Báo cáo khoa học: Functional association of the AAA complex and the peroxisomal importomer potx

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-

ger peroxins, Pex2p, Pex10p and Pex12p [5–7] as well
as the ubiquitin-conjugating protein Pex4p and its
membrane anchor Pex22p [8,9]. How these proteins
co-operate in the import of proteins across the
Keywords
AAA (ATPases associated with various
cellular activities) proteins; peroxisome
biogenesis; Pex1p; Pex6p; Pex15p
Correspondence
R. Erdmann, Institut fu
¨
r Physiologische
Chemie, Abteilung fu
¨
r Systembiochemie,
Medizinische Fakulta
¨
t der Ruhr-Universita
¨
t
Bochum, D-44780 Bochum, Germany
Fax: +49 234321 4266
Tel: +49 234322 4943
E-mail:
*These authors contributed equally to this
paper
†Present address
Institut fu
¨
r Klinische Biochemie und

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

has been provided that the receptors traverse the mem-
brane together with their cargo [10,11], and ubiquitina-
tion of import receptors has been reported, but the
physiological relevance awaits revelation. Finally, a
number of interacting proteins play a role in recycling
of the empty receptor to the cytosol, which has been
reported to be ATP-dependent [12]. These include the
AAA (ATPases associated with various cellular activit-
ies) peroxins Pex1p and Pex6p, two well-known ATP-
ases that interact with each other [13–17] and defects
of which are responsible for the majority of perox-
isome biogenesis disorders in man [18]. AAA ATPases
are involved in a diverse set of cellular processes, for
example, protein unfolding and refolding, or, like
N-ethylmaleimide-sensitive fusion protein, the disas-
sembly of protein complexes [19–23]. Besides their role
in peroxisomal protein import, Pex1p and Pex6p have
been reported to perform an essential role in priming
and docking of early peroxisomal vesicle populations
before fusion [24].
Pex1p and Pex6p are thought to be recruited to the
peroxisome membrane by the integral membrane pro-
teins, Pex15p in Saccharomyces cerevisiae [17,25] and
Pex26p in mammals, respectively [26]. Epistasis analy-
sis based on a decreased concentration of PEX5 in
PEX1-deficient and PEX6-deficient cells revealed the
proteins to play a role late in the peroxisomal protein
import pathway [27]. It has recently been demonstrated
that Pex1p and Pex6p indeed function as dislocases in
the release of the PTS1 receptor Pex5p from the per-

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-

ison of the cytosolic and membrane-bound AAA
complex. Interestingly, the cytosolic complex still con-
tained a small but significant amount of Pex5p, which
is in agreement with the function of AAA peroxins in
Pex5p release from the membrane. To determine the
size of the cytosolic AAA complex, it was subjected
to 2D electrophoresis with blue native (BN) PAGE as
a first and SDS ⁄ PAGE as a second dimension
(Fig. 1C). Immunoblot analysis of the isolated native
complex revealed a congruent profile of separation for
Pex1p and Pex6p. Two rather broad peaks were
observed, with the first being larger than 880 kDa
(position a) and a second predominant one of
 600 kDa (position b), both containing Pex1p and
Pex6p. These data indicate that Pex1p and Pex6p
form a higher-molecular-mass complex in the cytosol,
which seems to fall apart during BN-PAGE, resulting
in different subcomplexes which, however, always con-
tain Pex1p and Pex6p.
Pex1p, Pex6p and Pex15p form a complex at the
peroxisomal membrane
In addition to the interaction with Pex1p in S. cere-
visiae, Pex6p interacts with Pex15p, a tail-anchored
integral peroxisomal membrane protein proposed to
function in the recruitment of the AAA complex from
the cytosol to the peroxisomal membrane. This is
supported by the observation that the cytosolic region
of Pex15p (amino acids 1–315) interacts with Pex6p
[17]. This interaction is independent of Pex1p, raising
the question whether the Pex15p-bound Pex6p is still

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

peroxins in the late steps of peroxisomal protein
import raised the question whether there is a physical
elbul
os
-en
arbmem
dnuo
b
Pex1p-TEV-ProtA
eluate
Pex1p
Pex6p
Pex5p
Pex15p
Pex13p
Pex14p
B
C
Ato
r
P-VET-p
1
xeP
ep
y
t
-dli
w
At
o

