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: Ralf.Erdmann@rub.de *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 [...].. .AAA complex and peroxisomal importomer K Rosenkranz et al References 1 Gould SJ, Keller GA & Subramani S (1987) Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase J Cell Biol 105, 2923–2931 2 Osumi T, Tsukamoto T, Hata S, Yokota S, Miura S, Fujiki Y, Hijikata M, Miyazawa S & Hashimoto T (1991) Amino-terminal presequence of the precursor of peroxisomal. .. terminal steps of peroxisomal matrix protein import Mol Cell Biol 20, 7516–7526 Miyata N & Fujiki Y (2005) Shuttling mechanism of peroxisome targeting signal Type 1 receptor Pex5: ATPindependent import and ATP-dependent export Mol Cell Biol 25, 10822–10832 Birschmann I, Rosenkranz K, Erdmann R & Kunau W-H (2005) Structural and functional analysis of the interaction of the AAA- peroxins Pex1p and Pex6p FEBS... Brown LA & Baker A (2003) Peroxisome biogenesis and the role of protein import J Cell Mol Med 7, 388– 400 10 Dammai V & Subramani S (2001) The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol Cell 105, 187–196 11 Nair DM, Purdue PE & Lazarow PB (2004) Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae J Cell... proteins Curr Opin Struct Biol 12, 746–753 Titorenko VI & Rachubinski RA (2000) Peroxisomal membrane fusion requires two AAA family ATPases, Pex1p and Pex6p J Cell Biol 150, 881–886 Platta HW, Grunau S, Rosenkranz K, Girzalsky W & Erdmann R (2005) Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol Nat Cell Biol 7, 817–822 Matsumoto N, Tamura S, Furuki S, Miyata... Pex14p and Pex13p J Biol Chem 275, 4127–4136 5 Agne B, Meindl NM, Niederhoff K, Einwachter H, ¨ Rehling P, Sickmann A, Meyer HE, Girzalsky W & Kunau WH (2003) Pex8p An intraperoxisomal organizer of the peroxisomal import machinery Mol Cell 11, 635–646 6 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. .. in yeast In Molecular Genetics of Yeast: AAA complex and peroxisomal importomer 43 44 45 46 47 48 49 50 Practical Approaches (Johnston JA, ed.), pp 121–134 Oxford University Press, Oxford Rehling P, Marzioch M, Niesen F, Wittke E, Veenhuis M & Kunau W-H (1996) The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene EMBO J 15, 2901–2913... membrane-bound Pex15p and AAA peroxin complexes in cells lacking Pex5p ProtA-tagged Pex6p (A) and Pex15p (B) were immunoprecipitated from solubilized membranes of wild-type and pex5D mutant cells Precipitations from wild-type cells served as controls Immunoprecipitates were subjected to immunoblot analysis with the antibodies against the peroxins indicated This material is available as part of the online article... for peroxisomal targeting Biochem Biophys Res Commun 181, 947–954 3 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 4 Urquhart AJ, Kennedy D, Gould SJ & Crane DI (2000) Interaction of Pex5p, the Type 1 peroxisome targeting signal receptor, with the peroxisomal. .. motion and mechanism of action of p97 ⁄ VCP J Mol Biol 347, 437–452 Shiozawa K, Maita N, Tomii K, Seto A, Goda N, Akiyama Y, Shimizu T, Shirakawa M & Hiroaki H (2004) Structure of the N-terminal domain of PEX1 AAA- ATPase: Characterization of a putative adaptorbinding domain J Biol Chem 279, 50060–50068 Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth K & Schiebel E (1999) Epitope tagging of. .. AM, Pinto RASa, -Miranda C & Azevedo JE (2003) The energetics of Pex5p-mediated peroxisomal protein import J Biol Chem 278, 39483– 39488 13 Geisbrecht BV, Collins CS, Reuber BE & Gould SJ (1998) Disruption of a PEX1–PEX6 interaction is the most common cause of the neurologic disorders Zellwe- 3814 14 15 16 17 18 19 20 21 22 23 24 25 26 27 ger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum . 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. 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