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1.
Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.  相似文献   

2.
Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling   总被引:1,自引:0,他引:1  
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.  相似文献   

3.
Peroxisomal biogenesis disorders (PBDs) are caused by mutations in 12 distinct genes that encode the components of the peroxisome assembly machinery. Three mutations in the gene encoding Pex5p, the peroxisomal targeting signal type-1 (PTS1) receptor, have been reported, each associated with a disorder of the Zellweger spectrum of different severity. Here, we report studies of the affinities of mutated forms of Pex5p for a series of PTS1 peptides and conclude that PTS1-affinity reductions are correlated with disease severity and cell biological phenotype. A quantitative model has been developed that allows estimation of the dissociation constants for complexes with a wide range of PTS1 sequences bound to wild-type and mutant Pex5p. In the context of this model, the binding measurements suggest that no PTS1-containing proteins are targeted by Pex5p(N489K) and only a relatively small subset of PTS1-containing proteins with the highest affinity for Pex5p are targeted to peroxisomes by Pex5p(S563W). Furthermore, the results of the analysis are consistent with an approximate dissociation constant threshold near 500 nM required for efficient protein targeting to peroxisomes.  相似文献   

4.
Scavenger receptor class B type I (SR-BI) is an HDL receptor that mediates selective HDL lipid uptake. Peroxisomes play an important role in lipid metabolism and peroxisomal targeting signal type 1 (PTS1)-containing proteins are translocated to peroxisomes by the peroxisomal targeting import receptor, Pex5p. We have previously identified a PTS1 motif in the intracellular domain of rat SR-BI. Here, we examine the possible interaction between Pex5p and SR-BI. Expression of a Flag-tagged intracellular domain of SR-BI resulted in translocation to the peroxisome as demonstrated by double labeling with anti-Flag IgG and anti-catalase IgG analyzed by confocal microscopy. Immunoprecipitation experiments with anti-SR-BI antibody showed that Pex5p co-precipitated with SR-BI. However, when an antibody against Pex5p was used for immunoprecipitation, only the 57kDa, non-glycosylated form, of SR-BI co-precipitated. We conclude that the PTS1 domain of SR-BI is functional and can mediate peroxisomal interaction via Pex5p, in vitro.  相似文献   

5.
Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5. We found evidence that, at least the 12 C-terminal residues of a given substrate protein are implicated in PTS1 signal recognition. This motif can be structurally and functionally divided into three regions: (i) the C-terminal tripeptide, (ii) a region interacting with the surface of Pex5 (about four residues further upstream), and (iii) a polar, solvent-accessible and unstructured region with linker function (the remaining five residues). Specificity differences are confined to taxonomic subgroups (metazoa and fungi) and are connected with amino acid type preferences in region 1 and deviating hydrophobicity patterns in region 2.  相似文献   

6.
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.  相似文献   

7.
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9.
PEX5 acts as a cycling receptor for import of PTS1 proteins into peroxisomes and as a co-receptor for PEX7, the PTS2 receptor, but the mechanism of cargo unloading has remained obscure. Using recombinant protein domains we show PEX5 binding to the PEX14N-terminal domain (PEX14N) has no effect on the affinity of PEX5 for a PTS1 containing peptide. PEX5 can form a complex containing both recombinant PTS1 cargo and endogenous PEX7-thiolase simultaneously but isolation of the complex via the PEX14 construct resulted in an absence of thiolase, suggesting a possible role for PEX14 in the unloading of PTS2 cargos.  相似文献   

10.
Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.  相似文献   

11.
Peroxisomal matrix protein import requires PEX12, an integral peroxisomal membrane protein with a zinc ring domain at its carboxy terminus. Mutations in human PEX12 result in Zellweger syndrome, a lethal neurological disorder, and implicate the zinc ring domain in PEX12 function. Using two-hybrid studies, blot overlay assays, and coimmunoprecipitation experiments, we observed that the zinc-binding domain of PEX12 binds both PEX5, the PTS1 receptor, and PEX10, another integral peroxisomal membrane protein required for peroxisomal matrix protein import. Furthermore, we identified a patient with a missense mutation in the PEX12 zinc-binding domain, S320F, and observed that this mutation reduces the binding of PEX12 to PEX5 and PEX10. Overexpression of either PEX5 or PEX10 can suppress this PEX12 mutation, providing genetic evidence that these interactions are biologically relevant. PEX5 is a predominantly cytoplasmic protein and previous PEX5-binding proteins have been implicated in docking PEX5 to the peroxisome surface. However, we find that loss of PEX12 or PEX10 does not reduce the association of PEX5 with peroxisomes, demonstrating that these peroxins are not required for receptor docking. These and other results lead us to propose that PEX12 and PEX10 play direct roles in peroxisomal matrix protein import downstream of the receptor docking event.  相似文献   

