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 rece
Trang 1Biogenesis 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: ralf.erdmann@rub.de
(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.
Trang 2(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-X5HL [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
Trang 3Pex13p, 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
Trang 4Pex19p – 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.
Trang 5contribute 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
Trang 6post-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.
Trang 7ship This work was supported by grants from the
Deutsche Forschungsgesellschaft (Er178⁄ 2–4) and by
the Fond der Chemischen Industrie
References
1 Lazarow PB & Fujiki Y (1985) Biogenesis of
peroxi-somes Annu Rev Cell Biol 1, 489–530
2 DeDuve C & Baudhuin P (1966) Peroxisomes
(micro-bodies and related particles) Physiol Rev 46, 323–357
3 Wanders RJ (2004) Metabolic and molecular basis of
peroxisomal disorders: a review Am J Med Genet
126A, 355–375
4 Kovacs WJ, Olivier LM & Krisans SK (2002) Central
role of peroxisomes in isoprenoid biosynthesis Prog
Lipid Res 41, 369–391
5 Kovacs WJ, Krisans S, Hogenboom S, Wanders RJ &
Waterham HR (2003) Cholesterol biosynthesis and
reg-ulation: role of peroxisomes Adv Exp Med Biol 544,
315–327
6 Hogenboom S, Romeijn GJ, Houten SM, Baes M,
Wanders RJ & Waterham HR (2002) Absence of
func-tional peroxisomes does not lead to deficiency of
enzymes involved in cholesterol biosynthesis J Lipid
Res 43, 90–98
7 Weller S, Gould SJ & Valle D (2003) Peroxisome
bio-genesis disorders Annu Rev Genomics Hum Genet 4,
165–211
8 Holroyd C & Erdmann R (2001) Protein translocation
machineries of peroxisomes FEBS Lett 501, 6–10
9 Lazarow PB (2003) Peroxisome biogenesis: advances
and conundrums Curr Opin Cell Biol 15, 489–497
10 Marzioch M, Erdmann R, Veenhuis M & Kunau W-H
(1994) PAS7 encodes a novel yeast member of the
WD-40 protein family essential for import of
3-oxo-acyl-CoA thiolase, a PTS2-containing protein, into
per-oxisomes EMBO J 13, 4908–4918
11 Dodt G & Gould SJ (1996) Multiple PEX genes are
required for proper subcellular distribution and
stabi-lity of Pex5p, the PTS1 receptor: Evidence that PTS1
protein import is mediated by a cycling receptor J Cell
Biol 135, 1763–1774
12 van der Klei IJ & Veenhuis M (1996) Peroxisome
biogen-esis in the yeast Hansenula polymorpha: a structural and
functional analysis Ann New York Acad Sci 804, 47–59
13 Dammai V & Subramani S (2001) The human
peroxi-somal targeting signal receptor, Pex5p, is translocated
into the peroxisomal matrix and recycled to the
cyto-sol Cell 105, 187–196
14 Nair DM, Purdue PE & Lazarow PB (2004) Pex7p
translocates in and out of peroxisomes in
Saccharo-myces cerevisiae J Cell Biol 167, 599–604
15 Gould SJ, Keller GA, Hosken N, Wilkinson J &
Sub-ramani S (1989) A conserved tripeptide sorts proteins
to peroxisomes J Cell Biol 108, 1657–1664
16 Lametschwandtner G, Brocard C, Fransen M, Van Veldhoven P, Berger J & Hartig A (1998) The differ-ence in recognition of terminal tripeptides as peroxiso-mal targeting signal 1 between yeast and human is due
to different affinities of their receptor Pex5p to the cog-nate signal and to residues adjacent to it J Biol Chem
