+ model
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Biochimica et Biophysica Acta xx (2006) xxx – xxx
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Review
Peroxisomal matrix protein receptor ubiquitination and recycling
Sven Thoms, Ralf Erdmann ⁎
Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany
Received 10 May 2006; received in revised form 15 August 2006; accepted 23 August 2006
Abstract
The peroxisomal targeting signal type1 (PTS1) receptor Pex5 is required for the peroxisomal targeting of most matrix proteins. Pex5 recognises
target proteins in the cytosol and directs them to the peroxisomal membrane where cargo is released into the matrix, and the receptor shuttles back
to the cytosol. Recently, it has become evident that the membrane-bound Pex5 can be modified by mono- and polyubiquitination. This review
summarises recent results on Pex5 ubiquitination and on the role of the AAA peroxins Pex1 and Pex6 as dislocases required for the release of
Pex5 from the membrane to the cytosol where the receptor is either degraded by proteasomes or made available for another round of protein
import into peroxisomes.
© 2006 Elsevier B.V. All rights reserved.
Keywords: AAA protein; Peroxisome biogenesis; Pex1p; Pex6p; Pex5p; Pex15p; Peroxin; Ubiquitin
Ubiquitination plays a vital role in the physiology of cells
and organisms. Among the first functions of ubiquitination to be
discovered was its role in the targeting of proteins for
degradation by the proteasome. In the recent years, it has
become more and more evident that ubiquitin attachment to
proteins also serves other purposes like control of protein
activities or organellar targeting. In this review, we survey the
components of the ubiquitination machinery and their relevance
to peroxisome biogenetic functions. The ubiquitin-proteasome
system (UPS) comprises the core ubiquitination machinery E1,
E2, and E3, as well as the proteasome together with AAA
proteins and ubiquitin-binding and deubiquitinating proteins.
Several lines of evidence point to an involvement of
components of this extended UPS in peroxisome biogenesis:
(1) an E2 enzyme, Pex4, is essential for peroxisomal biogenesis.
(2) The AAA peroxins, Pex1 and Pex6, are likely to be
Abbreviations: AAA, ATPases associated with various cellular activities;
ERAD, ER associated degradation; NSF, N-ethylmaleimide-sensitive factor;
PTS, peroxisomal targeting signal; RING, really interesting new gene; Ubc,
ubiquitin conjugating enzyme; UPS, ubiquitin-proteasome system
⁎ Corresponding author. Institut für Physiologische Chemie, Abteilung für
Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D44780 Bochum, Germany. Tel.: +49 234 322 4943; fax: +49 234 321 4266.
E-mail address: [email protected] (R. Erdmann).
responsible for the ATP-dependency of peroxisomal protein
import. (3) RING proteins (Pex2, Pex10, and Pex12), which are
putative E3s, are required for protein import into peroxisomes.
(4) The matrix protein receptors Pex5 and Pex18 (Pex20) have
been demonstrated to be modified by ubiquitination.
We suggest a model in which Pex4-dependent receptor
mono- or polyubiquitination is required for functional AAAdependent receptor release followed by the recycling or
degradation of the receptors depending on the type of
ubiquitination.
1. Ubiquitination basics
The UPS involves ubiquitin attachment and proteasome
degradation of ubiquitinated proteins (reviewed in [1]). For
ubiquitin attachment, ubiquitin is activated in an ATPdependent manner by a ubiquitin-activating enzyme (E1). It
is transferred to a ubiquitin-conjugating enzyme (Ubc, E2)
that – supported by a ubiquitin-protein ligase (E3) – attaches
ubiquitin to ε-amino groups in target protein lysine residues or
the α-amino group of the N-terminal residue. The conjugated
ubiquitin itself may become an ubiquitination substrate in a
process called polyubiquitination. The attachment of at least
four ubiquitin units is required to target proteins for degradation
by the proteasome. Usually, lysine at position 48 of ubiquitin is
0167-4889/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2006.08.046
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ubiquitinated in polyubiquitination. The term ‘monoubiquitination’ refers to the modification of a protein by one or several
(‘multiubiquitination’) ubiquitin moieties which themselves are
not ubiquitinated.
