Molecular Mechanisms of Import of Peroxisome-targeting Signal Type 2 (PTS2) Proteins by PTS2 Receptor Pex7p and PTS1 Receptor Pex5pL*

In the present study, we investigated molecular mechanisms underlying the import of peroxisome-targeting signal type 2 (PTS2) proteins into peroxisomes. Purified Chinese hamster Pex7p that had been expressed in an Sf9/baculovirus system was biologically active in several assays such as those for PTS2 binding and assessing the restoration of the impaired PTS2 protein import in Chinese hamster ovary (CHO) pex7 mutant ZPG207. Pex7p was eluted as a monomer in gel filtration chromatography. Moreover, the mutation of the highly conserved cysteine residue suggested to be involved in the dimer formation did not affect the complementing activity in ZPG207 cells. Together, Pex7p more likely functions as a monomer. Together with PTS1 protein, the Pex7p-PTS2 protein complex was bound to Pex5pL, the longer form of Pex5p, which was prerequisite for the translocation of Pex7p-PTS2 protein complexes. Pex5pL-(Pex7p-PTS2 protein) complexes were detectable in wild-type CHO-K1 cells and were apparently more stable in pex14 CHO cells deficient in the entry site of the matrix proteins, whereas only the Pex7p-PTS2 protein complex was discernible in a Pex5pL-defective pex5 CHO mutant. Pex7p-PTS2 proteins bound to Pex14p via Pex5pL. In contrast, PTS2 protein-bound Pex7p as well as Pex7p directly and equally interacted with Pex13p, implying that the PTS2 cargo may be released at Pex13p. Furthermore, we detected the Pex13p complexes likewise formed with Pex5pL-bound Pex7p-PTS2 proteins. Thus, the Pex7p-mediated PTS2 protein import shares most of the steps with the Pex5p-dependent PTS1 import machinery but is likely distinct at the cargo-releasing stage.

protein. Pex7p-PTS2 protein complexes bind to Pex14p via Pex5pL in vitro, consistent with a view depicting the docking step on peroxisome membranes in the import of PTS cargoes. Contrary to Pex5p (16), Pex7p directly binds to Pex13p in the presence or the absence of PTS2 proteins, suggesting PTS2 cargo release at Pex13p. Thus, the Pex5pL-dependent import of Pex7p-PTS2 protein complexes shares most of the steps with the Pex5p-mediated PTS1 import machinery but is likely distinct at the cargo releasing stage.

Construction of Expression Plasmids in E. coli and Mammalian Cells-His
pcDNA3.1Zeo-HA 2 -PTS2-EGFP and pcDNA3.1Zeo-FLAG-PTS2-EGFP were constructed as follows. BamHI-NcoI fragment of the PCR product for PTS2 of pTZ18R-pTH using primers pThϩ2-Fw and pTHM8-Rv was ligated together with the NcoI-NotI fragment of pEGFP into the BamHI-NotI site of pcDNA3.1Zeo-HA 2 -ubiquitin and pcDNA3.1Zeo-FLAG-ubiquitin. FLAG-PEX7C199S was also constructed. The PCR products each with pairs of primers C199S-Fw and ClPEX7SalI-Rv (14) and ClPEX7-Fw (14) and C199S-Rv using a template pUcD2SRaMCSHyg-FLAG-PEX7 were mixed and used as a template in PCR with ClPEX7-Fw and ClPEX7SalI-Rv. The resulting product in pGEM T-Easy was digested with NotI and SalI. The NotI-SalI fragments was ligated into the NotI-SalI site of pUcD2SRaMCSHyg-FLAG vector (14).
Gel Filtration Chromatography-Five g of recombinant Pex7p was applied onto 1.6 ϫ 60-cm Superdex S-200 (Amersham Biosciences) column equilibrated with 20 mM Hepes-KOH, pH 7.4, 150 mM NaCl, and 1 mM DTT. Elution was done at 1 ml/min and monitored at 280 nm by an AKTA system (Amersham Biosciences). Pex7p in the eluate fractions was detected by immunoblotting with anti-Pex7p antibody.
Assessment of Disulfide Linkage by SDS-PAGE-HEK293 cells transiently expressing FLAG-Pex7p were lysed in 50 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol in the presence or absence of 10 mM DTT. Pex7p was assessed by SDS-PAGE and immunoblotting with anti-Pex7p antibody.
