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At least 19 recordsLinked to original sources
PubMed2004

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.

Amino Acid Sequence↗
PubMed2001

Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells.

We previously isolated and characterized a Chinese hamster ovary (CHO) cell mutant, ZPG207, that is defective in import of proteins carrying a peroxisome-targeting signal type 2 (PTS2) nonapeptide. Herein we have cloned Chinese hamster (Cl) PEX7 encoding the PTS2 receptor. ClPex7p consists of 318 amino acids, shorter than human Pex7p by 5 residues, showing 91 and 30% identity with Pex7p from humans and the yeast Saccharomyces cerevisiae, respectively. Expression of ClPEX7 rescued the impaired PTS2 import in pex7 ZPG207. Mutation in ZPG207 PEX7 was determined by reverse transcription PCR; a G-to-A transition caused a 1-amino acid substitution, W221ter. We investigated the molecular dysfunction of Pex7p variants in mammals, including Pex7p-W221ter and Pex7p with one site mutation at G217R, A218V, or L292ter, which frequently occurs in the human fatal genetic peroxisomal disease rhizomelic chondrodysplasia punctata, showing a cell phenotype of PTS2 import defect. All types of the mutations affected Pex7p in binding to both PTS2 cargo protein and the longer isoform of PTS1 receptor Pex5pL that is responsible for transport of the Pex7p-PTS2 complex. Subcellular fractionation and protease protection studies demonstrated bimodal distribution of Pex7p between the cytoplasm and peroxisomes in CHO and human cells. Moreover, expression of Pex5pL, but not of the shorter isoform Pex5pS, enhanced translocation of Pex7p-PTS2 proteins into peroxisomes, thereby implying that both PTS receptors shuttle between peroxisomes and the cytosol. Furthermore, a ClPex7p mutant with a deletion of 7 amino acids from the N terminus retained peroxisome-restoring activity, whereas an 11-amino acid truncation abrogated the activity. ClPex7p with a C-terminal 9- amino acid truncation, comprising residues 1--309, maintained the activity, whereas a 14-amino acid shorter form lacking several amino acids of the sixth WD motif lost the activity. Therefore, nearly the full length of Pex7p, including all WD motifs, is required for its function.

Amino Acid Sequence↗
PubMed2006

Molecular mechanisms of import of peroxisome-targeting signal type 2 (PTS2) proteins by PTS2 receptor Pex7p and PTS1 receptor Pex5pL.

In the present study, we investigated molecular mechanisms underlying the import of peroxisome-targeting signal type 2 (PTS2) proteins into peroxisomes. Purified Chinese hamster Pex7p that had been expressed in an Sf9/baculovirus system was biologically active in several assays such as those for PTS2 binding and assessing the restoration of the impaired PTS2 protein import in Chinese hamster ovary (CHO) pex7 mutant ZPG207. Pex7p was eluted as a monomer in gel filtration chromatography. Moreover, the mutation of the highly conserved cysteine residue suggested to be involved in the dimer formation did not affect the complementing activity in ZPG207 cells. Together, Pex7p more likely functions as a monomer. Together with PTS1 protein, the Pex7p-PTS2 protein complex was bound to Pex5pL, the longer form of Pex5p, which was prerequisite for the translocation of Pex7p-PTS2 protein complexes. Pex5pL-(Pex7p-PTS2 protein) complexes were detectable in wild-type CHO-K1 cells and were apparently more stable in pex14 CHO cells deficient in the entry site of the matrix proteins, whereas only the Pex7p-PTS2 protein complex was discernible in a Pex5pL-defective pex5 CHO mutant. Pex7p-PTS2 proteins bound to Pex14p via Pex5pL. In contrast, PTS2 protein-bound Pex7p as well as Pex7p directly and equally interacted with Pex13p, implying that the PTS2 cargo may be released at Pex13p. Furthermore, we detected the Pex13p complexes likewise formed with Pex5pL-bound Pex7p-PTS2 proteins. Thus, the Pex7p-mediated PTS2 protein import shares most of the steps with the Pex5p-dependent PTS1 import machinery but is likely distinct at the cargo-releasing stage.

Animals↗
PubMed1998

An isoform of pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes.

Mutations in the peroxisome targeting signal (PTS) 1 receptor gene, PEX5 , are responsible for complementation group (CG) 2 of the peroxisome biogenesis disorders (PBD). Of the two reported patients in this CG, cells from PBD018 (homozygous for the missense mutation N489K) are defective in the import of PTS1 proteins into peroxisomes, as expected. However, cells from PBD005 (homozygous for the nonsense mutation R390ter) are defective in the import of both PTS1 and PTS2 proteins, suggesting that the PTS1 receptor also mediates PTS2-targeted protein import. To investigate this possibility, we characterized PEX5 expression and found that it undergoes alternative splicing, producing two transcripts, one containing (PEX5L) and one lacking (PEX5S) a 111 bp internal exon. Fibroblasts from PBD005 have greatly reduced levels of PEX5 transcript and protein as compared with PBD018. Transfection of PBD005 cells with PEX5S cDNA restores PTS1 but not PTS2 import; transfection with PXR5L cDNA restores both PTS1 and PTS2 protein import. Furthermore, transfection of PBD005 cells with PEX5L cDNAs containing the patient mutations (which are located downstream of the additional exon) restores PTS2 but not PTS1 import. Taken together, these data provide an explanation for the different protein import defects in CG2 patients and show that the long isoform of the Pex5 protein is required for peroxisomal import of PTS2 proteins.

