Báo cáo khoa học: Biogenesis of peroxisomes Topogenesis of the peroxisomal membrane and matrix proteins ppt

REVIEW ARTICLE
Biogenesis of peroxisomes
Topogenesis of the peroxisomal membrane and matrix proteins
Ines Heiland and Ralf Erdmann
Ruhr-Universita
¨
t Bochum, Institut fu
¨
r Physiologische Chemie, Bochum, Germany
Introduction
Peroxisomes are ubiquitious, single membrane bound
organelles of eukaryotic cells [2]. They maintain various
functions that differ depending on the species and cell
type, as well as the environmental or developmental
conditions. Many metabolic pathways of peroxisomes
lead to the production of hydrogen peroxide. The
subsequent decomposition of this toxic compound by
catalase is a fundamental process that takes place in
almost all peroxisomes. Moreover, peroxisomes contrib-
ute to the b- and a-oxidation of fatty acids, synthesis of
ether lipids such as plasmalogens, and the oxidation of
bile acids and cholesterol [3–6]. Defects in the biogenesis
of peroxisomes are the molecular cause for severe inher-
ited diseases, called peroxisome biogenesis disorders
Keywords
peroxin, peroxisome, protein transport
Correspondence
R. Erdmann, Ruhr-Universita
¨
t Bochum,
Institut fu

¨
r Physiologische Chemie,
Abteilung fu
¨
r Systembiochemie,
44780 Bochum, Germany
Fax: +49 234 321 4266
Tel: +49 234 322 4943
E-mail:
(Received 10 February 2005, accepted 31
March 2005)
doi:10.1111/j.1742-4658.2005.04690.x
Genetic and proteomic approaches have led to the identification of 32 pro-
teins, collectively called peroxins, which are required for the biogenesis of
peroxisomes. Some are responsible for the division and inheritance of per-
oxisomes; however, most peroxins have been implicated in the topogenesis
of peroxisomal proteins. Peroxisomal membrane and matrix proteins are
synthesized on free ribosomes in the cytosol and are imported post-trans-
lationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu
Rev Cell Biol 1, 489–530 [1]). Progress has been made in the elucidation of
how these proteins are targeted to the organelle. In addition, the under-
standing of the composition of the peroxisomal import apparatus and the
order of events taking place during the cascade of peroxisomal protein
import has increased significantly. However, our knowledge on the basic
principles of peroxisomal membrane protein insertion or translocation of
peroxisomal matrix proteins across the peroxisomal membrane is rather
limited. The latter is of particular interest as the peroxisomal import
machinery accommodates folded, even oligomeric, proteins, which distin-
guishes this apparatus from the well characterized translocons of other
organelles. Furthermore, the origin of the peroxisomal membrane is still

enigmatic. Recent observations suggest the existence of two classes of per-
oxisomal membrane proteins. Newly synthesized class I proteins are
directly targeted to and inserted into the peroxisomal membrane, while
class II proteins reach their final destination via the endoplasmic reticulum
or a subcompartment thereof, which would be in accord with the idea
that the peroxisomal membrane might be derived from the endoplasmic
reticulum.
Abbreviations
APX, ascorbate peroxidase; mPTS, membrane protein targeting signals; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting
signal; TPR, tetratricopeptide repeat.
2362 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS
(PBD,) such as Zellweger syndrome, neonatal adreno-
leukodystrophy and Refsums disease [7].
Peroxisomal matrix protein import
Many investigations have focussed on the elucidation
of the import of peroxisomal matrix proteins, and the
mechanisms involved are becoming better understood
[8,9]. It is generally accepted that Pex5p and Pex7p,
the receptors for the proteins harboring peroxisomal
targeting sequences, cycle between the cytosol and the
peroxisome. This gave rise to the so-called model of
shuttling receptors [10,11]. According to this model,
the import receptors bind cargo proteins in the cytosol
and direct them to a docking and translocation com-
plex at the peroxisomal membrane. There, the cargo is
released and translocated across the peroxisomal mem-
brane while the receptor shuttles back to the cytosol in
a so-far unknown manner. The so-called extended
shuttle hypothesis is based on the assumption that the
import receptor does not stop at the peroxisomal

