PTS2 protein import into mammalian peroxisomes

Peroxisome targeting signal (PTS)2 directs proteins from their site of synthesis in the cytosol to the lumen of the peroxisome. Unlike PTS1 which is present in the great majority of peroxisomal matrix proteins and whose import mechanics have been dissected in considerable detail, PTS2 is a relatively rare topogenic signal whose import mechanisms are far less well understood. However, as is the case for PTS1 proteins, an inability to import PTS2 proteins leads to human disease. In this report, we describe the biochemical characterization of mammalian PTS2 protein import using a semi-permeabilized cell system. We show that a PTS2-containing reporter molecule is taken up by peroxisomes in a reaction that is time-, temperature-, ATP-, and cytosol-dependent. Furthermore, the import process is specific, saturable, and requires action of the chaperone Hsc70, the cochaperone Hsp40, and the peroxins Pex5p and Pex14p. We also demonstrate peroxisomal translocation of PTS2 reporter/antibody complexes confirming the import competence of higher order structures. Importantly, cultured fibroblasts from patients with the rhizomelic form of chondrodysplasia punctata (RCDP) which are deficient for the PTS2 receptor protein, Pex7p, are unable to import the PTS2 reporter in this assay. The ability to monitor PTS2 import in vitro will permit, for the first time, a detailed comparison of the biochemical properties of PTS1 and PTS2 protein import.     

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.     

Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.     

Differential protein import deficiencies in human peroxisome assembly disorders

Two peroxisome targeting signals (PTSs) for matrix proteins have been well defined to date. PTS1 comprises a COOH-terminal tripeptide, SKL, and has been found in several matrix proteins, whereas PTS2 has been found only in peroxisomal thiolase and is contained within an NH2- terminal cleavable presequence. We have investigated the functional integrity of the import routes for PTS1 and PTS2 in fibroblasts from patients suffering from peroxisome assembly disorders. Three of the five complementation groups tested showed a general loss of PTS1 and PTS2 import. Two complementation groups showed a differential loss of peroxisomal protein import: group I cells were able to import a PTS1- but not a PTS2- containing reporter protein into their peroxisomes, and group IV cells were able to import the PTS2 but not the PTS1 reporter into aberrant, peroxisomal ghostlike structures. The observation that the PTS2 import pathway is intact only in group IV cells is supported by the protection of endogenous thiolase from protease degradation in group IV cells and its sensitivity in the remaining complementation groups, including the partialized disorder of group I. The functionality of the PTS2 import pathway and colocalization of endogenous thiolase with the peroxisomal membranes in group IV cells was substantiated further using immunofluorescence, subcellular fractionation, and immunoelectron microscopy. The phenotypes of group I and IV cells provide the first evidence for differential import deficiencies in higher eukaryotes. These phenotypes are analogous to those found in Saccharomyces cerevisiae peroxisome assembly mutants.     

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.     

The cytosolic peroxisome-targeting signal (PTS)-receptors,Pex7p and Pex5pL,are sufficient to transport PTS2 proteins to peroxisomes

Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.     

Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: yes and no!

Previously, Waterham et al. [EMBO J. 12 (1993) 4785] reported that cytosolic oligomeric alcohol oxidase (AO) is not incorporated into peroxisomes after reassembly of the organelles in the temperature-sensitive peroxisome-deficient mutant pex1-6(ts) of Hansenula polymorpha shifted to permissive growth conditions. Here, we show that the failure to import assembled AO protein is not exemplary for other folded proteins because both an artificial peroxisomal matrix protein, PTS1-tagged GFP (GFP.SKL), and the endogenous dimeric PTS1 protein dihydroxyacetone synthase (DHAS) were imported under identical conditions. In vitro receptor-ligand binding studies using immobilised H. polymorpha Pex5p and crude extracts of methanol-induced pex1-6(ts) cells, showed that AO octamers did not interact with the recombinant PTS1 receptor, at conditions that allowed binding of folded GFP.SKL and dimeric DHAS. This shows that import of oligomeric proteins is not a universal pathway for peroxisomal matrix proteins.     