Amino Acid Sequence↗
PubMed1996

The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene.

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein beta-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired.

Acetyl-CoA C-Acetyltransferase↗
PubMed2000

Evidence for a novel pathway for the targeting of a Saccharomyces cerevisiae peroxisomal protein belonging to the isomerase/hydratase family.

We, and others, have identified a novel Saccharomyces cerevisiae peroxisomal protein that belongs to the isomerase/hydratase family. The protein, named Dci1p, shares 50% identity with Eci1p, a delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids. Both of these proteins are localized to peroxisomes, and both contain motifs at their amino- and carboxyl termini that resemble peroxisome targeting signals (PTS) 1 and 2. However, we demonstrate that the putative type 1 signaling motif is not required for the peroxisomal localization of either of these proteins. Furthermore, the correct targeting of Eci1p and Dci1p occurs in the absence of the receptors for the type 1 or type 2 peroxisome targeting pathway. Together, these data suggest a novel mechanism for the intracellular targeting of these peroxisomal proteins.

Amino Acid Sequence↗
PubMed1999

Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective patients.

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

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.

Base Sequence↗
PubMed2005

Peroxisomal matrix protein import: the transient pore model.

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a translocation pore by the import receptor, the ubiquitylation of the import receptors, and pore disassembly/ receptor recycling.

Humans↗
PubMed2006

Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors.

Based on earlier suggestions that peroxisomes may have arisen from endosymbionts that later lost their DNA, it was expected that protein transport into this organelle would have parallels to systems found in other organelles of endosymbiont origin, such as mitochondria and chloroplasts. This review highlights three features of peroxisomal matrix protein import that make it unique in comparison with these other subcellular compartments - the ability of this organelle to transport folded, co-factor-bound and oligomeric proteins, the dynamics of the import receptors during the matrix protein import cycle and the existence of a peroxisomal quality-control pathway, which insures that the peroxisome membrane is cleared of cargo-free receptors.

Cytoplasm↗
PubMed2000

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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

[Structural, functional and genetic aspects of peroxisome biogenesis].

Intracellular organelles, peroxisomes, occur in cells of most eukaryotic species. Human severe congenital disorders are associated with defective assembly and functioning of peroxisomes, which partly explains the attention of researchers paid to peroxisome biogenesis. It has been shown that peroxisomes are involved in the realization of eukaryotic developmental programs (in particular, neuroblast differentiation and postembryonic development). Cytobiochemical and electron-microscopic studies of mutations involving peroxisomes showed that the primary role in peroxisome biogenesis is played by synthesis of proteins (peroxins) and their transport and incorporation into peroxisome membranes. More than 30 peroxin-encoding genes have been examined. These genes are synthesized on free polysomes and transported into peroxisomes by means of specific signaling peptides, PTS1, PTS2, and PTS3. The import of matrix proteins depends on at least two shuttle receptor proteins, Pex5p and Pex7p. Some proteins regulating peroxisome proliferation in cells have been identified.

Animals↗
PubMed2004

Protein translocation across the peroxisomal membrane.

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs through a large membrane protein assembly.

Biophysics↗
PubMed2005

Identification of a novel, intraperoxisomal pex14-binding site in pex13: association of pex13 with the docking complex is essential for peroxisomal matrix protein import.

The peroxisomal docking complex is a key component of the import machinery for matrix proteins. The core protein of this complex, Pex14, is thought to represent the initial docking site for the import receptors Pex5 and Pex7. Associated with this complex is a fraction of Pex13, another essential component of the import machinery. Here we demonstrate that Pex13 directly binds Pex14 not only via its SH3 domain but also via a novel intraperoxisomal site. Furthermore, we demonstrate that Pex5 also contributes to the association of Pex13 with Pex14. Peroxisome function was affected only mildly by mutations within the novel Pex14 interaction site of Pex13 or by the non-Pex13-interacting mutant Pex5(W204A). However, when these constructs were tested in combination, PTS1-dependent import and growth on oleic acid were severely compromised. When the SH3 domain-mediated interaction of Pex13 with Pex14 was blocked on top of that, PTS2-dependent matrix protein import was completely compromised and Pex13 was no longer copurified with the docking complex. We conclude that the association of Pex13 with Pex14 is an essential step in peroxisomal protein import that is enabled by two direct interactions and by one that is mediated by Pex5, a result which indicates a novel, receptor-independent function of Pex5.

Amino Acid Sequence↗
PubMed2000

Biogenesis of nonspecific lipid transfer protein and sterol carrier protein x: studies using peroxisome assembly-defective pex cell mutants.

Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.

Acetyl-CoA C-Acetyltransferase↗