273, 33635–33643
17 Elgersma Y, Vos A, van den Berg M, van Roermund CWT, van der Sluijs P, Distel B & Tabak HF (1996) Analysis of the carboxyl-terminal peroxisomal targeting signal 1 in a homologous context in Saccharomyces cer-evisiae J Biol Chem 271, 26375–26382
18 Subramani S, Koller A & Snyder WB (2000) Import of peroxisomal matrix and membrane proteins Annu Rev Biochem 2000, 399–418
19 Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A
& Eisenhaber F (2003) Prediction of peroxisomal tar-geting signal 1 containing proteins from amino acid sequence J Mol Biol 328, 581–592
20 Reumann S (2004) Specification of the peroxisome tar-geting signals type 1 and type 2 of plant peroxisomes
by bioinformatics analyses Plant Physiol 135, 783–800
21 Swinkels BW, Gould SJ, Bodnar AG, Rachubinski RA
& Subramani S (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase EMBO J 10, 3255–3262
22 Klein AT, Barnett P, Bottger G, Konings D, Tabak
HF & Distel B (2001) Recognition of the peroxisomal targeting signal type 1 by the protein import receptor Pex5p J Biol Chem 276, 15034–15041
23 Gatto GJJ, Geisbrecht BV, Gould SJ & Berg JM (2000) Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5 Nat Struct Biol 7, 1091–1095
24 Gatto GJJ, Maynard EL, Guerrerio AL, Geisbrecht
BV, Gould SJ & Berg JM (2003) Correlating structure and affinity for PEX5: PTS1 complexes Biochemistry
42, 1660–1666
25 Klein AT, van Den Berg M, Bottger G, Tabak HF & Distel B (2002) Saccharomyces cerevisiae acyl-CoA oxi-dase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p J Biol Chem
277, 25011–25019
26 Purdue PE, Yang X & Lazarow PB (1998) Pex18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway J Cell Biol
143, 1859–1869
27 Stein K, Schell-Steven A, Erdmann R & Rottensteiner
H (2002) Interactions of Pex7p and Pex18p⁄ Pex21p with the peroxisomal docking machinery: Implications for the first steps in PTS2 protein import Mol Cell Biol
22, 6059–6069
28 Sichting M, Schell-Steven A, Prokisch H, Erdmann R
& Rottensteiner H (2003) Pex7p and Pex20p of Neuro-spora crassafunction together in PTS2-dependent
Trang 8protein import into peroxisomes Mol Biol Cell 14,
810–821
29 Einwa¨chter H, Sowinski S, Kunau WH & Schliebs W
(2001) Yarrowia lipolytica Pex20p, Saccharomyces
cere-visiaePex18p⁄ Pex21p and mammalian Pex5pL fulfil a
common function in the early steps of the peroxisomal
PTS2 import pathway EMBO Report 2, 1035–1039
30 Schafer A, Kerssen D, Veenhuis M, Kunau WH &
Schliebs W (2004) Functional similarity between the
peroxisomal PTS2 receptor binding protein Pex18p
and the N-terminal half of the PTS1 receptor Pex5p
Mol Cell Biol 24, 8895–8906
31 Matsumura T, Otera H & Fujiki Y (2000) Disruption
of the interaction of the longer isoform of Pex5p,
Pex5pL, with Pex7p abolishes peroxisome targeting
signal type 2 protein import in mammals Study with a
novel Pex5-impaired Chinese hamster ovary cell
mutant J Biol Chem 275, 21715–21721
32 Dodt G, Warren D, Becker E, Rehling P & Gould SJ
(2001) Domain mapping of human PEX5 reveals
functional and structural similarities to Saccharomyces
cerevisiaePex18p and Pex21p J Biol Chem 276,
41769–41781
33 Woodward AW & Bartel B (2005) The Arabidopsis
peroxisomal targeting signal type 2 receptor PEX7 is
