Functional association of the AAA complex and the
peroxisomal importomer
Katja Rosenkranz*, Ingvild Birschmann*
,†
, Silke Grunau, Wolfgang Girzalsky, Wolf-H. Kunau
and Ralf Erdmann
Abteilung fu
¨
r Systembiochemie, Medizinische Fakulta
¨
t der Ruhr-Universita
¨
t Bochum, Germany
Peroxisomes import folded, even oligomeric proteins,
but the basic principles of the import process are still a
mystery. Peroxisomal enzymes are synthesized in the
cytosol and delivered post-translationally to their tar-
get organelle by specific peroxisomal targeting signals
(PTSs), two of which are well characterized: a C-ter-
minal signal sequence related to the canonical SKL
sequence (PTS1) [1] and a signal located at the
N-terminus of proteins, which contains the consensus
sequence (R ⁄ K) ⁄ (L ⁄ V ⁄ I)X5(H ⁄ Q)(L ⁄ A) (PTS2) [2,3].
PTS1 and PTS2 sequences are recognized by the cyto-
solic receptors Pex5p and Pex7p, respectively. These
import receptors are thought to bind their cargo pro-
teins in the cytosol and direct them to the peroxisomal
membrane, where the receptor–cargo complex interacts
with components of a so-called docking complex
consisting of Pex13p, Pex14p and Pex17p [4,5]. It is
known that the following steps require the RING fin-

Pathobiochemie, Medizinische
Universita
¨
tsklinik, D-97078 Wu
¨
rzburg,
Germany
(Received 26 April 2006, revised 13 June
2006, accepted 20 June 2006)
doi:10.1111/j.1742-4658.2006.05388.x
The AAA peroxins, Pex1p and Pex6p, are components of the peroxisomal
protein import machinery required for the relocation of the import receptor
Pex5p from the peroxisomal membrane to the cytosol. We demonstrate
that Pex1p and Pex6p form a stable complex in the cytosol, which associ-
ates at the peroxisomal membrane with their membrane anchor Pex15p
and the peroxisomal importomer. The interconnection of Pex15p with the
components of the importomer was independent of Pex1p and Pex6p, indi-
cating that Pex15p is an incorporated component of the assembly. Further
evidence suggests that the AAA peroxins shuttle between cytosol and per-
oxisome with proper binding of the Pex15p–AAA complex to the impor-
tomer and release of the AAA peroxins from the peroxisomal membrane
depending on an operative peroxisomal protein import mechanism. Pex4p-
deficient cells exhibit a wild-type-like assembly of the importomer, which
differs in that it is associated with increased amounts of Pex1p and Pex6p,
in agreement with a function for Pex4p in the release of AAA peroxins
from the peroxisomal membrane.
Abbreviations
AAA, ATPases associated with various cellular activities; BN, blue native; PTS, peroxisomal targeting signal; TEV, tobacco etch virus.
3804 FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS
peroxisomal membrane is still unresolved. Evidence

oxisomal membrane and its shuttling back to the cyto-
sol [25,28].
Here we show by biochemical and structural charac-
terization that Pex1p and Pex6p form a stable complex
with Pex15p and the peroxisomal importomer. Analy-
sis of the composition of the membrane-bound com-
plexes in import mutants provides new insights into
the molecular dynamics of the importomer assembly
and function of peroxins involved.
Results
The AAA peroxins form a high-molecular-mass
complex in the cytosol
The interaction between the AAA peroxins Pex1p and
Pex6p and their association with the peroxisomal
membrane protein Pex15p has been thoroughly dem-
onstrated [13–17,25,29]. To study the association of
the AAA peroxins with each other and with compo-
nents of the peroxisomal protein import machinery in
more detail, we isolated the corresponding complexes
from both cytosol and peroxisomal membranes by
IgG affinity chromatography [5], using tobacco etch
virus (TEV)-protein A (ProtA)-tagged Pex1p, Pex6p
or Pex15p. As shown in Fig. 1A, immunoblot analysis
of the isolated cytosolic Pex1p complex revealed its
association with Pex6p; also the Pex6p complex con-
tained Pex1p. These data indicate that Pex1p and
Pex6p form a complex in the cytosol. The specifity of
the precipitation is confirmed by the lack of detection
of the abundant proteins Fbp1p and thiolase in the
eluates (data not shown). Figure 1B shows a compar-

