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PubMed2004

Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae.

Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.

Cytoplasm↗
PubMed2006

PTS2 co-receptors: diverse proteins with common features.

One feature of the PTS2 import pathway is the separation of the roles of the PTS receptor between two proteins. Pex7p alone is insufficient to act as the receptor for the import cycle for peroxisomal matrix proteins. In all cases, Pex7p needs a PTS2 co-receptor to form an import-competent PTS2 receptor complex together with the PTS2 cargo. We provide an overview of the proteins that have been identified as PTS2 co-receptors and discuss their proposed functions.

Amino Acid Sequence↗
PubMed2006

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.

Animals↗
PubMed2001

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.

Acetyl-CoA C-Acetyltransferase↗
PubMed2002

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.

Acetyl-CoA C-Acyltransferase↗
PubMed2001

Pex18p is constitutively degraded during peroxisome biogenesis.

Pex18p and Pex21p are structurally related yeast peroxins (proteins required for peroxisome biogenesis) that are partially redundant in function. One or the other is essential for the import into peroxisomes of proteins with type 2 peroxisomal targeting sequences (PTS2). These sequences bind to the soluble PTS2 receptor, Pex7p, which in turn binds to Pex18p (or Pex21p or possibly both). Here we show that Pex18p is constitutively degraded with a half-time of less than 10 min in wild-type Saccharomyces cerevisiae. This degradation probably occurs in proteasomes, because it requires the related ubiquitin-conjugating enzymes Ubc4p and Ubc5p and occurs normally in a mutant lacking the Pep4p vacuolar protease. The turnover of Pex18p stops, and Pex18p accumulates to a much higher than normal abundance in pex mutants in which the import of all peroxisomal matrix proteins is blocked. This includes mutants that lack peroxins involved in receptor docking at the membrane (Deltapex13 or Deltapex14), a mutant that lacks the peroxisomal member of the E2 family of ubiquitin-conjugating enzymes (Deltapex4), and others (Deltapex1). This stabilization in a variety of pex mutants indicates that Pex18p turnover is associated with its normal function. A Pex18p-Pex7p complex is detected by immunoprecipitation in wild type cells, and its abundance increases considerably in the Deltapex14 peroxisome biogenesis mutant. Cells that lack Pex7p fail to stabilize and accumulate Pex18p, indicating an important role for complex formation in the stabilization. Mono- and diubiquitinated forms of Pex18p are detected in wild-type cells, and there is no Pex18p turnover in a yeast doa4 mutant in which ubiquitin homeostasis is defective. These data represent, to the best of our knowledge, the first instance of an organelle biogenesis factor that is degraded constitutively and rapidly.

Animals↗
PubMed2003

Impaired neuronal migration and endochondral ossification in Pex7 knockout mice: a model for rhizomelic chondrodysplasia punctata.

Rhizomelic chondrodysplasia punctata is a human autosomal recessive disorder characterized by skeletal, eye and brain abnormalities. The disorder is caused by mutations in the PEX7 gene, which encodes the receptor for a class of peroxisomal matrix enzymes. We describe the generation and characterization of a Pex7 mouse knockout (Pex7(-/-)). Pex7(-/-) mice are born severely hypotonic and have a growth impairment. Mortality in Pex7(-/-) mice is highest in the perinatal period although some Pex7(-/-) mice survived beyond 18 months. Biochemically Pex7(-/-) mice display the abnormalities related to a Pex7 deficiency, i.e. a severe depletion of plasmalogens, impaired alpha-oxidation of phytanic acid and impaired beta-oxidation of very-long-chain fatty acids. In the intermediate zone of the developing cerebral cortex Pex7(-/-) mice have an increase in neuronal density. In vivo neuronal birthdating revealed that Pex7(-/-) mice have a delay in neuronal migration. Analysis of bone ossification in newborn Pex7(-/-) mice revealed a defect in ossification of distal bone elements of the limbs as well as parts of the skull and vertebrae. These findings demonstrate that Pex7 knockout mice provide an important model to study the role of peroxisomal functioning in the pathogenesis of the human disorder.

Acetyl-CoA C-Acetyltransferase↗
PubMed2003

Genetic heterogeneity of peroxisome biogenesis disorders among Japanese patients: evidence for a founder haplotype for the most common PEX10 gene mutation.

We, as the only diagnostic center for peroxisome biogenesis disorders (PBD) in Japan, identified a total of 31 Japanese patients with PBD during the last 20 years. They were 27 patients with Zellweger syndrome (ZS), including two sib cases, three with neonatal adrenoleukodystrophy (NALD) and one with rhizomelic type chondrodysplasia punctata (RCDP). No patient with infantile Refsum disease has been detected. These patients were genetically subdivided into complementation group A (five ZS and one NALD), B (11 ZS), C (four ZS), E (five ZS and two NALD), F (two ZS), and R (one RCDP). They were subjected to mutation analysis of PEX1, PEX2, PEX6, PEX7, and PEX10. All the 11 ZS patients with group-B PBD had a common mutation, i.e., a homozygous 2-base-pair deletion in PEX10. To determine whether this highly frequent mutation is due to a founder effect, we analyzed single nucleotide polymorphisms within PEX10 among patients and Japanese controls. The mutation apparently arose once on an ancestral chromosome in the Japanese population. Based on the value of 24 PBD patients identified during the last 10 years, we estimated the prevalence of PBD in Japan to be approximately one in 500,000 births.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

A new definition for the consensus sequence of the peroxisome targeting signal type 2.

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.

Amino Acid Sequence↗
PubMed2002

Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins.

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

Animals↗
PubMed2006

Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation.

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner.

Cytosol↗
PubMed2000

Caenorhabditis elegans has a single pathway to target matrix proteins to peroxisomes.

All eukaryotes so far studied, including animals, plants, yeasts and trypanosomes, have two pathways to target proteins to peroxisomes. These two pathways are specific for the two types of peroxisome targeting signal (PTS) present on peroxisomal matrix proteins. Remarkably, the complete genome sequence of Caenorhabditis elegans lacks the genes encoding proteins specific for the PTS2 targeting pathway. Here we show, by expression of green fluorescent protein (GFP) reporters for both pathways, that the PTS2 pathway is indeed absent in C. elegans. Lack of this pathway in man causes severe disease due to mislocalization of PTS2-containing proteins. This raises the question as to how C. elegans has accommodated the absence of the PTS2 pathway. We found by in silico analysis that C. elegans orthologues of PTS2-containing proteins have acquired a PTS1. We propose that switching of targeting signals has allowed the PTS2 pathway to be lost in the phylogenetic lineage leading to C. elegans.

Alkyl and Aryl Transferases↗
PubMed1998

Abnormality in catalase import into peroxisomes leads to severe neurological disorder.

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease.

Animals↗
PubMed1996

Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide.

Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16-amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.

Acetyl-CoA C-Acetyltransferase↗
PubMed2003

Identification of PEX7 as the second gene involved in Refsum disease.

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid alpha-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.

Acetyl-CoA C-Acetyltransferase↗