The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal.

The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.     

Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown. We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E. Monosov, and S. Subramani. 1993. J. Cell Biol. 121:761-774). The PTS1R mRNA is expressed in all human tissues examined. Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells. The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes. Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane. Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system. In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide. A PAS8-PTS1R fusion protein complements the P. pastoris pas8 mutant. The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome. The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map. Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.     

Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.     

Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome.

We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.     

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.     

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.     

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.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

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.     

A single peroxisomal targeting signal mediates matrix protein import in diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.     

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.     

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.     

The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino- terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.     

pex5 Mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This mutant also has delayed development and reduced fertility, although adult pex5-10 plants appear normal. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate also is disrupted. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins, and enzymatic processes that occur in peroxisomes are disrupted. To specifically study the import and importance of PTS1 proteins, we made a truncated PEX5 construct lacking the PTS1-binding region (PEX5(454)). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5(454)) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 and pex5-10 (PEX5(454)) phenotypes can separate the import mechanisms for enzymes acting in different peroxisomal processes, including indole-3-butyric acid/2,4-dichlorophenoxybutyric acid oxidation, isobutyrate and propionate metabolism, and photorespiration.     

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.     

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.     

Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis

Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.     

Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis. The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S. cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol. Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells. This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p. Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

AtPex14p maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes

We previously isolated an Arabidopsis: peroxisome-deficient ped2 mutant by its resistance to 2,4-dichlorophenoxybutyric acid. Here, we describe the isolation of a gene responsible for this deficiency, called the PED2 gene, by positional cloning and confirmed its identity by complementation analysis. The amino acid sequence of the predicted protein product is similar to that of human Pex14p, which is a key component of the peroxisomal protein import machinery. Therefore, we decided to call it AT:Pex14p. Analyses of the ped2 mutant revealed that AT:Pex14p controls intracellular transport of both peroxisome targeting signal (PTS)1- and PTS2-containing proteins into three different types of peroxisomes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. Mutation in the PED2 gene results in reduction of enzymes in all of these functionally differentiated peroxisomes. The reduction in these enzymes induces pleiotropic defects, such as fatty acid degradation, photorespiration and the morphology of peroxisomes. These data suggest that the AT:Pex14p has a common role in maintaining physiological functions of each of these three kinds of plant peroxisomes by determining peroxisomal protein targeting.