Biol 2004, 24:8895-8906.

58. Karnik SK, Trelease N: Arabidopsis Peroxin 16 coexists atsteady state in peroxisomes and endoplasmic reticulum.Plant Physiol 2005, 138:1967-1981.

59. Fan J, Quan S, Orth T, Awai C, Chory J, Hu J: The ArabidopsisPEX12 gene is required for peroxisome biogenesis and isessential for development. Plant Physiol 2005, 139: in press.


Peroxisome protein import: some answers, more questions

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