Target protein recognition relies on the interaction of one of a
few E2 enzymes with one of many E3 enzymes. Of the eleven
ubiquitin specific E2 enzymes in yeast, the similar proteins
Ubc4 and Ubc5 are least specialised and required for
degradation of unstable proteins [2]. Three types of E3 enzymes
are known so far. (a) HECT (homologous to E6-AP C-terminus) proteins, (b) RING (really interesting new gene) gene
products, or (c) U-box proteins (reviewed in [3]). HECT domain
proteins bind ubiquitin to a cystein residue in their HECT
domain before transferring it to the target molecule, whereas
RING and U-box domain proteins facilitate the interaction
between E2s and the target without previous covalent binding of
the ubiquitin.
Whereas polyubiquitination of proteins classically serves as
a degradation signal, monoubiquitination can be a targeting
signal (reviewed in [4]). Monoubiquitin attached to plasma
membrane proteins is a signal for their internalisation into the
endocytic pathway. Similarly, monoubiquitin is associated with
membrane protein uptake in late endosome vesicles (multivesicular bodies) for delivery into the lysosome or vacuole
(reviewed in [5]). Other examples are p53 and Rad18 that are
either monoubiquitinated for export from the nucleus, or they
are polyubiquitinated for proteasomal degradation [6,7].
Monoubiquitination of histones is required for mitotic cell
growth and meiosis and regulation of eukaryotic transcription
activation [8,9].
The identification of Pex4/Ubc10 (formerly Pas2) as an – at
the time putative – E2 in peroxisome biogenesis marked the
first association of the ubiquitination system with peroxisome
biogenesis [10]. Pex4 is membrane-anchored through Pex22 in
yeast and plant [11,12]. Cells lacking Pex4 are characterised by
the absence of peroxisomes with a typical morphology and
mislocalisation of peroxisomal matrix proteins to the cytosol.
Deletion of PEX4 or PEX22 also leads to a decrease in Pex5
abundance [11–14].
2. AAA-type ATPases
AAA proteins are ATPases that have originally been named
after the identification of Pex1 (previously called Pas1) as
“ATPases associated with various cellular activities” [15]. By
inclusion of a more varied number of metabolic and
transcriptional regulators, the classical AAA family has been
expanded and termed AAA+ family of ATPases [16,17].
AAA+ proteins are involved in protein degradation, DNA
replication, membrane fusion, or the movement of microtubule
motors in eukaryotes, or thermotolerance in bacteria, plant and
fungi (reviewed in [18,19]). The major characteristic of the
AAA family of proteins is the presence of one or two 200–250
amino-acid ATP-binding domain(s) that contain(s) Walker A
and B motifs, as well as other motifs that distinguish the AAA
proteins from other P-loop (phosphate-binding) NTPases. The
defining AAA domain is a structurally conserved ATPase
domain that assembles into oligomeric rings and undergoes
conformational changes during cycles of nucleotide binding and
hydrolysis [19].
Most of the AAA-proteins are involved in the manipulation
of proteins and protein complexes leading to their unfolding or
disassembly. Examples in this respect are NSF and Cdc48/p97.
NSF (N-ethylmaleimide-sensitive factor) is involved in the
disassembly of SNARE (α-SNAP receptor) complexes which is
essential in the process of intracellular membrane fusion [20].
NSF is ubiquitously expressed in eukaryotes, but most abundant
in the nervous system where it enables the membrane trafficking
required for synaptic exocytosis. Cdc48/p97 is required for the
dislocation of misfolded proteins from the ER substrates that are
exported from the ER for subsequent proteolytic degradation
through the ERAD (ER associated degradation) pathway
(reviewed in [21,22]). ERAD also regulates the degradation
of hydroxymethylglutaryl CoA-reductase [23] or the Δ9-fatty
acid desaturase in yeast [24]. ERAD substrates are usually
polyubiquitinated before they are recognised by Cdc48/p97.
Golgi reassembly [25] and nuclear fusion [26] are fusion
processes that are dependent on Cdc48/p97.