membrane but enters the peroxisomal lumen together
with its cargo [12–14]. In this case, cargo release
takes place in the peroxisomal matrix and the cargo-
unloaded receptors are transported back to the cytosol.
Peroxisomal targeting sequences and
their receptors
Peroxisomal matrix proteins are synthesized on free
ribosomes in the cytosol and are bound by the peroxi-
somal targeting sequence receptors Pex5p and Pex7p.
To date, two targeting sequences for peroxisomal mat-
rix proteins have been identified. The most abundant is
the peroxisomal targeting signal type I (PTS1), which
consists of a conserved tripeptide at the extreme C-ter-
minus of the protein and a less conserved upstream
region [15,16]. The consensus sequence of the C-ter-
minal tripeptide is S ⁄ A-K⁄ R-L ⁄ M, but not all varia-
tions are functional in all species [17–20]. The second
peroxisomal targeting signal (PTS2) is located close to
the N-terminus and is defined by the less conserved
consensus sequence R-L ⁄ I-X
5
HL [20,21].
The PTS1 receptor Pex5p contains seven tetratrico-
peptide repeat (TPR) domains, which are essential for
PTS1 binding [22]. Of these seven TPR domains six
interact directly with the tripeptide, whereas TPR4 is
important for the structural alignment of the other
TPR motifs [23,24]. Acyl-CoA oxidases from Saccharo-
myces cerevisiae, Hansenula polymorpha and Candida
tropicalis contain neither a PTS1 nor a PTS2 signal.

However, it has been shown that these proteins are
still targeted via the PTS1 receptor Pex5p, but bind to
regions of the protein distinct from the PTS1-recogni-
tion domain [25]. Pex7p is the cytosolic receptor for
PTS2 proteins and belongs to the family of WD40 pro-
teins that share a consensus sequence of 40 amino
acids, which contains a central tryptophan-aspartic
acid motif [10]. Pex7p contains six of these repeats. In
S. cerevisiae, Pex7p is associated with Pex18p ⁄ Pex21p
[26,27], proteins with redundant functions that are pre-
sumed to mediate the association of cargo-loaded
Pex7p with the docking complex. Whereas Pex7p is
present in nearly all species analysed, Pex18p
and Pex21p are evolutionarily less conserved. In
Neurospora crassa and Yarrowia lipolytica the function
of Pex18p ⁄ Pex21p is performed by Pex20p, suggesting
that the protein is a true orthologue of the yeast pro-
teins [28,29].
In addition to the fact that PTS1 and PTS2 protein
import pathways employ different components there
seems to be a common mechanism for both processes.
In support of this assumption, it has been shown that
Pex18p can functionally replace the N-terminal domain
of Pex5p [30]. Remarkably, in humans, Pex5p exists in
two isoforms, one characterized by a 37 amino acid
insertion that mediates binding of Pex7p to Pex5p and
therefore overcoming the requirement for Pex18p ⁄
Pex21p [31,32]. Thus, in mammalian cells, the PTS2
pathway depends on the presence of the long isoform
of PTS1 receptor Pex5p, which is required to direct

cargo-loaded Pex7p to the import machinery at the
peroxisomal membrane [29–32]. Furthermore, it has
been demonstrated recently that PTS1 and PTS2
import pathways are also coupled in plants [33].
The peroxisomal protein import
machinery
Upon the binding of PTS1 proteins, Pex5p depolym-
erizes [34] and is transported to the peroxisome
where it interacts with Pex14p [35–39] and Pex13p
[40–44], as well as Pex12p [45–48], leading to the
question of which of these proteins performs the
docking event. As Pex5p accumulates at the peroxi-
somal membrane in pex13-, pex2- and pex12- but
not in pex14-deficient cell lines [49] and as the bind-
ing affinity of cargo-loaded Pex5p is much higher
for Pex14p then for Pex13p [50,51], Pex14p is
believed to mediate peroxisomal membrane associ-
ation of Pex5p. At the peroxisomal membrane,
Pex14p is associated with Pex17p [52] and at least
temporally with Pex13p. The puative peroxisomal
import complex (importomer) is formed by the
RING-finger subcomplex containing Pex2p, Pex10p
and Pex12p, and the docking complex comprising
I. Heiland and R. Erdmann Biogenesis of peroxisomes
FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS 2363
Pex13p, Pex14p and Pex17p. Both subcomplexes are
linked via Pex8p [53], which contains both targeting
sequences for peroxisomal matrix protein import
(PTS1 and PTS2). However, the import of Pex8p
does not depend on these signals [54,55]. It is imagi-