associated with Pex1p. In agreement with such a
scenario, Platta and coworkers coprecipitated all three
proteins independent of whether Pex1p, Pex6p or
Pex15p was used as bait [25]. Here we tested the
organization of the membrane-bound Pex1p–Pex6p
complex by two-hybrid analysis. Full-length Pex15p
showed an interaction with Pex6p in the two-hybrid
assay that was weaker than that obtained with the
cytosolic fragment of Pex15p (amino acids 1–315)
(Fig. 2). This difference in intensity can be explained
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3805
by the fact that the presence of transmembrane
segments often interferes with the performance of
integral membrane proteins in two-hybrid assays.
Interestingly, Pex1p clearly interacted with the cytoso-
lic part of Pex15p. This interaction, however, did
depend on the presence of Pex6p, whereas other
import mutants (e.g. pex10D and pex14D; data not
shown) served as negative controls and showed no
effect on the interaction between Pex1p and Pex15p
(amino acids 1–315). The dependency of the Pex15p–
Pex1p interaction on Pex6p (Fig. 2) indicates that the
two-hybrid result is probably due to a bridging func-
tion of Pex6p. This in turn supports the idea that
Pex1p and Pex6p form a complex with Pex15p at the
peroxisomal membrane.
The core complex of Pex1p, Pex6p and Pex15p is
associated with the importomer
The recent discovery of the involvement of AAA

r
P-V
E
T
-p
6
xe
P
Pex1p
Pex1p-TEV-ProtA
A
Pex6p-TEV-ProtA
total
AtorP-
V
ET-p
1
xe
P
epyt-dliw
Ator
P-V
ET
-
p6x
e
P
eluate
Pex1p
Pex6p

Pex1p and Pex6p on the same blot.
AAA complex and peroxisomal importomer K. Rosenkranz et al.
3806 FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS
interaction of the membrane-bound AAA complex and
the importomer. Therefore, the protein complexes iso-
lated with protein A fusions of Pex1p, Pex6p and
Pex15p were tested for the presence of peroxins of the
peroxisomal protein import machinery. Aliquots of
TEV protease eluates, representing equal amounts of
membranes, were analyzed by SDS ⁄ PAGE and subse-
quent immunoblotting. These analyses revealed that
the eluates obtained with Pex1p, Pex6p and Pex15p
contained the same set of peroxins, although some of
these were recovered at various concentrations
(Fig. 3A). All three eluates covered the membrane-
bound AAA complex comprising Pex1p, Pex6p and
Pex15p. In addition, the RING finger protein Pex10p,
the docking complex components Pex13p, Pex14p,
Pex17p as well as Pex8p and the PTS1 receptor Pex5p
were identified in the eluates (Fig. 3A). In contrast, the
abundant peroxisomal membrane peroxin Pex11p, as
well as the peroxisomal catalase and thiolase and the
mitochondrial Tom40p and porin, were clearly detec-
ted in the detergent extracts, but did not contaminate
the eluates (Fig. 3A and data not shown), thereby con-
firming the specifity of the isolation. Taken together,
these data indicate a close association of the mem-
brane-bound AAA complex and the importomer.
The findings that Pex1p, Pex6p and Pex15p copuri-
fied with several peroxins raised the question whether

To determine whether the absence of Pex1p, Pex6p or
Pex15p has an influence on the association of the
AAA complex with the importomer, we analyzed the
composition of precipitates in the corresponding
knock-out strains. However, the amount of isolated
Pex1p complex in the pex6D strain and also the
amount of Pex6p complex in the pex1D strain was
drastically reduced, which made isolation of complexes
with Pex1p or Pex6p as baits virtually impossible. This
dependence on each other corroborates the assumption
that, for the formation of a stable complex of the
AAA peroxins, both proteins are needed. However,
the absence of Pex1p or Pex6p had no effect on
Pex15p, allowing analysis of its association with the
importomer. Interestingly, deletion of neither Pex1p
Gal-DB fused
to
Gal-AD fused
to
ß-galactosidase
filter assay
strain
PCY2
Pex15p(aa1-315)1 wild-type Pex6p
Pex15p(aa1-383)Pex6p
Pex15p(aa1-315)Pex1p
4
pex6
Δ Pex15p(aa1-315)Pex1p
Pex15p(aa1-315)5

