PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Δ3 and Δ19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

The Peroxisomal Matrix Import of Pex8p Requires Only PTS Receptors and Pex14p

Pichia pastoris (Pp) Pex8p, the only known intraperoxisomal peroxin at steady state, is targeted to peroxisomes by either the peroxisomal targeting signal (PTS) type 1 or PTS2 pathway. Until recently, all cargoes entering the peroxisome matrix were believed to require the docking and really interesting new gene (RING) subcomplexes, proteins that bridge these two subcomplexes and the PTS receptor-recycling machinery. However, we reported recently that the import of PpPex8p into peroxisomes via the PTS2 pathway is Pex14p dependent but independent of the RING subcomplex (Zhang et al., 2006 ). In further characterizing the peroxisome membrane-associated translocon, we show that two other components of the docking subcomplex, Pex13p and Pex17p, are dispensable for the import of Pex8p. Moreover, we demonstrate that the import of Pex8p via the PTS1 pathway also does not require the RING subcomplex or intraperoxisomal Pex8p. In receptor-recycling mutants (Δpex1, Δpex6, and Δpex4), Pex8p is largely cytosolic because Pex5p and Pex20p are unstable. However, upon overexpression of the degradation-resistant Pex20p mutant, hemagglutinin (HA)-Pex20p(K19R), in Δpex4 and Δpex6 cells, Pex8p enters peroxisome remnants. Our data support the idea that PpPex8p is a special cargo whose translocation into peroxisomes depends only on the PTS receptors and Pex14p and not on intraperoxisomal Pex8p, the RING subcomplex, or the receptor-recycling machinery.     

Interaction defect of the medium isoform of PTS1-receptor Pex5p with PTS2-receptor Pex7p abrogates the PTS2 protein import into peroxisomes in mammals

We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1'-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3'-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p.     

PTS1-independent sorting of peroxisomal matrix proteins by Pex5p

Most peroxisomal matrix proteins contain a peroxisomal targeting signal 1 (PTS1) for sorting to the correct organelle. This signal is located at the extreme C-terminus and generally consists of only three amino acids. The PTS1 is recognized by the receptor protein Pex5p. Several examples have been reported of peroxisomal matrix proteins that are sorted to peroxisomes via Pex5p, but lack a typical PTS1 tripeptide. In this contribution we present an overview of these so-called non-PTS1 proteins and discuss the current knowledge of the molecular mechanisms involved in their sorting.     

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.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

Distinct Modes of Ubiquitination of Peroxisome-targeting Signal Type 1 (PTS1) Receptor Pex5p Regulate PTS1 Protein Import

Peroxisome targeting signal type-1 (PTS1) receptor, Pex5p, is a key player in peroxisomal matrix protein import. Pex5p recognizes PTS1 cargoes in the cytosol, targets peroxisomes, translocates across the membrane, unloads the cargoes, and shuttles back to the cytosol. Ubiquitination of Pex5p at a conserved cysteine is required for the exit from peroxisomes. However, any potential ubiquitin ligase (E3) remains unidentified in mammals. Here, we establish an in vitro ubiquitination assay system and demonstrate that RING finger Pex10p functions as an E3 with an E2, UbcH5C. The E3 activity of Pex10p is essential for its peroxisome-restoring activity, being enhanced by another RING peroxin, Pex12p. The Pex10p·Pex12p complex catalyzes monoubiquitination of Pex5p at one of multiple lysine residues in vitro, following the dissociation of Pex5p from Pex14p and the PTS1 cargo. Several lines of evidence with lysine-to-arginine mutants of Pex5p demonstrate that Pex10p RING E3-mediated ubiquitination of Pex5p is required for its efficient export from peroxisomes to the cytosol and peroxisomal matrix protein import. RING peroxins are required for both modes of Pex5p ubiquitination, thus playing a pivotal role in Pex5p shuttling.     

Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.     

Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.     

The unique degradation pathway of the PTS2 receptor,Pex7, is dependent on the PTS receptor/coreceptor,Pex5 and Pex20

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.     

Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, which are both essential for PTS1 protein import

Pex14p is a peroxisomal membrane-associated protein involved in docking of both Pex5p and Pex7p to the peroxisomal membrane. Previous studies have shown that, in humans, the N-terminal region of Pex14p interacts with WxxxF/Y motifs in Pex5p. Here, we report that Saccharomyces cerevisiae Pex14p contains two independent Pex5p binding sites, one in the N- and one in the C-terminus. Using deletion analysis we show that, in vivo, both of these interactions are needed for PTS1 import. Furthermore, we show that the characterized WxxxF/Y motifs of Pex5p are not essential for binding to the N-terminus of Pex14p but do play a role in the interaction with the Pex14 C-terminus. Thus, the data suggest that the mechanism of the Pex14p-Pex5p interaction in yeast is different from that previously reported for humans.     

Interactions of Pex7p and Pex18p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13delta mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.     

The Cytosolic Domain of Pex22p Stimulates the Pex4p-Dependent Ubiquitination of the PTS1-Receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential.     

Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner

A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.     

Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.     

The import receptor Pex7p and the PTS2 targeting sequence

This chapter concerns one branch of the peroxisome import pathway for newly-synthesized peroxisomal proteins, specifically the branch for matrix proteins that contain a peroxisome targeting sequence type 2 (PTS2). The structure and utilization of the PTS2 are discussed, as well as the properties of the receptor, Pex7p, which recognizes the PTS2 sequence and conveys these proteins to the common translocation machinery in the peroxisome membrane. We also describe the recent evidence that this receptor recycles into the peroxisome matrix and back out to the cytosol in the course of its function. Pex7p is assisted in its functioning by several species-specific auxiliary proteins that are described in the following chapter.     

Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor

We have identified an S. cerevisiae integral peroxisomal membrane protein of M of 42,705 (Pex13p) that is a component of the peroxisomal protein import apparatus. Pex13p's most striking feature is an src homology 3 (SH3) domain that interacts directly with yeast Pex5p (former Pas10p), the recognition factor for the COOH-terminal tripeptide signal sequence (PTS1), but not with Pex7p (former Pas7p), the recognition factor for the NH2-terminal nonapeptide signal (PTS2) of peroxisomal matrix proteins. Hence, Pex13p serves as peroxisomal membrane receptor for at least one of the two peroxisomal signal recognition factors. Cells deficient in Pex13p are unable to import peroxisomal matrix proteins containing PTS1 and, surprisingly, also those containing PTS2. Pex13p deficient cells retain membranes containing the peroxisomal membrane protein Pex11p (former Pmp27p), consistent with the existence of independent pathways for the integration of peroxisomal membrane proteins and for the translocation of peroxisomal matrix proteins.     

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import

We have previously demonstrated that the targeting signal ofpumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targetingsignal 1)-like sequence, QKL, located at –13 to –11from the C-terminus, which is different from the typical PTS1SKL motif located in the C-terminus. Here we show that Cat1import into peroxisome is dependent on the cytosolic PTS receptor,Pex5p, in Arabidopsis, similar to typical PTS1 import, and thatother components for transport of peroxisomal matrix proteinssuch as Pex14p, Pex13p, Pex12p and Pex10p also contribute tothe import of Cat1. Interestingly, however, we found that Cat1interacts with the N-terminal domain of Pex5p, but not the C-terminaldomain for interaction with the typical PTS1, revealing thatPex5p recognizes Cat1 in a manner distinct from typical PTS1.