Protease Sensitivity Assay-Cargo-loaded Pex7p was prepared as follows. Purified Pex7p (0.1 g) was incubated with FLAG-PTS2-EGFP (0.8 g) bound to anti-FLAG-IgG agarose. After thorough washing, the complexes were eluted with FLAG peptide and treated with 1, 10, and 50 g/ml of Pronase on ice for 30 min (14). The reaction was terminated by boiling in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol. Pex7p and FLAG-PTS2-EGFP were detected using antibodies to Pex7p and GFP, respectively. Pex7p was also treated with Pronase in the presence of FLAG-EGFP-PTS1 equivalent to the amount to FLAG-PTS2-EGFP.
Morphological Analysis-Peroxisomes in CHO cells were visualized by indirect immunofluorescence light microscopy as described (27). Antigen-antibody complexes were detected using fluorescein isothiocyanate-labeled goat anti-rabbit IgG antibody (Alexa) and Texas Red-labeled goat anti-guinea pig IgG antibody (Alexa). GFP and EGFP fluorescence were directly observed by fluorescence light microscopy after fixation with 4% paraformaldehyde (26).
Expression, Purification, and Antibody Preparation of Pex13p SH3 Domain-EcoRI-SalI fragment of the PCR product using a template human (Hs) PEX13 (21) with primers, HsPex13SH3-Fw and HsPex13-Rv, was ligated into the EcoRI-SalI site of pGEX6P-1 (Amersham Biosciences). E. coli BL21 was transformed with pGEX6P-1-HsPex13pSH3 and grown according to the procedure recommended by the manufacture. A C-terminal part comprising amino acid residues 256 -403 of HsPex13p, termed Pex13pSH3, was recovered from cell lysates by incubation at 4°C for 2 h with glutathione-Sepharose (Amersham Biosciences). After thoroughly washing, Pex13pSH3 was isolated by cleaving the bound GST fusion protein with Prescission protease in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, and 1 mM EDTA. Rabbit anti-Pex13p antibody was raised by conventional subcutaneous injection of Pex13pSH3 (28).
Immunoprecipitation-Immunoprecipitation was performed in immunoprecipitation buffer with anti-FLAG IgGconjugated agarose or antibodies to Pex14p and Pex13p bound to protein A-Sepharose (Amersham Biosciences).
Other Methods-Western blot analysis was performed as described (14). In detection by immunoblot of protein import complexes in HEK293 cells, monoclonal anti-rabbit IgG conjugated with alkaline phosphatase (Sigma) and CDP-Star detection reagent (Amersham Biosciences) was used.

RESULTS
Expression and Purification of Pex7p Using Sf9 Cell/Baculovirus System-As a step to understanding at a molecular level the mechanisms of PTS2 import mediated by the PTS2 receptor, we attempted to express and purify recombinant Pex7p using an Sf9 cell/baculovirus expression system. Basically after two-step purification, soluble 40-kDa Pex7p was obtained (Fig.  1A). To assess the PTS2 receptor activity, the purified Pex7p was introduced into pex7 ZPG207 cells stably expressing PTS2-GFP (14,26). PTS2 import was restored in ZPG207 cells upon introduction of Pex7p, but not in mock-treated ZPG207 (Fig.  1B). Next, a binding assay was performed with peroxisomal matrix model proteins FLAG-EGFP-PTS1(SGLRSSKL) and FLAG-PTS2-EGFP containing a 36-amino acid presequence plus N-terminal 8-amino-acid residues (23) of rat thiolase A (24), on agarose beads. Pex7p was detected only in the fraction bound to FLAG-PTS2-EGFP, but not to FLAG-EGFP-PTS1 ( Fig. 2A). Pex7p binding to GST-Pex5p was also verified by GST pull-down assay. Pex7p bound to Pex5pL, not to Pex5pS, only in the presence of PTS2 cargo protein, FLAG-PTS2-EGFP (Fig.  2B), consistent with our earlier observation (14). Taken together, these results demonstrated that the purified recombinant Pex7p was biologically active. Furthermore, PTS2 cargodependent interaction between Pex7p and Pex5pL was assessed in vivo by co-immunoprecipitation assay upon their ectopic expression in CHO-K1 cells (Fig. 2C). HA 2 -Pex5pL was coimmunoprecipitated with FLAG-Pex7p and the endogenous thiolase precursor (Fig. 2C, lane 8) and more efficiently in the presence of excess amount of a PTS2 protein, HA 2 -PTS2-EGFP (lane 10), confirming the PTS2-dependent binding of Pex7p to Pex5pL.