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

Pex6p
Pex1p-TEV-ProtA
Pex6p-TEV-ProtA
Fig. 1. Pex1p and Pex6p form a stable com-
plex in the cytosol. (A) Composition of
Pex1p-TEV and Pex6p-TEV protease eluates.
Cytosolic protein complexes were isolated
from wild-type cells expressing either
Pex1p-TEV-ProtA (lane 2) or Pex6p-TEV-
ProtA (lane 3) via IgG–Sepharose and subse-
quent TEV protease cleavage. As a control,
wild-type cells expressing no protein A
fusion protein were treated in the same
way (lane 1). The corresponding whole cell
lysates (totals) are shown on the left panels.
The TEV protease eluates and totals were
subjected to immunoblot analysis with anti-
bodies against Pex1p (upper panels) and
Pex6p (lower panels), respectively. (B) Com-
position of the cytosolic and membrane-
bound AAA complexes. TEV protease
eluates of cytosolic and solubilized mem-
brane fractions isolated from wild-type cells
expressing Pex1p-TEV-ProtA were analyzed
for the presence of the peroxins as indica-
ted by immunoblot analysis. (C) Cytosolic
complexes isolated from wild-type cells
expressing Pex1p-TEV-ProtA were separ-
ated by BN-PAGE, subjected to SDS ⁄ PAGE
as a second dimension, and then probed for

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

they all assemble into a common complex or whether
they are associated in different subcomplexes. To test
these possibilities, we subjected the TEV protease elu-
ate obtained with Pex1p to 2D electrophoresis. Immu-
noblot analysis identified Pex15p along with Pex1p
and Pex6p in a complex of  880 kDa (Fig. 3B, posi-
tion c). These findings confirm the results of the yeast
two-hybrid assay and support the notion that Pex1p,
Pex6p and Pex15p functionally interact at the peroxi-
somal membrane. These proteins build one major
complex of  880 kDa. As previously observed for the
cytosolic AAA complex, both AAA peroxins also
smear through the gel, indicative of artificial complexes
of different sizes that might have formed by aggrega-
tion or the presence of a high-molecular-mass complex
that disaggregates during the preparation. Most inter-
estingly, Pex5p, Pex14p and Pex17p cosegregated in
a specific complex of  440 kDa (position d) which
agrees with the previously determined docking complex
of the importomer [5]. The complex observed in posi-
tion e may indicate an assembly of Pex5p with impor-
tomer components or oligomeric Pex5p that has fallen
off during the preparation. Probably because of detec-
tion limits, the RING finger peroxins Pex10p and
Pex12p could not be detected after 2D electrophoresis.
Taken together, the data demonstrate that the AAA
complex and the importomer do physically interact at
the peroxisomal membrane.
Association of the importomer and the AAA
complex in import mutants

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

pex10
Δ Pex1p
Pex15p(aa1-383)6 wild-type
3 wild-type
2 wild-type
Fig. 2. Pex6p-dependent interaction of
Pex1p and Pex15p. Two-hybrid interactions
of various Pex15p fragments with Pex6p or
Pex1p. Wild-type PCY2 or mutant strains
expressing the indicated fusion protein com-
bination of Pex6p, Pex1p or Pex15p were
analyzed for b-galactosidase activity by a
filter assay using X-Gal as substrate. Two
representative independent double
transformants are shown.
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3807
nor Pex6p influenced the composition of the complex
obtained with Pex15p-TEV-ProtA (Fig. 4). Thus, we
conclude that the association of Pex15p with the com-
ponents of the import machinery does not depend on
the AAA peroxins. Moreover, these data indicate that
at least one of the contact sites of the AAA complex
and the importomer is made by Pex15p.
To investigate the influence of individual peroxins of
the importomer on the composition of the membrane-
bound AAA complex, we selected gene deletion strains
affected in the docking complex (pex13D, pex14D) and
the RING finger complex (pex10D, pex12D) as well as
pex8D, and pex4D. ProtA fusion of Pex1p, Pex6p and

