Quantitative analysis of peroxisomal targeting signal type-1 binding to wild-type and pathogenic mutants of Pex5p supports an affinity threshold for peroxisomal protein targeting

Peroxisomal biogenesis disorders (PBDs) are caused by mutations in 12 distinct genes that encode the components of the peroxisome assembly machinery. Three mutations in the gene encoding Pex5p, the peroxisomal targeting signal type-1 (PTS1) receptor, have been reported, each associated with a disorder of the Zellweger spectrum of different severity. Here, we report studies of the affinities of mutated forms of Pex5p for a series of PTS1 peptides and conclude that PTS1-affinity reductions are correlated with disease severity and cell biological phenotype. A quantitative model has been developed that allows estimation of the dissociation constants for complexes with a wide range of PTS1 sequences bound to wild-type and mutant Pex5p. In the context of this model, the binding measurements suggest that no PTS1-containing proteins are targeted by Pex5p(N489K) and only a relatively small subset of PTS1-containing proteins with the highest affinity for Pex5p are targeted to peroxisomes by Pex5p(S563W). Furthermore, the results of the analysis are consistent with an approximate dissociation constant threshold near 500 nM required for efficient protein targeting to peroxisomes.     

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, resulting in its accumulation in peroxisomes. These results suggest an essential role of the cysteine residue in the export of Pex5p. Furthermore, domain mapping indicates that N-terminal 158-amino-acid region of Pex5p-C11A, termed 158-CA, is sufficient for such dominant-negative activity by binding to membrane peroxin Pex14p via its two pentapeptide WXXXF/Y motifs. Stable expression of either Pex5p-C11A or 158-CA likewise inhibits the wild-type Pex5p import into peroxisomes, strongly suggesting that Pex5p-C11A exerts the dominant-negative effect at the translocation step via Pex14p. Taken together, these findings show that the cysteine(11) of Pex5p is indispensable for two distinct steps, its import and export. The Pex5p-C11A would be a useful tool for gaining a mechanistic insight into the matrix protein import into peroxisomes.     

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.     

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.     

Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import

Two isoforms of the peroxisomal targeting signal type 1 (PTS1) receptor, termed Pex5pS and (37-amino-acid-longer) Pex5pL, are expressed in mammals. Pex5pL transports PTS1 proteins and Pex7p-PTS2 cargo complexes to the initial Pex5p-docking site, Pex14p, on peroxisome membranes, while Pex5pS translocates only PTS1 cargoes. Here we report functional Pex5p domains responsible for interaction with peroxins Pex7p, Pex13p, and Pex14p. An N-terminal half, such as Pex5pL(1-243), comprising amino acid residues 1 to 243, bound to Pex7p, Pex13p, and Pex14p and was sufficient for restoring the impaired PTS2 import of pex5 cell mutants, while the C-terminal tetratricopeptide repeat motifs were required for PTS1 binding. N-terminal Pex5p possessed multiple Pex14p-binding sites. Alanine-scanning analysis of the highly conserved seven (six in Pex5pS) pentapeptide WXXXF/Y motifs residing at the N-terminal region indicated that these motifs were essential for the interaction of Pex5p with Pex14p and Pex13p. Moreover, mutation of several WXXXF/Y motifs did not affect the PTS import-restoring activity of Pex5p, implying that the binding of Pex14p to all of the WXXXF/Y sites was not a prerequisite for the translocation of Pex5p-cargo complexes. Pex5p bound to Pex13p at the N-terminal part, not to the C-terminal SH3 region, via WXXXF/Y motifs 2 to 4. PTS1 and PTS2 import required the interaction of Pex5p with Pex14p but not with Pex13p, while Pex5p binding to Pex13p was essential for import of catalase with PTS1-like signal KANL. Pex5p recruited PTS1 proteins to Pex14p but not to Pex13p. Pex14p and Pex13p formed a complex with PTS1-loaded Pex5p but dissociated in the presence of cargo-unloaded Pex5p, implying that PTS cargoes are released from Pex5p at a step downstream of Pex14p and upstream of Pex13p. Thus, Pex14p and Pex13p very likely form mutually and temporally distinct subcomplexes involved in peroxisomal matrix protein import.     

Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.     

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.     

Interaction of Pex5p, the type 1 peroxisome targeting signal receptor, with the peroxisomal membrane proteins Pex14p and Pex13p

Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein. Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20-40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp(100)-Glu(213)) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.     

The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.     

Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p

The peroxisomal protein acyl-CoA oxidase (Pox1p) of Saccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2. Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor. The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins. The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found. By making use of pex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p.     

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed.In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.     

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5p_C6A, Pex5p_C6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5p_C6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5p_C6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.     

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor,Pex5

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1–110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.     

Pex5p, the peroxisomal cycling receptor, is a monomeric non-globular protein

In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed.     

Disruption of the interaction of the longer isoform of Pex5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals. Study with a novel Pex5-impaired Chinese hamster ovary cell mutant

We isolated peroxisome biogenesis-defective Chinese hamster ovary cell mutants from TKaG2 cells, wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal (PTS) type 2-tagged green fluorescent protein, by the 9-(1'-pyrene)nonanol/UV selection method. Ten mutant clones showed cytosolic PTS2-green fluorescent protein, indicative of a defect in PTS2 import, and were classified in five complementation groups, i.e. pex1, pex2, pex5, pex14, and group A. One PEX5-deficient mutant, ZPG231, showed a novel phenotype: PTS2 proteins in the cytosol, but PTS1 proteins and catalase in peroxisomes. In ZPG231, two isoforms of the PTS1 receptor Pex5p, a shorter Pex5pS and a longer Pex5pL, were expressed as in wild-type cells, but possessed the missense point mutation S214F in both Pex5p isoforms, termed Pex5pS-S214F and Pex5pL-S214F, respectively. The S214F mutation was located only one amino acid upstream of the Pex5pL-specific 37-amino acid insertion site. Pex5pS-S214F and Pex5pL-S214F interacted with peroxisomal proteins, including PTS1 protein, catalase, and Pex14p, as efficiently as normal Pex5p. In contrast, the S214F mutation severely affected the binding of Pex5pL to the PTS2 receptor Pex7p. Expression of Pex5pL-S214F in pex5 cell mutants defective in PTS1 and PTS2 transport restored peroxisomal import of PTS1, but not PTS2. Together, the results indicate that ZPG231 is the first cell mutant providing evidence that disruption of the Pex5pL-Pex7p interaction completely abolishes PTS2 import in mammals.     

Pex5p imports folded tetrameric catalase by interaction with pex13p

Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins.     

Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14

Import of matrix proteins into peroxisomes requires two targeting signal-specific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structural information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetramers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low nanomolar range. Stable complexes between recombinant PEX14-(1-78) and both the full-length and truncated versions of PEX5 were formed in vitro. Analysis of these complexes revealed that PEX5 possesses multiple binding sites for PEX14, which appear to be distributed throughout its N-terminal half. Coincidentally, this part of the molecule is also responsible for oligomerization, whereas the C-terminal half with its seven tetratricopeptide repeats has been reported to bind PTS1-proteins. A pentapeptide motif that is reiterated seven times in PEX5 is proposed as a determinant for the interaction with PEX14.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.     

Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome- associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.