necessary for peroxisome function and dependent on
PEX5 Mol Biol Cell 16, 573–583
34 Madrid KP, De Crescenzo G, Wang S & Jardim A
(2004) Modulation of the Leishmania donovani peroxin
5 quaternary structure by peroxisomal targeting signal
1 ligands Mol Cell Biol 24, 7331–7344
35 Albertini M, Rehling P, Erdmann R, Girzalsky W,
Kiel JAKW, Veenhuis M & Kunau W-H (1997)
Pex14p, a peroxisomal membrane protein binding both
receptors of the two PTS-dependent import pathways
Cell 89, 83–92
36 Fransen M, Terlecky SR & Subramani S (1998)
Identi-fication of a human PTS1 receptor docking protein
directly required for peroxisomal protein import Proc
Natl Acad Sci USA 95, 8087–8092
37 Schliebs W, Saidowsky J, Agianian B, Dodt G,
Herberg FW & Kunau WH (1999) Recombinant
human peroxisomal targeting signal receptor PEX5
Structural basis for interaction of PEX5 with PEX14
J Biol Chem 274, 5666–5673
38 Will GK, Soukupova M, Hong X, Erdmann KS, Kiel
JA, Dodt G, Kunau WH & Erdmann R (1999)
Identi-fication and characterization of the human orthologue
of yeast Pex14p Mol Cell Biol 19, 2265–2277
39 Saidowsky J, Dodt G, Kirchberg K, Wegner A,
Nasta-inczyk W, Kunau WH & Schliebs W (2001) The
di-aromatic pentapeptide repeats of the human
peroxi-some import receptor PEX5 are separate high affinity
binding sites for the peroxisomal membrane protein
PEX14 J Biol Chem 276, 34524–34529
40 Elgersma Y, Kwast L, Klein A, Voorn-Brouwer T, van den Berg M, Metzig B, America T, Tabak HF & Distel
B (1996) The SH3 domain of the Saccharomyces cerevi-siaeperoxisomal membrane protein Pex13p functions
as a docking site for Pex5p, a mobile receptor for the import of PTS1 containing proteins J Cell Biol 135, 97–109
41 Erdmann R & Blobel G (1996) Identification of Pex13p
a peroxisomal membrane receptor for the PTS1 recog-nition factor J Cell Biol 135, 111–121
42 Gould SJ, Kalish JE, Morrell JC, Bjorkman J, Urquhart AJ & Crane DI (1996) Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTS1 receptor J Cell Biol 135, 85–95
43 Douangamath A, Filipp FV, Klein AT, Barnett P, Zou
P, Voorn-Brouwer T, Vega MC, Mayans OM, Sattler
M, Distel B & Wilmanns M (2002) Topography for independent binding of alpha-helical and PPII-helical ligands to a peroxisomal SH3 domain Mol Cell 10, 1007–1017
44 Pires JR, Hong X, Brockmann C, Volkmer-Engert R, Schneider-Mergener J, Oschkinat H & Erdmann R (2003) The ScPex13p SH3 domain exposes two distinct binding sites for Pex5p and Pex14p J Mol Biol 326, 1427–1435
45 Chang CC, Warren DS, Sacksteder KA & Gould SJ (1999) PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import J Cell Biol 147, 761–774
46 Gouveia AM, Reguenga C, Oliveira ME, Sa-Miranda
C & Azevedo JE (2000) Characterization of peroxiso-mal Pex5p from rat liver: Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein J Biol Chem 275, 32444–32451
47 Fransen M, Brees C, Ghys K, Amery L, Mannaerts
GP, Ladant D & Van Veldhoven PP (2002) Analysis of mammalian peroxin interactions using a non-transcrip-tion-based bacterial two-hybrid assay Mol Cell Proteo-mics 1, 243–252
48 Albertini M, Girzalsky W, Veenhuis M & Kunau W-H (2001) Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import Eur J Cell Biol 80, 257–270