Pex15p was used to isolate membrane-bound com-
plexes, and peroxins were detected by immunoblot
analysis (Fig. 5). As the results for Pex1p and Pex6p
were the same, only the data for one of them (Pex6p;
Fig. 5A) are presented. When isolated from wild-type
cells, independent of whether Pex1p, Pex6p or Pex15p
was used as bait, the complexes contained all the
Pex1p
Pex6p
Pex15p
Pex5p
Pex13p
Pex14p
Pex17p
Pex10p
Pex8p-HA
6
Pex11p
eluate
Pex1p
Pex6p
Pex5p
Pex13p
Pex14p
Pex17p
Pex10p
Pex11p
Pex8p-HA
6
solubilisate

as indicated. Expression of Pex8p was
followed by using strains coexpressing
Pex8p-HA
6
together with Pex1p-TEV-ProtA,
Pex6p-TEV-ProtA or Pex15p-TEV-ProtA.
*Pex6p-TEV-ProtA; #Pex1p-TEV-ProtA.
(B) Native complexes isolated from solubi-
lized membranes of wild-type cells expres-
sing Pex1p-TEV-ProtA were separated by
BN-PAGE (1st dimension) and further
resolved into their components by
SDS ⁄ PAGE (2nd dimension). Proteins were
immunodecorated with specific antibodies
against peroxins as indicated.
AAA complex and peroxisomal importomer K. Rosenkranz et al.
3808 FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS
tested components of the import machinery, i.e. Pex1p,
Pex6p, Pex15p, Pex5p, Pex13p, Pex14p, Pex17p and
Pex10p (Fig. 5A,B). Pex11p, the dominant protein of
the peroxisomal membrane, was not detected in the
precipitates and thereby served as an internal control
for the specifity and purity of the precipitation. Except
for pex4D, deletion of either component of the import
machinery significantly decreased the amount of perox-
ins of the importomer pulled down by the AAA perox-
ins, whereas the amount of components of the AAA
complex that coprecipitated remained unchanged or
even increased, as in the case of Pex15p in pex8D cells.
However, despite the decrease in amount, Pex1p and

tomer in a wild-type-like manner but showed a dra-
matic increase in association with the AAA peroxins,
which would be explained by a defect in the release of
the AAA peroxins from the peroxisomal membrane.
Discussion
The AAA peroxins have been reported to play a role
in late steps in peroxisomal protein import [30,31].
Pex1p
Pex6p
- Pex15p-
TEV-ProtA
solubilisate
eluate
Pex15p
Pex1p
Pex6p
Pex5p
Pex13p
Pex14p
Pex17p
Pex10p
Pex11p
- Pex15p-
TEV-ProtA
Fig. 4. Association of Pex15p with the
importomer does not depend on Pex1p or
Pex6p. Analysis of the composition of TEV
protease eluates (left panel) isolated from
wild-type, pex1D and pex6D cells expressing
Pex15p-TEV-ProtA. A TEV protease eluate

Pex14p
Pex5p
Pex6p
Pex1p
detergent extract
- Pex6p-TEV-Pro
A
tA
Pex6p-TEV-ProtA
Pex11p
Pex10p
Pex17p
Pex13p
Pex14p
Pex5p
Pex15p
Pex1p
*
detergent extract
- Pex6p-TEV-ProtA
Pex6p
Pex11p
Pex10p
Pex17p
Pex13p
Pex14p
Pex5p
Pex15p
Pex1p
eluate