AAA proteins are often hexamers in their physiologically
active form. In the hexameric configuration, the ATP-binding
site is positioned at the interface between the subunits. The
hexameric enzymes have a central pore or cavity whose
function is still a matter of debate. Upon ATP binding and/or
hydrolysis, AAA enzymes undergo conformational changes in
the AAA-domains as well as in the adjacent N-domains that
can be transmitted to substrate proteins and might lead to their
unfolding or disassembly [19]. One of best-studied examples
in that respect is HslU for which four conformations of the
AAA+ domain could be delineated from several X-ray
structures [27–29]. In HslU, the N- and C-terminal subdomains move towards each other upon nucleotide binding
and hydrolysis. In the nucleotide-free state, they are most
distant, whereas in the ADP-bound state, they are closest.
Nucleotide binding thereby affects the opening of the central
cavity of HslU, a feature that might be common to all
hexameric AAA-ATPases.
AAA proteins are also crucial components of 19S cap of the
26S proteasome. The cap consists of a lid that recognises and
deubiquitinates polyubiquitin-tagged substrates. The cap
ATPases initiate unfolding of the substrate proteins at
unstructured regions and deliver them to the central chamber
of the 20S core for degradation [30,31]. AAA proteins are also
involved in the disassembly of protein aggregates. Proteins that
fail to fold properly often form aggregates, which might become
toxic. Such aggregates can be disassembled by Hsp104/ClpB
and then become substrates for refolding by the Hsp70 system
[32, 33].
3. The AAA peroxins Pex1 and Pex6
It was recognised from early on that peroxisomal protein
import is energy dependent. In permeabilised CHO cell
systems, PTS1 import was demonstrated to depend on ATP,
but not on GTP or the presence of a membrane potential [34,35].
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ATP-requirement was also found for import of firefly luciferase
into mammalian peroxisomes [36] or of glycolate oxidase into
glycosomes [37].
The peroxins Pex1 and Pex6 were identified as two ATPases
of the AAA-protein family that are required for peroxisomal
matrix protein import [15, 38]. Because they are the only
ATPases among the known peroxins, it is likely that they are
responsible for the overall ATP-requirement of peroxisomal
matrix protein import [39]. Other ATP-consuming factors as
well as GTPases have been found to be associated with
peroxisome maintenance and movement. Dynamin-like proteins are GTPases that are proposed to play a role in peroxisome
fission and inheritance [40], the Rho1 GTPase might link
peroxisome to the cytoskeleton [41]. A dynein AAA+ protein as
well as a kinesin have been demonstrated to work together in
moving peroxisomes within the cell [42,43]. Also an Hsp70
family member and J-domain proteins are functionally
associated with peroxisomes [44,45].
In their domain structure, Pex1 and Pex6 are similar to other
AAA proteins with two C-terminal AAA cassettes. In both
AAA peroxins, the second AAA domain (D2) is evolutionarily
better conserved than the first one, which exhibits less sequence
similarity to the D1 of other AAA family members. The largest
differences in the two AAA peroxins can be found in their
N-terminal regions. The mouse Pex1 N-domain consists of two
structurally independent lobes separated by a shallow groove
[46], which might provide a protein binding site.
Pex1 and Pex6 have been demonstrated to interact in an
ATP-dependent manner [47–50]. However, the exact molecular
constitution of this Pex1–Pex6-heterooligomer is still unclear.
Cells lacking Pex1 or Pex6 are characterised by the mislocalisation of peroxisomal matrix proteins to the cytosol
[15,51,52] and the presence of peroxisomal membrane ghosts
which contain residual amounts of matrix proteins [53].
Interestingly, mutations in the AAA peroxins represent by far
the most frequent cause of human peroxisomal biogenesis
disorders (PBDs) [54].
Pex15 and Pex26 have been identified as peroxisomal
membrane anchors for Pex6 in yeast [55] and mammalian cells
[56], respectively. Mutational analysis of the Walker A and B
motifs of Pex6 indicated that ATP-hydrolysis in the conserved
AAA-domain is required to disconnect Pex6 from Pex15. Based
on these data, it has been proposed that Pex6 might exert its
function by an ATP-dependent cycle of recruitment and release
to and from Pex15 [55]. In the Yarrowia lipolytica and Pichia
pastoris, it was found that Pex1 and Pex6 are associated with
peroxisomal precursors [57] or different cellular structures [49],
respectively. In human fibroblasts – but not in yeast –
peroxisomal localisation of PEX26 and PEX6 has been reported
not to be absolutely required for AAA peroxin function [58].