nable that these targeting signals are bound by the
import receptors after cargo release to prevent reas-
sociation with cargo proteins and evidence has been
provided for Pex8p being directly involved in cargo–
receptor dissociation [56]. The functions of other
components of the import complex are still
unknown. Whether the RING-finger complex is
really involved in peroxisomal matrix protein import
or rather in the re-export of the PTS1 receptor
Pex5p still has to be investigated.
It has been demonstrated that Pex5p becomes ubi-
quitinated during import [57–59]. Furthermore,
Pex18p, a component of the signal recognition com-
plex in the PTS2-pathway, becomes mono- and diubiq-
uitinated during import and is degraded in a
proteasome-dependent manner [60]. Polyubiquitination
of Pex5p is detectable in pex1, pex6, pex4 and pex22
mutants of S. cerevisiae and requires a functional
import complex. The physiological relevance of Pex5p
ubiquitination, however, remains to be shown. It is
possible that import receptors that remained in the
import pathway are polyubiquitinated and subse-
quently directed to proteasomal degradation as a form
of quality control [58]. However, it is also conceivable
that ubiquitination of Pex5p and Pex18p serves as a
signal for their export back to the cytosol [57,59]. As
RING-finger proteins often function as E3–ubiquitin
protein ligases in ubiquitin and ubiquitin-like conjuga-
tions [61], Pex2p, Pex10p and Pex12p might be
involved in the ubiquitination of the import receptor.

Pex5p recycling to the cytosol has been demonstrated
to be accompanied by ATP hydrolysis and to require
the N-terminus of the receptor [62,63].
The current understanding of the organization of
the peroxisomal import machinery for PTS1 proteins is
summarized in Fig. 1. In the absence of cargo protein,
Pex5p is retained in the cytosol in a tetrameric com-
plex. Upon PTS1–protein binding, Pex5p disaggregates
into dimers [34] and is transported in a currently
unknown manner to the peroxisome. At the peroxi-
somal membrane, Pex5p binds to the docking complex,
presumably mediated by Pex14p. How the cargo or
the cargo–receptor complex is translocated across the
peroxisomal membrane is completely unknown. Eluci-
dation of this cellular process is a particular challenge,
as the proteins are transported in a folded or even
oligomeric conformation. Pex8p triggers the associ-
ation of the docking and the RING-finger complex
and might contribute to cargo release. At the end of
the pathway, Pex5p is recycled back to the cytosol in
an ATP-dependent manner.
Lipid transport to peroxisomes
The major lipid components of peroxisomal mem-
branes are phosphatidylcholine and phosphatidyletha-
nolamine [64–66]. Most enzymes involved in the
synthesis of polar lipids are localized in the endoplas-
mic reticulum (ER), and the peroxisome is not capable
of synthesizing these lipids [65,67]. Therefore the lipids
have to be tranported from the ER to the peroxisome,
which might require the employment of specialized ves-

icles as postulated by Purdue and Lazarow [68]. As an
alternative, membrane constituents might flip from the
ER membrane at contact sites between ER and peroxi-
somes. Evidence has been provided that the latter
mechanism is employed for the transport of phospho-
lipids from the ER to mitochdondria [69–71]. How
peroxisomes gain their phospholipids remains to be
investigated.
Peroxisomal membrane protein import
Most mutants that are defective for the import of
PTS1 and PTS2 proteins still import peroxisomal
membrane proteins. Thus, the import of peroxisomal
membrane and matrix proteins is independent
[41,42,72]. The peroxisomal membrane protein target-
ing signals (mPTS) were identified for several peroxi-
somal membrane proteins (PMPs). These targeting
sequences contained a basic amino acid sequence in
conjunction with at least one transmembrane region
[73–77].
Some PMPs have been shown to posses multiple tar-
geting signals [55,78,79]. One possible reason for the
existence of multiple mPTS might be that they are
required to distinguish targeting to different peroxi-
some populations [55]. This might be of particular
interest for higher eukaryotes such as plants, which
generate different types of peroxisomes during their
development.
Only three of the 32 peroxins identified so far –
Pex3p, Pex16p and Pex19p – have been shown to be
involved in peroxisomal membrane protein import