protein was used as bait and they cosegregate on
BN-PAGE (Fig. 3). Finally and perhaps most intrigu-
ingly, when the AAA complex is isolated from import
mutants, the three proteins are still associated whereas
in most cases the association with other components of
the import machinery is lost or diminished (Fig. 5).
Remarkably, the membrane-bound AAA complex
also contained Pex5p, components of the docking and
RING finger complexes, as well as Pex8p (Fig. 3A). The
coprecipitation of these components with the AAA
peroxins and Pex15p clearly indicates an association of
the AAA complex with the importomer (Fig. 3A,B).
This is also supported by the BN-PAGE of the precipi-
tated membrane-bound AAA complex, which revealed
the presence of the 440-kDa Pex14p complex and a
200-kDa complex containing Pex5p. We could not
detect RING finger peroxins after BN-PAGE, probably
because they were below the level of detection. The iden-
tified complexes resemble subcomplexes of the impor-
tomer of the peroxisomal protein import machinery
originally described by Agne et al. [5]. The importomer
is a large complex consisting of the docking complex,
the RING finger complex, and Pex5p and Pex8p, which
is known to disassemble during BN-PAGE. A similar
observation has been described recently for the impor-
tomer of mammalian cells [28]. Although we cannot rule
out that the AAA peroxins also form separate subcom-
plexes, the presence of the components of the importom-
er in the precipitates of the AAA peroxins as well as
the appearance of the typical subcomplexes during

an N-terminal domain that contributes to the binding of
adaptors, targets and ⁄ or effectors [33]. Furthermore, the
N-terminal fragment of Pex6p has been demonstrated to
bind to Pex15p [17], and possible adaptor binding has
been proposed for the N-terminal domain of Pex1p on
the basis of structural properties [34].
The peroxisomal protein import machinery exhibits
dynamic properties indicated by changes in the protein
composition of its subcomplexes in import mutants,
schematically represented in Fig. 6. The comparison of
protein complexes from different mutants (Fig. 5A,B)
indicates that Pex15p is allied with the importomer in
wild-type cells, whereas only a fraction of the Pex15p-
carrying importomer is associated with AAA peroxins.
On deletion of components of the importomer, the
amount of AAA proteins associated with Pex15p is
dramatically increased (Figs 5B and 6). These data
emphasize the specificity of the AAA–peroxin–Pex15p
association and are in agreement with the idea of shut-
tling of the AAA peroxins between cytosol and perox-
isome with release from the peroxisomal membrane
depending on an operative peroxisomal protein import
mechanism. Moreover, despite the presence of excess
AAA peroxins, the association of Pex15p with the
other importomer components is drastically reduced
on deletion of Pex8p or components of the docking
and RING finger complexes (Figs 5B and 6). These
data indicate that proper binding of the Pex15p–AAA
complex is only to a functional importomer. In fact,
these import defects seem to result in the accumulation

corresponding pex6D (this study, primers KU230 ⁄ KU231)
and pex10D (this study, primers KU562 ⁄ KU699) deletion
strains. Strains in which the genomic copies of genes
express proteins fused to TEV-ProtA or HA
6
[5] were pro-
duced by transforming haploid yeast cells with the PCR
products as described previously [35]. The sequences of the
primers used to amplify the integration cassettes are presen-
ted in Table 2. PCR products for Pex1p-TEV-ProtA,
Pex6p-TEV-ProtA and Pex15p-TEV-ProtA were obtained
with the primer pairs KU1009 ⁄ KU1010, KU1011 ⁄ KU1012
and KU1013 ⁄ KU1014, respectively. The PCR products
were transformed in the corresponding strains as described
[36]. Transformants were selected for geneticin resistance as
a marker, and proper integration was confirmed by PCR
and immunodetection of the fusion protein.
Complete and minimal media used for yeast culturing
have been described elsewhere [37]. YNDO medium con-
tained 0.1% oleic acid, 0.1% glucose, 0.05% Tween 40,
0.1% yeast extract and 0.67% yeast nitrogen base without
amino acids, adjusted to pH 6.0.
Plasmids
The two-hybrid plasmids used have been described previ-
ously [17,29]. For two-hybrid studies, the PEX15 ORF
was amplified by PCR using primers KU296 ⁄ KU1168
(pWG15 ⁄ 1 as template) and subcloned EcoRI ⁄ NotI into
the transcription activation domain containing plasmid
pPC86. Recombinant DNA techniques, including enzymatic
modification of DNA, fragment purification, bacterial