49 Otera H, Harano T, Honsho M, Ghaedi K, Mukai S, Tanaka A, Kawai A, Shimizu N & Fujiki Y (2000) The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p-PTS2 protein com-plex into peroxisomes via its initial docking site, Pex14p J Biol Chem 275, 21703–21714
50 Urquhart AJ, Kennedy D, Gould SJ & Crane DI (2000) Interaction of Pex5p, the type 1 peroxisome tar-geting signal receptor, with the peroxisomal membrane
Trang 9proteins Pex14p and Pex13p J Biol Chem 275, 4127–
4136
51 Otera H, Setoguchi K, Hamasaki M, Kumashiro T,
Shimizu N & Fujiki Y (2002) Peroxisomal targeting
signal receptor Pex5p interacts with cargoes and import
machinery components in a spatiotemporally
differen-tiated manner: Conserved Pex5p WXXXF⁄ Y motifs
are critical for matrix protein import Mol Cell Biol 22,
1639–1655
52 Huhse B, Rehling P, Albertini M, Blank L, Meller K
& Kunau W-H (1998) Pex17p of Saccharomyces
cerevi-siaeis a novel peroxin and component of the
peroxiso-mal protein translocation machinery J Cell Biol 140,
49–60
53 Agne B, Meindl N, Niederhoff K, Einwa¨chter H,
Rehling P, Sickmann A, Meyer HE, Girzalsky W &
Kunau W-H (2003) Pex8p, an intraperoxisomal
organi-zer of the peroxisomal import machinery Mol Cell 11,
635–646
54 Rehling P, Skaletz-Rorowski A, Girzalsky W,
Voorn-Brouwer T, Franse MM, Distel B, Veenhuis M, Kunau
W-H & Erdmann R (2000) Pex8p, an intraperoxisomal
peroxin of Saccharomyces cerevisiae required for
pro-tein transport into peroxisomes binds the PTS1
recep-tor Pex5p J Biol Chem 275, 3593–3602
55 Wang X, McMahon MA, Shelton SN, Nampaisansuk
M, Ballard JL & Goodman JM (2004) Multiple
targeting modules on peroxisomal proteins are not
redundant: discrete functions of targeting signals
within Pmp47 and Pex8p Mol Biol Cell 15, 1702–
1710
56 Wang D, Visser NV, Veenhuis M & Van Der Klei IJ
(2003) Physical interactions of the peroxisomal
target-ing signal 1-receptor, Pex5p, studied by fluorescence
correlation spectroscopy J Biol Chem 278, 43340–
43345
57 Platta HW, Girzalsky W & Erdmann R (2004)
Ubiqui-tination of the peroxisomal import receptor Pex5p
Biochem J 384, 37–45
58 Kiel JA, Emmrich K, Meyer HE & Kunau WH (2004)
Ubiquitination of the PTS1 receptor, Pex5p, suggests
the presence of a quality control mechanism during
peroxisomal matrix protein import J Biol Chem 280,
1921–1930
59 Kragt A, Voorn-Brouwer TM, Van den Berg M,
Distel B, Kiel JA, Emmrich K, Meyer HE & Kunau
WH (2005) The Saccharomyces cerevisiae peroxisomal
import receptor Pex5p is monoubiquitinated in wild
type cells J Biol Chem 280, 7867–7874
60 Purdue PE & Lazarow PB (2001) Pex18p is
constitu-tively degraded during peroxisome biogenesis J Biol
Chem 276, 47684–47689
61 Schwartz DC & Hochstrasser M (2003) A superfamily
of protein tags: ubiquitin, SUMO and related
modi-fiers Trends Biochem Sci 28, 321–328
62 Costa-Rodrigues J, Carvalho AF, Gouveia AM, Fran-sen M, Sa-Miranda C & Azevedo JE (2004) The N-ter-minus of the peroxisomal cycling receptor, Pex5p, is required for redirecting the peroxisome-associated per-oxin back to the cytosol J Biol Chem 279, 46573– 46579
63 Gouveia AM, Guimaraes CP, Oliveira ME, Reguenga
C, Sa-Miranda C & Azevedo JE (2003) Characteriza-tion of the peroxisomal cycling receptor Pex5p import pathway Adv Exp Med Biol 544, 213–220
64 Fujiki Y, Hubbard AL, Fowler S & Lazarow PB (1982) Isolation of intracellular membranes by means