Conformational Change of Pex7p Is Required for the Interaction with Pex5pL-How does Pex7p interact with Pex5pL in a PTS2-dependent manner? One possibility is that conformational change of Pex7p is induced when it binds to PTS2 protein. To verify such possibility, cargo-free Pex7p and PTS2 cargo-loaded Pex7p were treated with Pronase (14). Pex7p loaded with FLAG-PTS2-EGFP was detectable at up to 10 g/ml of Pronase and completely digested with 50 g/ml Pronase (Fig. 3, lanes 5-8), whereas cargo-free 40-kDa Pex7p was digested to a 35-kDa fragment in a manner dependent on Pronase concentration, in the presence of FLAG-EGFP-PTS1 (lanes 1-4), EGFP-PTS1 (data not shown), or EGFP (data not shown). Detection of the 35-kDa fragment with antibody against the C-terminal peptide of Pex7p (14) suggested that Pex7p was proteolytically cleaved at an N-terminal site. This difference in the sensitivity to the protease suggests that the conformational change occurs in Pex7p upon the cargo loading. It is also possible that FLAG-PTS2-EGFP binding to Pex7p protects the Pronase-sensitive site.
Conserved Cysteine Residue at 199 of Pex7p Is Not Critical for Function-In Pex7p from yeast to mammals including Chinese hamster (14,29), only one cysteine residue located in the fourth WD region is conserved. Functional importance in vivo of this cysteine residue remained unclear, although it was shown to be required for dimerization of Pex7p in vitro (29). FLAG-Pex7p with mutation at the conserved cysteine at 199 to serine (C199S) was expressed in pex7 ZPG207. Wild-type FLAG-PEX7 (WT) and FLAG-PEX7G212R (G212R) (14) were also transfected to ZPG207 as positive and negative controls, respectively. WT, C199S, and G212R were comparable in the expression level (Fig. 4A, panel d). C199S complemented the impaired PTS2 import in ZPG207 cells as efficiently as WT (Fig.  4A, panels c and a), whereas G212R failed to restore the PTS2 protein import (Fig. 4A, panel b). 44-kDa thiolase precursor was processed to the matured 41-kDa form in the ZPG207 cells each expressing C199S and WT, but not in those expressing G212R (Fig. 4A, panel d), implying the PTS2 import into peroxisomes. Next, recombinant Pex7p was analyzed by gel filtration chromatography (Fig. 4B). Pex7p was eluted as a single peak corresponding to ϳ40 kDa in size. Furthermore, cell lysates of HEK293 cells transfected with WT were processed in the presence and absence of a reducing reagent, DTT, for SDS-PAGE (Fig. 4C). Endogenous Pex7p and WT were each detected as a single band with ϳ40 and 41 kDa, respectively, in both conditions (Fig. 4C). A minor band with ϳ70 kDa was likely a nonspecific one, because no additional band appeared with WT expression. Accordingly, these results strongly suggest that the conserved cysteine is not essential for the physio- Quaternary Complexes Formed in the Cytosol in Matrix Protein Import-The C-terminal half of Pex5p including TPR motifs binds PTS1 of its cargo, and the N-terminal half interacts with Pex14p, Pex13p, and Pex7p (16). Pex5pL also plays an essential role in PTS2 protein import (15). However, it remains unclear whether Pex5pL simultaneously loads PTS1 and Pex7p-PTS2 complexes. To address this issue, a binding assay was performed with PTS1-loaded Pex5pL and PTS2-saturated Pex7p (Fig.  5A). Soluble Pex7p-(His 6 -PTS2-EGFP) complexes were added to the assay mixture containing Pex5pLbound FLAG-EGFP-PTS1 on FLAG-IgG beads (Fig. 