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

*
Pex1p-TEV-ProtA
Pex6p-TEV-ProtA#
Pex15p-TEV-ProtA
Pex15p
A
c
d
1st d im.
BN-PAGE
e
Pex6p
Pex1
B
p
Pex14p
Pex5p
Pex17p
Pex15p
Fig. 3. Association of the AAA peroxins with
Pex15p and the peroxisomal importomer.
(A) Native complexes were isolated from
solubilized membrane fractions from wild-
type cells expressing Pex1p-TEV-ProtA,
Pex6p-TEV-ProtA or Pex15p-TEV-ProtA as
indicated and eluted from IgG–Sepharose
with TEV protease. An eluate of wild-type
cells served as control. The composition of
the complexes was analyzed with a range
of specific antibodies against yeast peroxins

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

Pex6p pulled down almost the same set of peroxins,
excluding the deleted one and peroxins that are unsta-
ble in these deletion strains (e.g. Pex17p in pex14D or
Pex10p in pex12D, see detergent extracts; Fig. 5A).
Pex10p and Pex17p were below the detection level.
The same association of the AAA peroxins or Pex15p
with components of the importomer was observed in
cells lacking Pex5p, indicating that the discovered
physical contact between components of the AAA
complex and importomer components is not due to
bridging by the PTS1 receptor (Supplementary Mater-
ial Fig. S1). Deletion of PEX4 gave the same set of
peroxins as isolated from wild-type cells, and the
amount coprecipitating was only slightly reduced
(Fig. 5A). Interestingly, in the absence of Pex8p or the
RING finger components Pex10p or Pex12p, both
AAA peroxins and Pex15p still pulled down the com-
ponents of the docking complex. As Pex8p is thought
to connect docking and RING finger complex, these
results indicate the existence of direct or indirect links
between the membrane-bound AAA complex and the
docking as well as the RING finger complex.
Most interestingly, the amount of Pex1p and Pex6p
that copurified with Pex15p in import mutants was
much higher than in wild-type cells, whereas the
amount of the other peroxins was as much reduced
(Fig. 5B), suggesting that the efficient association of
the Pex15p-bound AAA peroxins with the importomer
requires a functional import mechanism. Remarkably,
in pex4D cells, Pex15p was associated with the impor-

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

derived from wild-type cells served as a
control. The corresponding solubilisates
(right panel) were analyzed for the presence
of Pex1p and Pex6p as indicated. The defici-
ency of either protein of the AAA peroxin
couple in the absence of its partner indi-
cates cross-stabilization.
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3809
Recently, it was proposed that their role in the import
process is the relocation of the import receptor Pex5p
from the peroxisomal membrane back to the cytosol,
where the receptor is available for cargo recognition
and another round of import [25,28,32]. In line with
such a function, both proteins show a bipartite
localization in the cytosol and at the peroxisomal
membrane. Both proteins are thought to form a core
complex in the cytosol which is recruited to the perox-
isome membrane by binding to the integral membrane
proteins Pex15p in S. cerevisiae [14,17,25] and Pex26p
in mammals, respectively [26]. Our studies revealed
Pex1p and Pex6p to form a complex of  600 kDa in
the cytosol (Fig. 1). The dramatically reduced amount
B
- Pex15p-TEV-ProtA
Pex15p-TEV-ProtA
Pex11p
Pex10p
Pex17p
Pex13p

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

- Pex15p-TEV-ProtA
Pex15p
Pex11p
Pex10p
Pex17p
Pex13p
Pex14p
Pex5p
Pex6p
Pex1p
eluate
Fig. 5. Composition of the membrane-bound Pex15p and AAA peroxin complexes in peroxisomal import mutants. ProtA-tagged Pex6p (A)
and Pex15p (B) were immunoprecipitated from solubilized membranes of wild-type or different pex mutant cells. Precipitations from wild-
type cells served as controls. Immunoprecipitates and detergent extracts were subjected to immunoblot analysis with the antibodies against
the peroxins indicated. *Pex6p-TEV-ProtA.
AAA complex and peroxisomal importomer K. Rosenkranz et al.
3810 FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS
of isolated Pex1p complex in the pex6D strain and,
vice versa, the reduced amount of Pex6p complex in
the pex1D strain (Fig. 4) indicates that, for the forma-
tion of a stable AAA complex, both proteins are nee-
ded. Three lines of evidence indicate that Pex1p, Pex6p
and Pex15p associate to form a membrane-bound com-
plex. First, our two-hybrid data indicate an association
of Pex1p with Pex15p that depends on the presence of
Pex6p (Fig. 2). This result can be explained by the
binding of a heteromeric AAA complex to Pex15p,
with Pex6p making the direct contact to Pex15p [29],
thereby providing a bridge to Pex1p. Secondly, Pex1p,
Pex6p and Pex15p coprecipitate irrespective of which