of sodium carbonate treatment: application to endo-plasmic reticulum J Cell Biol 93, 97–102
65 Schneiter R, Brugger B, Sandhoff R, Zellnig G, Leber
A, Lampl M, Athenstaedt K, Hrastnik C, Eder S, Daum G et al (1999) Electrospray ionization tandem mass spectrometry (ESI-MS⁄ MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting⁄ remodeling
of distinct molecular species en route to the plasma membrane J Cell Biol 146, 741–754
66 Zinser E, Sperka-Gottlieb CD, Fasch EV, Kohlwein
SD, Paltauf F & Daum G (1991) Phospholipid synth-esis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae
J Bacteriol 173, 2026–2034
67 Natter K, Leitner P, Faschinger A, Wolinski H, McCraith S, Fields S & Kohlwein SD (2005) The spa-tial organization of lipid synthesis in the yeast Sacchar-omyces cerevisiae derived from large-scale green fluorescent protein tagging and high-resolution micro-scopy Mol Cell Proteomics, 00, 000–000 [Epub ahead
of print]
68 Purdue PE & Lazarow PB (2001) Peroxisome biogen-esis Annu Rev Cell Dev Biol 17, 701–752
69 Achleitner G, Gaigg B, Krasser A, Kainersdorfer E, Kohlwein SD, Perktold A, Zellnig G & Daum G (1999) Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact Eur J Biochem 264, 545–553
70 Ardail D, Gasnier F, Lerme F, Simonot C, Louisot P
& Gateau-Roesch O (1993) Involvement of mitochon-drial contact sites in the subcellular compartmentaliza-tion of phospholipid biosynthetic enzymes J Biol Chem 268, 25985–25992
71 Shiao YJ, Lupo G & Vance JE (1995) Evidence that phosphatidylserine is imported into mitochondria via a mitochondria-associated membrane and that the major-ity of mitochondrial phosphatidylethanolamine is derived from decarboxylation of phosphatidylserine
J Biol Chem 270, 11190–11198
72 Santos MJ, Imanaka T, Shio H, Small GM & Lazarow
PB (1988) Peroxisomal membrane ghosts in Zellweger
Trang 10syndrome-aberrant organelle assembly Science 239,
1536–1538
73 Dyer JM, McNew JA & Goodman JM (1996) The
sorting sequence of the peroxisomal integral membrane
protein PMP47 is contained within a short hydrophilic
loop J Cell Biol 133, 269–280
74 Pause B, Saffrich R, Hunziker A, Ansorge W & Just
WW (2000) Targeting of the 22 kDa integral
peroxiso-mal membrane protein FEBS Lett 471, 23–28
75 Honsho M & Fujiki Y (2001) Topogenesis of
peroxiso-mal membrane protein requires a short, positively
charged intervening-loop sequence and flanking
hydro-phobic segments J Biol Chem 276, 9375–9382
76 Wang X, Unruh MJ & Goodman JM (2001) Discrete
targeting signals direct Pmp47 to oleate-induced
peroxi-somes in Saccharomyces cerevisiae J Biol Chem 276,
10897–10905
77 Honsho M, Hiroshige T & Fujiki Y (2002) The
mem-brane biogenesis peroxin Pex16p: Topogenesis and
functional roles in peroxisomal membrane assembly
J Biol Chem 277, 44513–44524
78 Jones JM, Morrell JC & Gould SJ (2001) Multiple
distinct targeting signals in integral peroxisomal
mem-brane proteins J Cell Biol 153, 1141–1150
79 Brosius U, Dehmel T & Ga¨rtner J (2002) Two different
targeting signals direct human PMP22 to peroxisomes
J Biol Chem 277, 774–784
80 South ST & Gould SJ (1999) Peroxisome synthesis in
the absence of preexisting peroxisomes J Cell Biol 144,
255–266
81 Hettema EH, Girzalsky W, van Den Berg M, Erdmann
R & Distel B (2000) Saccharomyces cerevisiae Pex3p