5B). Pex7p-(His 6 -PTS2-EGFP) was detected in the fraction bound to (FLAG-EGFP-PTS1)-Pex5pL complexes, not to FLAG-EGFP-PTS1 (Fig. 5B,  lane 1), and was elevated with the loaded amount (lanes 2-5). To assess this finding in vivo, CHO pex14 ZP161 cells stably expressing FLAG-EGFP-PTS1 were transfected with HA 2 -EGFP-PTS1 or HA 2 -PTS2-EGFP. Expression of HA 2 -EGFP-PTS1 and HA 2 -PTS2-EGFP did not alter the endogenous level of Pex5p and Pex7p to any greater extent (Fig. 5C, lanes 1-3). In the immunoprecipitates of FLAG-EGFP-PTS1, Pex5p was reduced when HA 2 -EGFP-PTS1 was expressed (Fig. 5C, lanes 4 and 5), apparently because of the competition in binding to Pex5p between two types of PTS1 cargoes. In contrast, HA 2 -PTS2-EGFP expression gave rise to an apparent increase in the amount of Pex5p, presumably Pex5pL, in the immunoprecipitates, where HA 2 -PTS2-EGFP and Pex7p were concomitantly co-immunoprecipitated (Fig. 5C, lane 6), hence suggesting that the complexes of (FLAG-EGFP-PTS1)-Pex5p-(HA 2 -PTS2-EGFP)-Pex7p were formed. It is also likely that (HA 2 -PTS2-EGFP-Pex7p)-bound Pex5pL was elevated. Co-immunoprecipitation of Pex7p in a PTS2 protein-dependent manner was consistent with our earlier finding (14). We therefore interpreted these results to mean that the quaternary complexes consisting of PTS1, Pex5pL, Pex7p, and PTS2 are formed in the cytosol. Thus, the Pex7p-PTS2 complexes are likely imported to peroxisomes with PTS1 via Pex5pL.

Pex7p-mediated Transport of PTS2 Proteins
PTS2 Import Complexes at the First Docking Site-To dissect the steps as well as the interacting peroxins involved in the PTS2 import pathway, we carried out GST pull-down assays using recombinant proteins. All of the recombinant proteins, including FLAG-EGFP-PTS1, FLAG-PTS2-EGFP, Pex7p, Pex5pL, and Pex13p, were purified nearly to the homogeneity (Fig. 6A). GST-Pex13p and GST-Pex14p were similar to those used in our earlier studies (16,30). Pex5pL and Pex5pL plus FLAG-EGFP-PTS1 were detected in fractions each bound spe-cifically to GST-Pex14p (Fig. 6B,  lanes 2-4), indicating the binding of Pex5pL and Pex5pL-PTS1 complexes to Pex14p, consistent with the report by Otera et al. (16). In contrast, neither Pex7p nor Pex7p plus FLAG-PTS2-EGFP were detectable in fractions bound to GST-Pex14p, suggesting no direct interaction of these proteins with Pex14p (Fig. 6B,  lanes 5 and 6). Binding of Pex7p and FLAG-PTS2-EGFP to GST-Pex14p occurred only in the presence of Pex5pL (lane 7), possibly representing the initial docking step on peroxisome membranes in the PTS2 protein import. Binding of Pex7p, FLAG-PTS2-EGFP, Pex5pL, and Pex13p to GST-Pex14p was likewise detected (Fig. 6B, lane 8), suggesting that the hetero-pentameric complexes comprising Pex14p, Pex13p, Pex5pL, Pex7p, and PTS2 cargo were formed.
We verified the PTS protein import complexes formed in vivo by immunoprecipitating Pex14p from HEK293 cells transiently transfected with HA 2 -EGFP-PTS1 and HA 2 -PTS2-EGFP. Together with Pex5p, Pex13p, and Pex7p, both types of PTS cargoes were co-immunoprecipitated (Fig. 6C, lane 3), whereas none of these proteins was detectable with preimmune serum (lane 2). These findings strongly suggest that the peroxin complexes consisting of Pex14p, Pex13p, Pex5p, and Pex7p, together with both types of PTS cargoes are formed during the PTS protein import on peroxisome membranes, namely as the first docking event in mammalian cells.