12.
Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins.  相似文献   

13.
Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called “peroxins,” which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the αL conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.  相似文献   

14.
During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.  相似文献   

15.
Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD+ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.  相似文献   

16.
Arabidopsis carries three receptor genes for the phytohormone gibberellin (GA), AtGID1a, AtGID1b and AtGID1c. Expression of each gene in the rice gid1-1 mutant for GA receptors causes reversion of its severely dwarfed phenotype and GA insensitivity to a normal level, even though each loss-of-function mutant shows no clear phenotype in Arabidopsis (Nakajima et al., 2006). In this paper, we report the functional redundancy and specificity of each AtGID1 by analyzing the multiple mutants for loss of function. Seeds of the double knockout mutants atgid1a atgid1b, atgid1a atgid1c and atgid1b atgid1c germinated normally. The double knockout mutant atgid1a atgid1c showed a dwarf phenotype, while other double mutants were of normal height compared to the wild-type. The stamens of the double knockout mutant atgid1a atgid1b were significantly shorter than those of the wild-type, and this leads to low fertility. A severe disarrangement of the pattern on its seed surface was also observed. The triple knockout mutant atgid1a atgid1b atgid1c did not germinate voluntarily, and only started to grow when the seed coat was peeled off after soaking. Seedlings of the triple knockout mutants were severe dwarfs, only a few millimeters high after growing for 1 month. Moreover, the triple knockout seedlings completely lost their ability to respond to exogenously applied GA. These results show that all AtGID1s function as GA receptors in Arabidopsis, but have specific role(s) for growth and development.  相似文献   

17.
Using the rice PEX14 cDNA as a bait in a yeast two-hybrid assay, two splice variants of the type I peroxisomal targeting signal (PTS1) receptor, OsPex5pL and OsPex5pS, were cloned from a pathogen-treated rice leaf cDNA library. The proteins were produced from a single gene by alternative splicing, which generated a full-length variant, OsPEX5L, and a variant that lacked exon 7, OsPEX5S. OsPex5pL contained 11 copies of the pentapeptide motif WXXXF/Y in its N-terminus, and seven tetratricopeptide repeats in its C-terminus. Expression of OsPEX5L and OsPEX5S predominantly occurred in leaf tissues, and was induced by various stresses, such as exposure to the pathogen Magnaporthe grisea, and treatment with fungal elicitor, methyl viologen, NaCl or hydrogen peroxide. The Arabidopsis T-DNA insertional pex5 mutant, Atpex5, which does not germinate in the absence of sucrose and was resistant to indole-3-butyric acid (IBA), was perfectly rescued by over-expression of OsPex5pL, but not by OsPex5pS. Using transient expression of OsPex5pL and OsPex5pS in the Atpex5 mutant, we show that OsPex5pL translocates both PTS1- and PTS2-containing proteins into the peroxisome by interacting with OsPex7p, whereas OsPex5pS is involved only in PTS1-dependent import in Arabidopsis.  相似文献   

18.
19.
The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1. We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms. Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner. In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction. From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.  相似文献   

20.
Two members of the ‘AhR family’ (a family which is part of the bHLH-PAS superfamily), aryl hydrocarbon receptor (AhR) and AhR repressor (AhRR), originated from a common ancestor and form a regulatory circuit in xenobiotic signal transduction. AhRR is a nucleocytoplasmic shuttle protein, harboring both a nuclear localization signal (NLS) and a nuclear export signal (NES). Because NLS is dominant over NES, AhRR resides predominantly in the nuclear compartment. The NES of AhRR resembles that of AhR in sensitivity to leptomycin B, whereas the NLS of AhRR is monopartite and is, therefore, distinguished from the reported bipartite NLS of AhR. The NLS deletion mutant of GFP-AhRR was transported into the nuclear compartment in the presence of AhR nuclear translocator (Arnt), suggesting the assembly of an AhRR/Arnt heterodimer complex in the cytoplasmic compartment and Arnt-dependent nuclear translocation of this complex.  相似文献   

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