The following functions have been attributed to the AAA
peroxins [39]: (1) separation of the import receptor from its
cargo immediately before import (‘preimplex’ model) [59], (2)
dislocation of the PTS1 receptor from the peroxisomal
membrane to the cytosol for recycling or degradation [60], (3)
peroxisome fusion [61], (4) lipid ferries on their way to the
peroxisome [62]. These suggestions are, however, not all
3
mutually exclusive. It would be especially rewarding to
conciliate the function of AAA peroxins in peroxisome fusion
with their function in import receptor recycling, on which recent
effort has concentrated.
4. Ubiquitin-binding proteins and deubiquitination
In the recent years, many ubiquitin-binding proteins have
been identified. These include the UBA (ubiquitin-associated)
domain-containing proteins as the largest family. UIM (ubiquitin-interacting motif), GAT (GGA and TOM1 proteins), CUE
(coupling of ubiquitin conjugation to endoplasmic reticulum
degradation), and NZF (nuclear protein localisation gene 4 zinc
finger) domain-containing proteins appear to have more
specialised functions (reviewed in [63]). The requirement for
ubiquitin binding factors is illustrated by the AAA chaperone
Cdc48/p97. To perform its function, it associates with one of
several UBX (ubiquitin-related domain) proteins [64] or with
the cofactors Ufd1–Npl4, Shp1/p47, Rad23, or VCIP135. The
basic recognition of unfolded or unstructured domains by
Cdc48 does not require these cofactors [65]. Recent evidence
suggests that ubiquitinated proteins are escorted to the
proteasome through a pathway that involves sequential
interactions with the Cdc48 cofactors Ufd1/Npl4, Ufd2, and
Rad23 or Dsk2 [66].
Eventually, ubiquitin is removed from proteins by deubiquitination. There are about 17 deubiquitinating proteases (DUBs)
in Saccharomyces cerevisiae [67] and about 79 functional
DUBs in humans [68]. DUBs are grouped into ubiquitin
carboxy-terminal hydrolases (UCHs) and ubiquitin-specific
processing enzymes (UBPs). Some DUBs constitutively
remove ubiquitin from substrates, whereas others have more
specialised functions. At the proteasome, DUBs cleave
polyubiquitin chains from proteins. The Drosophila DUB Fat
facets regulates endocytosis by deubiquitinating the epsin
homologue Liquid facets, a component of the clathrin-based
endocytosis machinery [69]. The p97–p47 protein complex
associates with the DUB VCIP135 that is needed for Golgi
reassembly [70,71]. So far, no specific ubiquitin binding
proteins or DUBs have been found in association with
peroxisome function or biogenesis.
5. Pex5 receptor ubiquitination
Peroxisomal import of matrix proteins can be conceptually
divided in (1) binding of the peroxisomal targeting signals of
import substrates to their receptors in the cytosol; (2) binding of
these cargo–receptor complexes to a docking complex at the
peroxisomal membrane; (3) dissociation of the receptor–cargo
complexes and translocation of the cargo proteins across the
peroxisomal membrane, (4) recycling or removal of the
receptors. This conceptualisation might reflect a protein cascade
of temporal and/or spatial steps in matrix protein import [39,72]
and much recent work has been devoted on proving its validity.
Pex5 and Pex7 are the import receptors for peroxisomal
targeting signals type1 and 2, respectively. Pex7 in fungi acts in
concert with a Pex20 protein (Pex18–Pex21 in S. cerevisiae,
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Pex20 in Y. lipolytica, Pichia pastoris or Neurospora crassa)
which is similar to the N-terminus of Pex5 that is required for
the association with the membrane-bound components of the
peroxisomal protein import machinery [73–77]. The import
receptors shuttle between the peroxisomal membrane and the
cytosol [80,81] or the receptors might even enter the
peroxisome [78,79] lending support to an 'extended shuttle
model'. Evidence that Pex5 itself is integrated into the
membrane [82] prompted the proposal of the ‘transient pore
hypothesis’ [60], suggesting that the import receptor itself
becomes an integral part of a peroxisomal translocon and that a
cascade of protein–protein interactions leads to receptor
ubiquitination and its ATP- and AAA-peroxin dependent
recycling [60,83–85].