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

BN-PAGE provides conclusive evidence for the, at least
transient, association of the Pex1p–Pex6p–Pex15p com-
plex with the peroxisomal importomer. Interestingly,
the comparison of the composition of membrane-bound
complexes (Fig. 3A) revealed that the Pex15p complex
is associated with importomer components whereas the
relative amount of AAA peroxins is drastically reduced.
In fact, precipitation of Pex15p pulled down compo-
nents of the importomer even in the absence of Pex1p
and Pex6p (Fig. 4), indicating that Pex15p itself is part
of the importomer. Moreover, in the absence of Pex8p
or the RING finger components, Pex10p or Pex12p,
both AAA peroxins as well as Pex15p pulled down the
components of the docking complex. As Pex8p is
thought to connect the docking and RING finger com-
plexes [5], these data point to the existence of direct
or indirect connections between the membrane-bound
AAA complex and the docking complex, as well as the
RING finger complex. In this respect, it is interesting to
note that the increased amount of importomer compo-
nents isolated with Pex1p as bait in relation to the
amount isolated with Pex6p (Fig. 3A) is difficult to
reconcile with the idea that Pex1p only makes contact
with the importomer indirectly via Pex6p and Pex15p.
Thus, we have to consider that the AAA–peroxin–
importomer association is a dynamic assembly most
likely pieced together by several sites of contact that
might also involve Pex1p. The idea of a Pex1p-based
association is also supported by its domain structure. It
is a common theme of many AAA proteins to possess

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

of the AAA peroxins at Pex15p, which, however, is no
longer associated with appreciable amounts of impor-
tomer components. The molecular reason for this
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3811
failed association of Pex15p with the importomer is
not yet clear. Most interestingly, in contrast with the
lack of other components of the peroxisomal protein
import machinery, the deficiency of Pex4p does not
interfere with the assembly of the components of
the importomer. In fact, cells lacking Pex4p show a
wild-type-like assembly of the importomer, which dif-
fers in that it is associated with increased amounts of
Pex1p and Pex6p (Figs 5A,B and 6). This opens up the
possibility of a function for Pex4p in the release of
AAA peroxins from the peroxisomal membrane, which
would agree with an epistasis analysis demonstrating
that Pex4p plays a functional role late in the peroxi-
somal protein import pathway [27] and the function of
the AAA peroxins in the ATP-dependent relocation of
the PTS1 receptor from the peroxisomal membrane
to the cytosol [25]. These possibilities provide fertile
ground for future research aimed at understanding the
mechanistic principles and physiological relevance of
the observed dynamic assembly and disassembly of the
peroxisomal importomer.
Experimental procedures
Strains, media and culture conditions
Yeast strains used in this study are listed in Table 1. The
strains used for two-hybrid experiments were PCY2 and the

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

transformation and plasmid isolation, were performed as
described previously [38,39].
Immunopurification of native complexes using
IgG–Sepharose
Immunopurification of proteins was performed as described
previously [5]. Sedimented membranes (200 mg protein)
wild-type:
p
5
1xe
P
remotropmI
p5
1xeP
r
e
motropmI
r
e
m
o
tr
o
pmI
p51xeP
Pex6p
Pex1p
pex8
Δ
, pex10

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

Pex6p
Pex1p
p
5
1xeP
Pex6p
Pex1p
cytoplasm
peroxisomal membrane
peroxisomal lumen
pex4
Δ:
p
51x
e
P
Pex6p
Pex1p
remotr
op
mI
p5
1
xeP
Pex6p
Pex1p
remo
t
r
o