and Pex19p are required for proper localization and
stability of peroxisomal membrane proteins EMBO J
19, 223–233
82 Shimozawa N, Nagase T, Takemoto Y, Suzuki Y,
Fujiki Y, Wanders RJ & Kondo N (2002) A novel
aberrant splicing mutation of the PEX16 gene in two
patients with Zellweger syndrome Biochem Biophys
Res Commun 292, 109–112
83 Lin Y, Sun L, Nguyen LV, Rachubinski RA &
Good-man HM (1999) The Pex16p homolog SSE1 and
sto-rage organelle formation in Arabidopsis seeds Science
284, 328–330
84 Lin Y, Cluette-Brown JE & Goodman HM (2004) The
peroxisome deficient Arabidopsis mutant sse1 exhibits
impaired fatty acid synthesis Plant Physiol 135, 814–
827
85 Kammerer S, Arnold N, Gutensohn W, Mewes HW,
Kunau WH, Hofler G, Roscher AA & Braun A (1997)
Genomic organization and molecular characterization
of a gene encoding HsPXF, a human peroxisomal
far-nesylated protein Genomics 45, 200–210
86 Go¨tte K, Girzalsky W, Linkert M, Baumgart E,
Kam-merer S, Kunau WH & Erdmann R (1998) Pex19p, a
farnesylated protein essential for peroxisome biogen-esis Mol Cell Biol 18, 616–628
87 Hazra PP, Suriapranata I, Snyder WB & Subramani S (2002) Peroxisome remnants in pex3Delta cells and the requirement of Pex3p for interactions between the per-oxisomal docking and translocation subcomplexes Traffic 3, 560–574
88 Sacksteder KA, Jones JM, South ST, Li X, Liu Y & Gould SJ (2000) PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and
is required for peroxisome membrane synthesis J Cell Biol 148, 931–944
89 Fransen M, Vastiau I, Brees C, Brys V, Mannaerts GP
& Van Veldhoven PP (2004) Potential role for Pex19p
in assembly of PTS-receptor docking complexes J Biol Chem 279, 12615–12624
90 Fransen M, Wylin T, Brees C, Mannaerts GP & Van Veldhoven PP (2001) Human Pex19p binds peroxiso-mal integral membrane proteins at regions sistinct from their sorting sequences Mol Cell Biol 21, 4413–4424
91 Rottensteiner H, Kramer A, Lorenzen S, Stein K, Landgraf C, Volkmer-Engert R & Erdmann R (2004) Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals (mPTS) Mol Biol Cell 7, 3406–3417
92 Jones JM, Morrell JC & Gould SJ (2004) PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins J Cell Biol
164, 57–67
93 Fang Y, Morrell JC, Jones JM & Gould SJ (2004) PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins
J Cell Biol 164, 863–875
94 Eckert JH & Erdmann R (2003) Peroxisome biogenesis Rev Physiol Biochem Pharmacol 147, 75–121
95 Shibata H, Kashiwayama Y, Imanaka T & Kato H (2004) Domain architecture and activity of human Pex19p, a chaperone-like protein for intracellular traf-ficking of peroxisomal membrane proteins J Biol Chem
279, 38486–38494
96 Muntau AC, Roscher AA, Kunau WH & Dodt G (2003) The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis Eur J Cell Biol 82, 333–342
97 Faber KN, Haan GJ, Baerends RJ, Kram AM & Veenhuis M (2002) Normal peroxisome development from vesicles induced by truncated Hansenula polymor-phaPex3p J Biol Chem 277, 11026–11033
98 Baerends RJS, Rasmussen SW, Hilbrands RE, van der Heide M, Faber KN, Reuvekamp PTW, Kiel JAKW, Cregg JM, van der Klei IJ & Veenhuis M (1996) The Hansenula polymorphaPER9 gene encodes a peroxiso-mal membrane protein essential for peroxisome assem-bly and integrity J Biol Chem 271, 8887–8894