[80,81]. PEX16-deficient cell lines lack detactable per-
oxisomal membrane structures [77,80,82]. Moreover,
Arabidopsis thaliana pex16 mutants show defects in oil
body and fatty acid synthesis [83,84]. How Pex16p par-
ticipates in peroxisomal membrane biogenesis is not
known. The function and characteristics of Pex3p and
Pex19p are discussed below.
Biogenesis of peroxisomes I. Heiland and R. Erdmann
2364 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS
Pex19p – chaperone, import receptor
or both?
The functional role of Pex19p in peroxisome biogenesis
has been controversial. Pex19p is a predominantly
cytosolic protein that can be farnesylated [85,86]. In
cells lacking Pex19p, peroxisomal membrane proteins
are unstable or mislocalized [81,87]. Pex19p is known
to bind multiple PMPs [88], but whether it binds to
the targeting signals of these proteins and therefore
functions as cytosolic receptor or whether Pex19p
binds unspecifically to hydrophobic regions – similar
to chaperones – is still a matter of debate [89,90].
However, using in vitro binding studies and bioinfor-
matic approaches Rottensteiner et al. [91] recently
identified a consensus sequence for the binding sites of
Pex19p. These binding sites were demonstrated to be
required for peroxisomal membrane protein targeting.
Moreover, in conjunction with an adjacent transmem-
brane domain, these sites proved to be sufficient for
the peroxisomal membrane targeting of an otherwise
mislocalized fusion protein. Thus, the mPTS is formed

by the Pex19p binding site together with an adjacent
transmembrane segment. In this assembly, the Pex19p
binding site is proposed to contain the required
targeting information, while the transmembrane seg-
ment is required for the permanent insertion of the
protein into the peroxisomal membrane. The fact that
the Pex19p binding site is an integral part of the mPTS
also demonstrates that Pex19p functions as a targeting
sequence receptor for peroxisomal membrane proteins.
There is, however, one exception. Pex3p targeting is
not dependent on Pex19p, and Pex19p binds to Pex3p
in regions different from its targeting signal [90,92].
Therefore, the existence of distinct classes of peroxi-
somal membrane proteins have been postulated
[93,94]. Class I PMPs are synthesized on free ribo-
somes in the cytosol and require Pex19p for their post-
translational import into the peroxisome. Class II
PMPs, such as Pex3p, are targeted to the peroxisome
independent of Pex19p [92].
The function of Pex19p as an mPTS receptor does
not exclude that binding could contribute to the stabil-
ity of the proteins [95]. In fact, Pex19p has been shown
to increase the half-life of newly synthesized membrane
proteins in vivo [78], and it has been demonstrated to
bind to in vitro synthesized Pmp22p and thereby main-
tain its solubility [92]. This could be explained by
mPTS itself being rather hydrophobic, and thus, if not
shielded from hydrophobic environment, it might
Fig. 1. PTS1-import model. Newly synthes-
ized peroxisomal matrix proteins are recog-

nized by receptors in the cytosol. Upon
PTS1–protein binding, the tetrameric Pex5p
disaggregates into dimers and is transported
to the peroxisome. At the peroxisomal
membrane, Pex5p binds to the docking
complex comprising Pex13p, Pex14p and
Pex17p. How the cargo is translocated
across the peroxisomal membrane is com-
pletely unknown. Pex8p triggers the associ-
ation of the docking and the RING-finger
complex (Pex2p, Pex10p and Pex12p) and
may contribute to cargo release. The func-
tion of the RING-finger complex is still
unknown. At the end of the import cascade,
Pex5p is recycled back to the cytosol in an
ATP-dependent manner.
I. Heiland and R. Erdmann Biogenesis of peroxisomes
FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS 2365
contribute to misfolding and aggregation. In some
cases, the Pex19p binding site may even overlap with
transmembrane regions of PMPs. Therefore, Pex19p
could indeed play a dual role in peroxisomal mem-
brane protein import – as a general import receptor
for PMPs and, probably as a consequence of mPTS
binding, also as a PMP-specific chaperone.
Pex3p – anchor protein for Pex19p at
the peroxisomal membrane
Pex3p is a peroxisomal membrane protein that interacts
with Pex19p at the peroxisomal membrane [96]. The
N-terminal region of Pex3p contains its peroxisomal