Pex5pL Binds to Pex13p via Pex7p-PTS2 in PTS2 Cargo Import Complex-To investigate whether Pex13p interacts with Pex7p, pull-down assays were likewise performed using GST-Pex13p. Pex7p as well as Pex7p plus FLAG-PTS2-EGFP specifically bound to GST-Pex13p (Fig. 7A, lanes 2, 5, and 6), whereas only Pex5pL bound to GST-Pex13p when incubated with Pex5pL or Pex5pL plus FLAG-EGFP-PTS1 (lanes 3 and 4), in good agreement with the result of Otera et al. (16). We interpreted this finding to imply that PTS2 cargo is likely released at Pex13p in the PTS2 protein import pathway. It is noteworthy that in the yeast two-hybrid assay PTS2 thiolase did not interact with Pex13p in the wild-type S. cerevisiae (19). Pex5pL bound to GST-Pex13p in the presence of Pex7p and to a higher level with Pex7p plus FLAG-PTS2-EGFP (Fig. 7A, lanes 7 and 8). The difference in the amount of Pex5pL in the fractions bound to Pex13p more likely reflected the Pex5pL bound to Pex7p-FLAG-PTS2-EGFP complexes. The addition of FLAG-EGFP-  lanes 1 and 2) or the presence of 10 mM DTT (lanes 3 and 4) and immunoblotting with anti-Pex7p antibody. Open and solid arrowheads indicate Pex7p and FLAG-Pex7p, respectively.

Pex7p-mediated Transport of PTS2 Proteins
PTS1 to such a reaction mixture did not alter the result obtained in the assay without the PTS1 cargo (lane 9), thereby strongly suggesting that Pex5pL bound to Pex13p via the Pex7p-PTS2 complex in the PTS2 protein import pathway.

DISCUSSION
In the present study, we succeeded in expressing and isolating mammalian Pex7p using the Sf9/baculovirus system. Purified recombinant Pex7p was active in several assays including re-establishment of PTS2 protein import upon introduction of Pex7p into pex7 ZPG207 cells and in vitro binding to PTS2 proteins and Pex5pL. Therefore, this preparation is the first recombinant Pex7p shown to possess biological activities. In this regard, it was possible that the cytosolic chaperonin TCP1 (t-complex protein 1)/TRiC/CCT (31) played a role in proper folding of Pex7p during its expression and purification, as reported for the yeast Pex7p as a model WD protein in S. cerevisiae (32).
We also attempted to address the molecular mechanisms underlying Pex7p-mediated PTS2 protein import, primarily using the highly purified Pex7p. PTS2 protein import is divided into several steps: Pex7p binding to PTS2 and formation of PTS2-Pex7p-Pex5pL complexes in the cytosol, their transloca-tion to peroxisomes, and the release of PTS2 cargoes. Consistent with our earlier findings by GST pull-down assay using cell lysates (14), the isolated Pex7p readily bound to Pex5pL only in the presence of PTS2 protein in in vitro assays, presumably representing the initial step in the cytosol for the import of PTS2 proteins to peroxisomes. Moreover, the difference in the sensitivity to the protease digestion of Pex7p between the PTS2 cargo-free and cargo-loaded states suggested a conformational change of Pex7p upon its binding to the cargoes. Such a difference may also represent the specific interaction between Pex5pL and Pex7p in the cytosol at the early steps in the PTS2 protein import pathway. Apparently two domains of Pex7p are likely involved in conformational changes: one responsible for the protease sensitivity of the N-terminal 5-kDa domain and the other conferring a compact folding of the entire protein, i.e. folding of all six WD40 domains. Pex7p can be superimposed over G␤1 of the G protein heterotrimer G i ␣ 1 ␤ 1 ␥ 2 (33,34). The N-terminal region comprising ϳ50 amino acids shows an ␣-helical domain plus a linker region, and the following six WD domains fit on the blades of propeller structures of G␤1 (34). Such a model in regard to the "three-dimensional structure" FIGURE 6. Pex5pL-mediated interaction of Pex7p-PTS2 complexes with import machinery components. A, recombinant proteins used were analyzed by SDS-PAGE and staining with Coomassie Blue. Arrowheads indicate respective proteins. B, GST pull-down assays were performed using GST-Pex14p (50 ng) with several combinations of proteins indicated at the top, including Pex5pL (2 g), Pex7p (5 g), FLAG-EGFP-PTS1 (10 g), FLAG-PTS2-EGFP (10 g), and Pex13p (50 ng). After thoroughly washing, the bound proteins were analyzed in immunoblotting using antibodies to Pex5p, Pex13p, Pex7p, and FLAG. Recovered GST and GST-Pex14p were also assessed with anti-GST antibody. One-thirtieth of the input was loaded in lane 1. C, endogenous Pex14p was immunoprecipitated from HEK293 cells transiently expressing HA 2 -EGFP-PTS1 and HA 2 -PTS2-EGFP with preimmune serum and anti-Pex14p antibody. Immunoprecipitates (IP) were analyzed by immunoblotting with antibodies to Pex5p, Pex7p, HA, Pex13p, and Pex14p. Lane 1, one-sixth of the input; lanes 2 and 3, immunoprecipitation with preimmune and anti-Pex14p sera, respectively. Both Pex5pS and Pex5pL were discernible in the immunoprecipitates. of Pex7p supports the findings in the protease digestion experiment.