Δ
, pex12
Δ
, pex13
Δ
, pex14
Δ:
rem
o
tr
op
m
I
r
em
otrop
m
I
r
em
otrop
m
I
p
5
1xeP
Pex6p
Pex1p
p5
1xeP

p
mI
p51xeP
Pex6p
Pex1p
remotropmI
cytoplasm
peroxisomal membrane
peroxisomal lumen
cytoplasm
peroxisomal membrane
peroxisomal lumen
pex1
Δ:
p5
1x
eP
r
e
m
o
t
r
opmI
p
51
x
e
P
r

coupled Sepharose were eluted by cleavage with TEV prote-
ase. For SDS ⁄ PAGE and subsequent immunoblot analyses,
equal volumes of the eluates were analyzed.
BN-PAGE
BN-PAGE and second dimension SDS ⁄ PAGE (2D) was
carried out as described previously [5]. For BN-PAGE and
2D analyses, TEV protease eluates of an entire preparation
were electrophoresed on the corresponding gels.
Antibodies
⁄
immunoblots
Immunoblot analyses were performed according to stand-
ard protocols [40]. Immunoblots were incubated with poly-
clonal rabbit antibodies raised against the HA epitope
(12CA5; Roche, Penzberg, Germany), Pex1p, Pex5p, Pex6p,
Pex10p, Pex11p, Pex13p, Pex14p, Pex15p and Pex17p (all
raised in our laboratory). Anti-rabbit coupled horseradish
peroxidase (Sigma-Aldrich, Taufkirchen, Germany) was
used as secondary antibody, and blots were developed using
the ECL system (Amersham Buchler GmbH, Braunschweig,
Germany). In general, 10% of the eluates of a complex
isolation were investigated by immunoblot analysis. Because
of the lower sensitivity of the Pex17p and Pex10p antibod-
ies, 30% of the eluates were subjected to immunodetection.
Two-hybrid analysis
The two-hybrid assay was based on a previous method [41].
Cotransformation of two-hybrid vectors into the strain
PCY2 was performed as described [42]. Transformed yeast
cells were plated on to synthetic dextrose (SD) medium
without tryptophan and leucine. b-Galactosidase filter

KU231 5¢-ATATATTTACAAATTTACCTATACGCTCTGAGTTGATATTACTTAATCGATGAATTCGAGCTCG-3¢
KU562 5¢-CAGGGCGAAGTAGGTATTAGCCGTTTACATTAGAAAATAAGGTAGCGTACGCTGCAGGTCGAC-3¢
KU699 5¢-GGCCTGTGGACAATGCTAAAAGAGTAGTCAAATTATTGATTAATAGGCCACTAGTGGATCTG-3¢
KU1009 5¢-GCCCAATGGTGAGAATTCCATCGACATTGGTAGCCGACTCTCCCTTATGCGTACGCTGCAGGTCGAC-3¢
KU1010 5¢-CCCTTTAAAGGGAAACGCGCTTTGTTCTTTTCTTCTTCCTTTATCGATGAATTCGAGCTCG-3¢
KU1011 5¢-GAATCATTATGAAGCGGTGAGAGCTAATTTTGAAGGTGCTCGTACGCTGCAGGTCGAC-3¢
KU1012 5¢-TATTTACAAATTTACCTATACGCTCTGAGTTGATATTACATCGATGAATTCGAGCTCG-3¢
KU1013 5¢-CCCCCAGATTGTAGGGTTGCTAAAACTTCTAGCGAGTATACGTACGCTGCAGGTCGAC-3¢
KU1014 5¢-AAATAAGTAGGTAGGGTTTTATAAACTATTCAAATATTTCATCGATGAATTCGAGCTCG-3¢
KU296 5¢-ACCCCGGGTTGAATTCAGATGGCTGCAAGTGAGATA-3¢
KU1168 5¢-AACTCGAGGCGGCCGCTCATATACTCGCTAGAAGTTTTAGC-3¢
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3813
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