PTS1 Proteins Research Articles

Fusarium verticillioides Pex7/20 mediates peroxisomal PTS2 pathway import, pathogenicity, and fumonisin B1 biosynthesis.

Fusarium verticillioides, a well-known fungal pathogen that causes severe disease in maize and contaminates the grains with fumonisin B1 (FB1) mycotoxin, affects the yield and quality of maize worldwide. The intrinsic roles of peroxisome targeting signal (PTS)-containing proteins in phytopathogens remain elusive. We therefore explored the regulatory role and other biological functions of the components of PTS2 receptor complex, FvPex7 and FvPex20, in F. verticillioides. We found that FvPex7 directly interacts with the carboxyl terminus of FvPex20 in F. verticillioides. PTS2-containing proteins are recognized and bound by the FvPex7 receptor or the FvPex7-Pex20 receptor complex in the cytoplasm, but the peroxisome localization of the PTS2-Pex7-Pex20 complex is only determined by Pex20 in F. verticillioides. However, we observed that some putative PTS2 proteins that interact with Pex7 are not transported into the peroxisomes, but a PTS1 protein that interacts with Pex5 was detected in the peroxisomes. Furthermore, ΔFvpex7pex20 as well as ΔFvpex7pex5 double mutants exhibited reduced pathogenicity and FB1 biosynthesis, along with defects in conidiation. The PTS2 receptor complex mutants (ΔFvpex7pex20) grew slowly on minimal media and showed reduced sensitivity to cell wall and cell membrane stress-inducing agents compared to the wild type. Taken together, we conclude that the PTS2 receptor complex mediates peroxisome matrix proteins import and contributes to pathogenicity and FB1 biosynthesis in F. verticillioides. KEY POINTS: • FvPex7 directly interacts with FvPex20 in F. verticillioides. • vThe PTS2 receptor complex is essential for the importation of PTS2-containing matrix protein into peroxisomes in F. verticillioides. • Fvpex7/pex20 is involved in pathogenicity and FB1 biosynthesis in F. verticillioides.

Translating the Arabidopsis thaliana Peroxisome Proteome Insights to Solanum lycopersicum: Consensus Versus Diversity.

Peroxisomes are small, single-membrane specialized organelles present in all eukaryotic organisms. The peroxisome is one of the nodal centers of reactive oxygen species homeostasis in plants, which are generated in a high amount due to various stress conditions. Over the past decade, there has been extensive study on peroxisomal proteins and their signaling pathways in the model plant Arabidopsis thaliana, and a lot has been deciphered. However, not much impetus has been given to studying the peroxisome proteome of economically important crops. Owing to the significance of peroxisomes in the physiology of plants during normal and stress conditions, understating its proteome is of much importance. Hence, in this paper, we have made a snapshot of putative peroxisomal matrix proteins in the economically important vegetable crop tomato (Solanum lycopersicum, (L.) family Solanaceae). First, a reference peroxisomal matrix proteome map was generated for Arabidopsis thaliana using the available proteomic and localization studies, and proteins were categorized into various groups as per their annotations. This was used to create the putative peroxisomal matrix proteome map for S. lycopersicum. The putative peroxisome proteome in S. lycopersicum retains the basic framework: the bulk of proteins had peroxisomal targeting signal (PTS) type 1, a minor group had PTS2, and the catalase family retained its characteristic internal PTS. Apart from these, a considerable number of S. lycopersicum orthologs did not contain any “obvious” PTS. The number of PTS2 isoforms was found to be reduced in S. lycopersicum. We further investigated the PTS1s in the case of both the plant species and generated a pattern for canonical and non-canonical PTS1s. The number of canonical PTS1 proteins was comparatively lesser in S. lycopersicum. The non-canonical PTS1s were found to be comparable in both the plant species; however, S. lycopersicum showed greater diversity in the composition of the signal tripeptide. Finally, we have tried to address the lacunas and probable strategies to fill those gaps.