targeting signal, whereas its C-terminus binds Pex19p
at regions distinct from the PMP binding site. The
interaction of Pex19p with Pex3p is essential for peroxi-
somal membrane protein import, suggesting that Pex3p
functions as a receptor for Pex19p at the peroxisomal
membrane [92,93]. It is now thought that Pex19p recog-
nizes newly synthesized PMPs in the cytosol and directs
them to the peroxisomal membrane, probably via bind-
ing to Pex3p. How peroxisomal membrane proteins
insert into the membrane remains to be investigated.
As outlined above, the topogenesis of Pex3p seems
to be different from that of other PMPs. The N-ter-
minal 50 amino acids of Pex3p have been shown to be
associated with vesicles that are located close to the
nucleus in Dpex3 mutants of H. polymorpha. Further-
more, these vesicles are reported to be capable of
forming mature peroxisomes after complementation
with full length Pex3p [97]. The first 16 amino acid of
Pex3p lead to targeting of reporter constructs to the
ER [98]. Whether this targeting sequence is functional
in the endogenous Pex3p is not known.
Involvement of the endoplasmic
reticulum in peroxisome biogenesis
In early years, it was assumed that peroxisomes origin-
ate through budding from the endoplasmic reticulum
[99]. In 1984, however, Fujiki and coworkers demon-
strated that the peroxisomal membrane protein
Pmp22p is synthesized on free ribosomes in the cytosol
and imported post-translationally directly into peroxi-
somes [100]. Based on these and other data, the

‘growth and division model’ was postulated by Laza-
row and Fujiki in 1985 [1]. The model postulates that
all peroxisomal matrix as well as peroxisomal mem-
brane proteins are synthesized on free ribosomes in the
cytosol and are imported post-translationally into pre-
existing peroxisomes which then start to grow and
multiply by division. A major implication of this
model is that peroxisomes cannot originate de novo as
known for mitochondria and chloroplasts. However,
based on data difficult to reconcile with this model,
the involvement of the ER in peroxisome biogenesis
was reconsidered. For example, treatment of H. poly-
morpha cells with Brefeldin A (a fungal toxin that
interferes with ER-to-Golgi transport) led to the accu-
mulation of peroxins in ER-like structures [101]. In
plants treated with Brefeldin A, ascorbate peroxidase
(APX) accumulates in a reticular circular network that
resembles the ER but does not contain typical ER-resi-
dent proteins such as calreticulin, BiP2 and calnexin
[102]. In human fibroblasts, however, treatment with
Brefeldin A has no effect on peroxisome biogenesis
and localization of peroxisomal membrane proteins in
ER-like structures has never been observed [103,104].
Inactivation of the endoplasmic reticulum protein
translocation factor, Sec61p, or its homologue Ssh1p
from S. cerevisiae, did not lead to defects in the target-
ing of Pex3p or peroxisome biogenesis ([105]; I. Hei-
land & R. Erdmann, unpublished data), while
Titorenko and Rachubinski detected a transient colo-
calization of peroxins with the ER marker protein