Pex5 can be ubiquitinated in vivo [86–88]. Polyubiquitination of Pex5 has been demonstrated to take place at the
membrane and depends on Ubc4–Ubc5. Current evidence
suggests that Pex5 polyubiquitination is not a prerequisite for
Pex5 function in peroxisomal protein import. It is likely part of a
quality control system that withdraws a non-functional fraction
of the membrane-accumulated Pex5 from cycling by targeting it
to the proteasome for degradation. It can be imagined that
failure in the removal of Pex5 form the membrane would
negatively affect import to some extent. As an alternative
description of this process, the acronym RADAR for ‘receptor
accumulation and degradation in the absence of recycling’ has
been proposed [93]. In fact, deficiency in Ubc4 and Ubc5 leads
to a partial import defect of peroxisomal matrix proteins [87],
and overproduction of UbK48R in Hansenula polymorpha has
even a more severe effect on the protein import into the
organelle [89]. Interestingly, in a Pex5 K21R mutant,
ubiquitination and degradation were abolished, suggesting
that this conserved lysine 21 is ubiquitinated in Pex5 [89].
Polyubiquitination of Pex5 is enhanced in cells lacking ‘late’
import pathway components like the AAA peroxins or Pex4 or
its membrane anchor Pex22. It had been noticed earlier that
Pex5 levels are decreased when these factors are deleted
[11,80,90,91]. This observation was used to establish an
epistasis analysis of matrix protein import that placed the
AAA peroxins as well as the Ubc Pex4 at the very end of the
peroxisomal protein import pathway [13].
In S. cerevisiae pex4, pex22, pex1 and pex6 mutants, Pex5
becomes polyubiquitinated and accumulates at the peroxisomal
membrane. This phenomenon facilitated the discovery and
functional characterisation of Pex5 ubiquitination [83,86,87].
Apparently the proteasomal disposal of polyubiquitinated Pex5
is less efficient in S. cerevisiae than in the other organisms,
where AAA peroxin defects lead to a more pronounced
reduction in Pex5 levels.
Pex18, which is part of the Pex7 receptor complex in S.
cerevisiae, is ubiquitinated and becomes a substrate for a
continuous proteasomal turnover [92]. It is thus a likely
candidate for a similar ubiquitin-dependent regulation. In
analogy, Pex18 turnover also requires the ubiquitin conjugating enzymes Ubc4 and Ubc5 [92]. Interestingly, Pex18
accumulates in Δpex4 and Δpex1 mutants, which might
indicate that the turnover of Pex18 is associated with its
normal function rather than abortive degradation [92]. Also in
Y. lipolytica, interference with polyubiquitination or removal
causes accumulation of Pex20 (a Pex18 orthologue) in
peroxisomes [93].
The similarities in ubiquitination of the Pex5 and the Pex7/
Pex20 receptor complex are striking [93]. In the absence of
either Pex4, Pex1, or Pex6 also Pex20 is degraded after
polyubiquitination at residue K19 [93]. This position can be
aligned to K21 in Pex5 [76]. Thus, it seems likely that
ubiquitination of Pex5 and Pex20 family members plays a
similar role in peroxisome biogenesis.
In S. cerevisiae wild-type cells, Pex5 has also been
demonstrated to be monoubiquitinated. Monoubiquitinated
Pex5 is localised to peroxisomes and requires the presence of
functional docking and RING finger complexes [88],
suggesting that it serves a non-degradative function that is a
late event in peroxisomal matrix protein import. In search for
an E2 for Pex5 ubiquitination, Pex4 is an obvious candidate.
Monoubiquitinated Pex5 can only be detected in the presence
of NEM, which might inhibit deubiquitinating enzymes [88],
and therefore suggests that under wild-type conditions Pex5p
is only transiently modified.
Based on the finding that the Pex5 level in a Δpex1Δpex4
double deletion strain were reduced to that in a the Δpex1 single
mutant strain, it was concluded, that Pex4 acts downstream of
the AAA peroxins [13]. This proposal will need re-evaluation, if
Pex4 turns out to provide the AAA substrates.