With respect to the molecular form of Pex7p, human Pex7p was reported to form a dimer via cysteine residue 204 (29). In the present study, however, Pex7p was eluted as a single peak with a mass of ϳ40 kDa in size exclusion chromatography, strongly suggesting that Pex7p is in a monomeric form. Endogenous human Pex7p and FLAG-tagged CHO Pex7p expressed in HEK293 cells were both detected in a monomeric form in SDS-PAGE under the nonreducing condition. Moreover, CHO Pex7pC199S equivalent to human Pex7pC204S showed the PTS2 import restoring activity as the normal Pex7p in pex7 ZPG207 cells. Therefore, it is less likely that Pex7p forms a dimer by disulfide linkage.
We demonstrated here that Pex7p-PTS2 protein complexes bound to PTS1 cargo-loaded Pex5pL. The complexes comprising four distinct proteins may well be categorized as one of "cytosolic preimplexes" (35). Such quaternary complexes are of physiological consequence and are envisaged as follows. Pex5pS and Pex5pL form homomeric and heteromeric oligomers and are present at nearly equal levels and more abundant than Pex7p in mammalian cells (14,18). PEX7 mRNA is elevated ϳ13-fold in the liver of a rat fed with peroxisomeproliferators such as clofibrate, a hypolipidemic drug, whereas the PEX5 mRNA level is not apparently altered (14). The newly synthesized PTS1 and PTS2 proteins at an induced level are to be transported to peroxisomes. Pex7p-PTS2 protein complexes are readily loaded onto Pex5pL that is PTS1-unloaded or occasionally carrying a part, if not all, of the increased level of PTS1 cargoes; therefore Pex5pL reconciles such situations together with Pex5pS solely for PTS1 proteins.
In pex14 ZP161 cells lacking the Pex5p-docking site, Pex5pL forms stable complexes with Pex7p and PTS2 cargoes, whereas such complexes are less abundant in CHO-K1, presumably because of constant recycling of the mobile PTS receptors. Similarly, in S. cerevisiae Pex18p and Pex18p-Pex21p complexes promote the formation of import competent Pex7p-PTS2 protein complexes in the cytosol (17,19). In Pichia pastoris, Pex20p was very recently proposed to act as a chaperone for Pex7p-PTS2 protein complexes (36). In our current model for Pex5p-mediated PTS1 import to peroxisomes (16,30), Pex5p with PTS1 cargoes docks with Pex14p and then forms four-component complexes together with Pex13p via Pex14p. After releasing cargo proteins, cargofree Pex5p binds to Pex13p and then recycles back to the cytosol for another round of the protein import. The recycling processes apparently involve RING peroxin complexes and Pex26p complexes with the AAA peroxins, Pex1p and Pex6p (37). In the present work, we addressed the PTS2 protein import pathway ( Fig. 9): 1) Pex7p recognizes PTS2 proteins in the cytosol, and then the Pex7p-PTS2 protein complexes bind to the cargo-free as well as PTS1-loaded Pex5pL; 2) these ternary and quaternary complexes dock onto Pex14p associated with Pex13p, as proposed for the transport of Pex7p-PTS2 protein complexes mediated by Pex18p-Pex21p complexes in S. cerevisiae (19); 3) PTS2 cargo is likely released at Pex13p; and 4) Pex7p and Pex5pL shuttle back to the cytosol for another round of the PTS2 protein import. Together, the PTS2 proteins are more likely released from Pex7p at Pex13p, whereas the PTS1 proteins are unloaded from Pex5p before the step at Pex13p in mammalian cells (16). Successful expression and purification of recombinant peroxins such as Pex7p and PTS cargoes would define more detailed mechanisms underlying the protein import to peroxisomes.