Kar2p and a cytosolic mislocalization of thiolase and
alcohol oxidase in secretory pathway mutants (sec-
mutants) of Yarrowia lipolytica [107]. Furthermore,
evidence for involvement of the ER in peroxisome bio-
genesis was provided by Mullen and coworkers, who
demonstrated that tail-anchored peroxisomal mem-
brane proteins such as APX and Pex15p are imported
into plant microsomes in vitro, whereas Pmp45p is
imported directly into peroxisomes [102,108]. Further-
more, Tabak and coworkers reported on reticular
structures observed in untreated mouse dendritic cells
that contained PMPs and were connected to the
smooth ER [109,110].
Taken together, there is striking evidence for an
involvement of the ER in peroxisome biogenesis. How-
ever, the data are clear in that the standard secretion
pathway is not involved. The only way to reconcile
these facts seems to propose the existence of a new
route for the insertion of peroxisomal proteins into the
ER membrane. In this respect, it is interesting to note
that several new routes for protein transport into the
ER have been identified in recent years that do not or
only partially employ the standard secretion pathway.
One of these novel import pathways into the ER is the
topogenesis of Ist2p. The import of Ist2p is mRNA-
dependent and takes place at the cortical ER of the
daughter cell [111]. Whether this process requires
Sec61p is unknown. An example of sec-independent
import into the ER is the sorting of Nyv1p. This
tail-anchored protein has been shown to be imported

Biogenesis of peroxisomes I. Heiland and R. Erdmann
2366 FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS
post-translationally into the ER independent of
the sec-machinery [112]. The mechanisms employed for
tail-anchored proteins have not yet been identified. It
has been shown recently that the signal recognition
particle can bind tail-anchored proteins, but the func-
tional significance for the insertion process remains to
be demonstrated. However, in contrast to the import
of secretory proteins, tail-anchored proteins are bound
post-translationally by the signal recognition particle
[113]. Interestingly, Pex15p and APX have been shown
to contain their targeting signal within their C-terminal
tails [108,114] and their targeting sequences have char-
acteristics of tail-anchored proteins [112]. Moreover,
APX has been shown to colocalize with tail-anchored
green fluorescent protein [115]. It will be interesting to
investigate whether PMPs are transported into the ER
via one of these novel routes or whether they employ a
novel, unidentified transport pathway into the ER
membrane.
Two distinct import pathways for
PMPs?
Taken together the results obtained on the import of
peroxisomal membrane proteins suggest that there are
at least two distinct classes of peroxisomal membrane
proteins (Fig. 2). The first, class I PMPs, are post-
translationally directly inserted into the peroxisomal
membrane in a Pex19p- and Pex3p-dependent manner.
The second are class II PMPs, such as Pex3p and tail-

anchored peroxisomal membrane proteins (e.g. Pex15p
and APX) that are supposed to be targeted to a thus
far uncharacterized circular reticular membrane com-
partment, namely peroxisomal ER or peroxisomal
reticulum. These reticular structures may, at least
temporally, be connected to the ER or may even repre-
sent an ER subdomain [110]. Consequently, newly syn-
thesized proteins of class II might first be inserted into
the ER membrane before they reach their final destina-
tion in the peroxisomal membrane in an unknown
fashion. Nevertheless, in the presence of mature per-
oxisomes these proteins might also behave like PMPs
of type I and thus be imported preferentially directly
into peroxisomes. In the absence or deficiency of per-
oxisomal membranes, these proteins might be imported
into the reticular structures and contribute to the
de novo synthesis of peroxisomes. Whether the topo-
genesis pathway of these PMPs shares components
with other sec-independent transport pathways remains
to be investigated.
Acknowledgements
We thank Hanspeter Rottensteiner and Wolfgang
Schliebs for reading the manuscript. Ines Heiland was
supported by a Boehringer Ingelheim Fonds fellow-
Fig. 2. Model of peroxisomal membrane
biogenesis. Peroxisomal class I membrane
proteins are synthesized on free ribosomes
in the cytosol, where they are recognized by
the import receptor Pex19p that directs
them to the peroxisomal membrane. Mem-

brane association of the Pex19p receptor–
cargo complex is mediated by Pex3p. How
membrane protein insertion takes place still
remains to be investigated. Topogenesis of
class II PMPs is independent of Pex19p.
Accumulating evidence suggests that PMPs
class II might be targeted to the ER prior to
their transport to peroxisomes. Again, how
these proteins reach the ER and their final
destination in the peroxisomal membrane is
unknown.
I. Heiland and R. Erdmann Biogenesis of peroxisomes
FEBS Journal 272 (2005) 2362–2372 ª 2005 FEBS 2367
ship. This work was supported by grants from the
Deutsche Forschungsgesellschaft (Er178 ⁄ 2–4) and by
the Fond der Chemischen Industrie.
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