6. The peroxisomal RING complex
The three peroxins Pex2, Pex10, and Pex12 are conserved
peroxisomal integral membrane proteins with RING domains
in their cytoplasmatically exposed C-termini. RING fingers
bind zinc ions through their characteristic conserved cysteine
and histidine residues. Zinc binding has been shown only for
Pex10 [94], whereas part of the RING motifs in Pex2 and
Pex12 are degenerate, so they might not coordinate zinc ions.
The RING peroxins are required for peroxisome biogenesis in
all species analysed [95–99]. A defect in PEX10 from
Arabidopsis leads to a defective peroxisome and lipid droplet
formation [100].
The RING peroxins interact with each other and with Pex5
[95,101–103]. Based on Pex5 stability in mutant strains, the
RING complex peroxins have been placed downstream of the
docking complex [13, 101], fostering the idea of an import
cascade, in which Pex5 is handed down from the docking
complex to the RING complex [72]. The RING complex is itself
associated with the docking complex through Pex8 [104].
Recently, it was shown that RING peroxins are required for
Pex5 import in an in vitro system [85].
Pex5 ubiquitination has been reported to require the RING
peroxins [86–88]. Thus, Pex22 might recruit the putative E2
Pex4 to the peroxisomal membrane and to the RING finger
peroxins which might function as E3s in poly- and/or
monoubiquitination of Pex5 or other substrates [72]. In line
with this assumption, an interaction between Pex4 and Pex10
has been observed [105].
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7. Functional role of AAA peroxins in Pex5 recycling
A ‘shuttling receptor model’ as proposed for the peroxisomal protein import process [80, 81] implies that receptor
release is part of receptor cycling. Release is understood here
as the removal of the unloaded cycling receptor from the
peroxisomal membrane, a process that itself might be coupled
to cargo release if the ‘transient pore hypothesis’ [60] holds
true.
Experiments using a refined in vitro import system in which
radiolabeled Pex5 is incubated with rat liver post-nuclear
supernatants indicated that ATP is needed predominantly for the
recycling of the import receptor at the end of the cycle rather
than for its insertion into the membrane [84, 106]. Two different
populations of Pex5 could be identified: the ‘stage 2’ form in the
presence of ATP, in which Pex5 adopts the properties of a
transmembrane protein with a short N-terminal domain
accessible from the cytosolic site; and the ‘stage 3’ appearing
under ATP-limiting conditions in which Pex5 is resistant to
protease treatment [84]. It was further shown that stage 2
precedes stage 3 and that both Pex5 populations can be
precipitated with anti-Pex14-antibodies [84]. Thus, binding and
import of Pex5 seemingly does not need ATP hydrolysis,
whereas the export of the receptor does [83–85]. Membranebound Pex5 is a target for an ATP-dependent component of the
peroxisomal protein–import machinery, which mediates its
release from the membrane. This membrane associated
5
chaperone machinery could be identified as the AAA peroxins
complex [83, 85].
In yeast mutants with defective AAA peroxins or a
deficiency in their membrane anchor Pex15, polyubiquitinated
Pex5 accumulates at the peroxisomal membrane, while in cells
affected in proteasomal degradation the polyubiquitinated
species also appear in the cytosol [83]. These data support the
idea that the polyubiquitinated Pex5-species are designated for
proteasomal degradation. (Fig. 1; [83,86,88]. If, however, the
AAA mutant and the proteasomal mutant are combined,
polyubiquitinated Pex5 is again found exclusively in the
membrane fraction where it is neither removed nor degraded.
This indicates (1) that the release of polyubiquitinated Pex5
depends on the presence of the AAA peroxins and (2) that Pex5
polyubiquitination and AAA-dependent release are indeed
sequential steps in a degradative pathway [83]. Finally, in an
in vitro assay, the purified Pex1–Pex6-complex was demonstrated to function as a dislocase in the ATP-dependent removal
of polyubiquitinated Pex5 from the peroxisomal membrane
[83]. As these polyubiquitinated Pex5 species proved to be
carbonate-resistant, these data indicated that the AAA peroxins
perform the dislocation of polyubiquitinated integral Pex5
which then is directed to proteasomal disposal. However, the
polyubiquitinated Pex5 species only represent a minor fraction
of the total Pex5 at the peroxisomal membrane and it was
demonstrated that also the non-ubiquitinated Pex5 pool is
released from the membrane in an AAA peroxin and ATP-
Fig 1. Model for Pex5 ubiquitination and recycling. Peroxisomal PTS1 proteins are recognised by the import receptor Pex5. Upon association of the cargo-loaded
receptor with docking complex subunits (not shown), Pex5 might insert into the peroxisomal membrane. Pex5 can be modified by poly- as well as monoubiquitination.