The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5

Plant peroxisomal proteins catalyze key metabolic reactions. Several peroxisome biogenesis PEROXIN (PEX) genes encode proteins acting in the import of targeted proteins necessary for these processes into the peroxisomal matrix. Most peroxisomal matrix proteins bear characterized Peroxisomal Targeting Signals (PTS1 or PTS2), which are bound by the receptors PEX5 or PEX7, respectively, for import into peroxisomes. Here we describe the isolation and characterization of an Arabidopsis peroxin mutant, pex7-1, which displays peroxisome-defective phenotypes including reduced PTS2 protein import. We also demonstrate that the pex5-1 PTS1 receptor mutant, which contains a lesion in a domain conserved among PEX7-binding proteins from various organisms, is defective not in PTS1 protein import, but rather in PTS2 protein import. Combining these mutations in a pex7-1 pex5-1 double mutant abolishes detectable PTS2 protein import and yields seedlings that are entirely sucrose-dependent for establishment, suggesting a severe block in peroxisomal fatty acid beta-oxidation. Adult pex7-1 pex5-1 plants have reduced stature and bear abnormally shaped seeds, few of which are viable. The pex7-1 pex5-1 seedlings that germinate have dramatically fewer lateral roots and often display fused cotyledons, phenotypes associated with reduced auxin response. Thus PTS2-directed peroxisomal import is necessary for normal embryonic development, seedling establishment, and vegetative growth.     

Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis

Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.     

Contribution of peroxisome-specific isoform of Lon protease in sorting PTS1 proteins to peroxisomes

Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in beta-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the beta-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.     

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.     

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.     

Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p

Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids. It has been reported, as well as confirmed in this study, to be a peroxisomal protein. Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL). We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor). This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting. Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL. Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway. Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own. By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting. This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p. Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p. Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp. These data demonstrate a novel, redundant targeting pathway for Eci1p.     

The plant PTS1 receptor: similarities and differences to its human and yeast counterparts

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon ( Citrullus vulgaris ) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X₂-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.     

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.     

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.     

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.     

The relevance of the non-canonical PTS1 of peroxisomal catalase

Catalase is sorted to peroxisomes via a C-terminal peroxisomal targeting signal 1 (PTS1), which binds to the receptor protein Pex5. Analysis of the C-terminal sequences of peroxisomal catalases from various species indicated that catalase never contains the typical C-terminal PTS1 tripeptide-SKL, but invariably is sorted to peroxisomes via a non-canonical sorting sequence. We analyzed the relevance of the non-canonical PTS1 of catalase of the yeast Hansenula polymorpha (-SKI). Using isothermal titration microcalorimetry, we show that the affinity of H. polymorpha Pex5 for a peptide containing -SKI at the C-terminus is 8-fold lower relative to a peptide that has a C-terminal -SKL. Fluorescence microscopy indicated that green fluorescent protein containing the -SKI tripeptide (GFP-SKI) has a prolonged residence time in the cytosol compared to GFP containing -SKL. Replacing the -SKI sequence of catalase into -SKL resulted in reduced levels of enzymatically active catalase in whole cell lysates together with the occurrence of catalase protein aggregates in the peroxisomal matrix. Moreover, the cultures showed a reduced growth yield in methanol-limited chemostats. Finally, we show that a mutant catalase variant that is unable to properly fold mislocalizes in protein aggregates in the cytosol. However, by replacing the PTS1 into -SKL the mutant variant accumulates in protein aggregates inside peroxisomes. Based on our findings we propose that the relatively weak PTS1 of catalase is important to allow proper folding of the enzyme prior to import into peroxisomes, thereby preventing the accumulation of catalase protein aggregates in the organelle matrix.     

PAS7 encodes a novel yeast member of the WD-40 protein family essential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes.

To identify components of the peroxisomal import pathway in yeast, we have isolated pas mutants affected in peroxisome biogenesis. Two mutants assigned to complementation group 7 define a new gene, PAS7, whose product is necessary for import of thiolase, a PTS2-containing protein, but not for that of SKL (PTS1)-containing proteins, into peroxisomes. We have cloned PAS7 by complementation of the oleic acid non-utilizing phenotype of the pas7-1 strain. The DNA sequence predicts a 42.3 kDa polypeptide of 375 amino acids encoding a novel member of the beta-transducin related (WD-40) protein family. A Myc epitope-tagged Pas7p, expressed under the control of the CUP1 promotor, was functionally active. Subcellular localization studies revealed that in the presence of thiolase this epitope-tagged Pas7p in part associates with peroxisomes. However, in a thiolase-deficient mutant, Pas7p was entirely found in the cytoplasm. We suggest that Pas7p mediates the binding of thiolase to these organelles.     

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.