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

e
motrop
m
I
pex6
Δ:
cytoplasm
peroxisomal membrane
peroxisomal lumen
Pex6p
Fig. 6. Schematic presentation of the composition of Pex15p and
AAA peroxin complexes in wild-type cells and the indicated peroxi-
somal protein import mutants. In wild-type cells, Pex15p is associ-
ated with the importomer, but only a fraction of the Pex15p
importomer complexes is associated with the AAA peroxins.
Pex15p is still associated with the importomer even in the absence
of the AAA peroxins, indicating that Pex15p is an integral part of the
importomer. In pex8D, pex10D, pex12D, pex13D or pex14D mutant
cells, association of the importomer with the Pex15p–AAA peroxin
complex is diminished. In these mutants, however, we have to con-
sider that the importomer might be partially disassembled. Cells
lacking Pex4p show a wild-type-like assembly of the importomer
which differs in that it is associated with increased amounts of
Pex1p and Pex6p, opening up the possibility of a function for Pex4p
in the release of AAA peroxins from the peroxisomal membrane.
AAA complex and peroxisomal importomer K. Rosenkranz et al.
3812 FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS
were solubilized with 1% (w ⁄ v) digitonin (Calbiochem,
Merck Biosciences, Darmstadt, Germany) and subjected to
affinity chromatography. Protein complexes bound to IgG-

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

assays were performed as described elsewhere [43].
Acknowledgements
We thank Uta Ricken for technical assistance and
Wolfgang Schliebs and Marion Witt Reinhardt for
reading the manuscript. This work was supported by
the Deutsche Forschungsgemeinschaft (SFB480, SFB642
and ER178 ⁄ 2–4) and by the Fonds der Chemischen
Industrie.
Table 1. Yeast strains used in this study.
Name Genotype Source or reference
UTL-7 A MATa, ura3–52, trp1, leu2–3,112 W. Duntze, Bochum
pex1D MATa, ura3–52, trp1, leu2–3,112, pex1::loxP [44]
pex4D MATa, ura3–52, trp1, leu2–3,112, pex4::LEU2 [45]
pex5D MATa, ura3–52, trp1, leu2–3,112, pex5::LEU2 [25]
pex6D MATa, ura3–52, trp1, leu2–3,112, pex6::loxP This study
pex8D MATa, ura3–52, trp1, leu2–3,112, pex8::LEU2 [46]
pex10D MATa, ura3–52, trp1, leu2–3,112, pex10::loxP [47]
pex12D MATa, ura3–52, trp1, leu2–3,112, pex12::LEU2 [6]
pex13D MATa, ura3–52, trp1, leu2–3,112, pex13::URA3 [48]
pex14D MATa, ura3–52, trp1, leu2–3,112, pex14::LEU2 [49]
PCY2 MATa, gal4D, gal80D, URA3::GAL1-lacZ,
lys2-801
amber
, his3-D200, trp1-D63, leu2 ade2-101
ochre
[50]
PCY2-pex6D As PCY2 plus pex6::kanMX4 This study
PCY2-pex10D As PCY2 plus pex10::kanMX4 This study
Table 2. Primers used in this study.
KU230 5¢-TTTGCATACCCTCCAAAAGAAAGCGATTATAGTAACATTAATATGCGTACGCTGCAGGTCGAC-3¢

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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Supplementary material
The following supplementary material is available
online:
Fig. S1. Composition of the membrane-bound Pex15p
and AAA peroxin complexes in cells lacking Pex5p.
ProtA-tagged Pex6p (A) and Pex15p (B) were immuno-
precipitated from solubilized membranes of wild-type
and pex5D mutant cells. Precipitations from wild-type
cells served as controls. Immunoprecipitates were sub-
jected to immunoblot analysis with the antibodies
against the peroxins indicated.
This material is available as part of the online article
from .
K. Rosenkranz et al. AAA complex and peroxisomal importomer
FEBS Journal 273 (2006) 3804–3815 ª 2006 The Authors Journal compilation ª 2006 FEBS 3815