The receptor polyubiquitination depends on the E2 enzymes Ubc4 and Ubc5 and concerns only a small fraction of Pex5. E3 enzymes for polyubiquitination have not
been identified; the RING peroxins Pex10, Pex12, and Pex2 are candidates. Polyubiquitinated Pex5 can be recognised by the AAA peroxins Pex1 and Pex6 and can be
targeted to the proteasome for degradation. This might be an abortive reaction as part of a quality control system that withdraws Pex5 from the import cycle. In contrast,
monoubiquitination of Pex5 might be mediated by the E2 enzyme Pex4, and the RING peroxins as possible E3 enzymes. Monoubiquitinated Pex5 is also recognised
by the AAA peroxins Pex1 and Pex6 and released from peroxisomal membrane to the cytosol, where the receptor becomes available for further rounds of matrix
protein import. The AAA dependent receptor recognition is likely to work in the absence of ubiquitination, albeit at lower efficiency. Ubiquitination of Pex5 might
facilitate the recruitment of the AAA machinery, possibly together with ubiquitin binding proteins as adaptors. Additionally, structural changes induced on the
Pex5 N-terminus by ubiquitination might ease recognition by the AAA peroxins.
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dependent manner [83]. Thus, polyubiquitination does not
seem to be a precondition for the removal of Pex5 from the
peroxisomal membrane and it is tempting to speculate that the
observed monoubiquitination of Pex5 might play a role as an
export signal [83, 88] (Fig. 1). However, this remains to be
investigated. When the localisation of the remaining Pex5 after
the export assay was tested, most of the carbonate resistant
Pex5 was removed from the membrane [83], indicating that
membrane-integrated Pex5 is a major target for Pex1–Pex6dependent export. In vitro studies with CHO cells also showed
that ATP was not required for Pex5 import but was indispensable for its export [84,85]. Moreover, it was demonstrated
that Pex5 was imported into the peroxisome remnants of cells
that were defective in Pex1, Pex6, or Pex26 in an ATPindependent manner but that these cells were defective in Pex5
release, thereby providing evidence that the AAA peroxins and
Pex26 are essential for the Pex5 export/release from the
peroxisome to the cytosol also in higher eukaryotes [85].
Evidence for a role of Pex6 in Pex5 recycling has also been
found in Arabidopsis, where a pex6 mutant has reduced levels
of PEX5, and defects in PEX6 can be partially rescued by
overexpression of PEX5 [91].
Taken together, the published data clearly show that Pex5 is
mono- and polyubiquitinated at the peroxisomal membrane and
dislocated to the cytosol by the AAA peroxins in an ATPdependent manner. In the cytosol, the released receptor is either
degraded by the proteasome as in the case for the polyubiquitinated forms or made available for another round of import as
part of the receptor cycle (Fig. 1). A major remaining question is
the nature of the signal by which the membrane-bound import
receptor is recognised by the AAA-peroxins. As outlined above,
it might well be that Pex4-dependent monoubiquitination of
Pex5 generates the recognition signal. Though there is no
conclusive evidence for a direct interaction between the import
receptor and the AAA peroxins, the N-terminal region of Pex5
is predestined for recognition by the AAA peroxins. This region
proved to be required for the ATP-dependent recycling [107]
and has recently shown to be unstructured [108]. We speculate
that, in analogy to Cdc48 [65], the AAA peroxins recognise the
unstructured N-terminus of Pex5 in a reaction that is specified
by ubiquitination and possibly with the help of ubiquitinbinding adaptor proteins.
8. Conclusions and perspectives
A model is emerging for the recycling of Pex5 in which
ubiquitination of Pex5 and its recognition by the AAA complex
play a central role. In this model, peroxisomal matrix proteins
are recognised by cytosolic import receptors, which direct them
to a docking complex at the peroxisomal membrane. Upon
association of cargo-loaded receptors with docking complex
subunits, the receptors might insert into the peroxisomal
membrane. A cascade of protein–protein interactions at the
peroxisomal membrane leads to (mono)-ubiquitination and
AAA peroxin-dependent release of the now unloaded receptor
from the peroxisomal membrane to the cytosol, where the
receptor becomes available for further rounds of matrix protein
import (Fig. 1). Part of the receptor is polyubiquitinated,
recognised and released by the AAA peroxins and degraded by
the proteasome. It is likely that this double function in Pex5 fate
is regulated by adaptors interacting with the AAA peroxins. It is
also likely that the process of AAA dependent receptor release
can also happen in the absence of ubiquitination, albeit at lower
efficiency. Ubiquitination of Pex5 might facilitate the recruitment of the AAA machinery, possibly together with ubiquitin
binding proteins as adaptors, or structural changes induced on
Pex5 by ubiquitination might facilitate recognition by the AAA
complex. These possibilities would bear resemblance to ERAD,
where Cdc48 can directly recognise unstructured proteins, but
needs adaptors to do so in vivo. The analogy to ERAD is indeed
striking [109,110] and it can be expected that more similarities
will be found in future.
In recent years, much has been learned on Pex5 ubiquitination and its peroxisomal release to the cytosol by the AAA
peroxins. We wish to conclude by mentioning six areas for
future investigation:
(1) To fully appreciate the role of Pex5 release in the process
of matrix protein import, it will be necessary to
understand the function of Pex5 in matrix protein import.
Is Pex5 a cycling receptor that crosses the peroxisomal
membrane, as suggested by the extended receptor shuttle
model? Can Pex5 functionally integrate into lipid
membranes, as has been suggested by the transient pore
hypothesis?
(2) To better understand the release of Pex5, we will have to
advance the in vitro reconstitution of Pex5 import and
release by purified recombinant proteins with the goal of
developing a cell- and ultimately cytosol-free system of
Pex5 receptor release.
(3) The E2 enzymes for Pex5 ubiquitination are now coming
into focus. The respective E3 enzymes are still elusive.
The three peroxisomal RING-finger proteins are obvious
candidates for E3s for peroxisomal receptor ubiquitination. It will be necessary to analyse their role and to
elucidate whether they are involved in mono- and/or
polyubiquitination of the import receptors.
(4) The suggestion of a link between Pex5 ubiquitination and
Pex5 release by the AAA peroxins is compelling, yet there
is no direct evidence for such an association. Knowledge
of more factors involved in receptor release, for instance
by the identification of a functional ubiquitin-binding
protein or a deubiquitinating enzyme that interacts with
the AAA peroxins would support the idea of an ubiquitindependent Pex5 recognition by the AAA peroxins.
(5) Another goal for the future is the elucidation of the role of
receptor ubiquitination and possible AAA-peroxinsdependent recycling in the PTS2-dependent protein
import pathway.
(6) The elucidation of how receptor membrane integration,
ubiquitination and release are mechanistically integrated
into the translocation of folded proteins across the
peroxisomal membrane is one of the fascinating challenges for future research.
Please cite this article as: Sven Thoms, Ralf Erdmann, Peroxisomal matrix protein receptor ubiquitination and recycling, Biochimica et Biophysica Acta (2006),
doi:10.1016/j.bbamcr.2006.08.046
ARTICLE IN PRESS
S. Thoms, R. Erdmann / Biochimica et Biophysica Acta xx (2006) xxx–xxx
Acknowledgements
We are grateful to Wolfgang Girzalsky, Harald W. Platta and
Wolfgang Schliebs, for reading of the manuscript. This work
was supported by the Deutsche Forschungsgemeinschaft
(Er178/2-4, SFB642), the FP6 European Union Project 'Peroxisome' (LSHG-CT-2004-512018) and by the Fonds der
Chemischen Industrie.
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Please cite this article as: Sven Thoms, Ralf Erdmann, Peroxisomal matrix protein receptor ubiquitination and recycling, Biochimica et Biophysica Acta (2006),
doi:10.1016/j.bbamcr.2006.08.046