Two Pex5 Proteins With Different Cargo Specificity Are Critical for Peroxisome Function in Ustilago maydis.

Peroxisomes are dynamic multipurpose organelles with a major function in fatty acid oxidation and breakdown of hydrogen peroxide. Many proteins destined for the peroxisomal matrix contain a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by tetratricopeptide repeat (TPR) proteins of the Pex5 family. Various species express at least two different Pex5 proteins, but how this contributes to protein import and organelle function is not fully understood. Here, we analyzed truncated and chimeric variants of two Pex5 proteins, Pex5a and Pex5b, from the fungus Ustilago maydis. Both proteins are required for optimal growth on oleic acid-containing medium. The N-terminal domain (NTD) of Pex5b is critical for import of all investigated peroxisomal matrix proteins including PTS2 proteins and at least one protein without a canonical PTS. In contrast, the NTD of Pex5a is not sufficient for translocation of peroxisomal matrix proteins. In the presence of Pex5b, however, specific cargo can be imported via this domain of Pex5a. The TPR domains of Pex5a and Pex5b differ in their affinity to variations of the PTS1 motif and thus can mediate import of different subsets of matrix proteins. Together, our data reveal that U. maydis employs versatile targeting modules to control peroxisome function. These findings will promote our understanding of peroxisomal protein import also in other biological systems.

Pex7 selectively imports PTS2 target proteins to peroxisomes and is required for anthracnose disease development in Colletotrichum scovillei

Pex7 is a shuttling receptor that imports matrix proteins with a type 2 peroxisomal targeting signal (PTS2) to peroxisomes. The Pex7-mediated PTS2 protein import contributes to crucial metabolic processes such as the fatty acid β-oxidation and glucose metabolism in a number of fungi, but cellular roles of Pex7 between the import of PTS2 target proteins and metabolic processes have not been fully understood. In this study, we investigated the functional roles of CsPex7, a homolog of the yeast Pex7, by targeted gene deletion in the pepper anthracnose fungus Colletotrichum scovillei. CsPex7 was required for carbon source utilization, scavenging of reactive oxygen species, conidial production, and disease development in C. scovillei. The expression of fluorescently tagged PTS2 signal of hexokinases and 3-ketoacyl-CoA thiolases showed that peroxisomal localization of the hexokinase CsGlk1 PTS2 is dependent on CsPex7, but those of the 3-ketoacyl-CoA thiolases are independent on CsPex7. In addition, GFP-tagged CsPex7 proteins were intensely localized to the peroxisomes on glucose-containing media, indicating a role of CsPex7 in glucose utilization. Collectively, these findings indicate that CsPex7 selectively recognizes specific PTS2 signal for import of PTS2-containing proteins to peroxisomes, thereby mediating peroxisomal targeting efficiency of PTS2-containing proteins in C. scovillei. On pepper fruits, the ΔCspex7 mutant exhibited significantly reduced virulence, in which excessive accumulation of hydrogen peroxide was observed in the pepper cells. We think the reduced virulence results from the abnormality in hydrogen peroxide metabolism of the ΔCspex7 mutant. Our findings provide insight into the cellular roles of CsPex7 in PTS2 protein import system.

Peroxisomal PEX7 Receptor Affects Cadmium-Induced ROS and Auxin Homeostasis in Arabidopsis Root System

Peroxisomes are important in plant physiological functions and stress responses. Through the production of reactive oxygen and nitrogen species (ROS and RNS), and antioxidant defense enzymes, peroxisomes control cellular redox homeostasis. Peroxin (PEX) proteins, such as PEX7 and PEX5, recognize peroxisome targeting signals (PTS1/PTS2) important for transporting proteins from cytosol to peroxisomal matrix. pex7-1 mutant displays reduced PTS2 protein import and altered peroxisomal metabolism. In this research we analyzed the role of PEX7 in the Arabidopsis thaliana root system exposed to 30 or 60 μM CdSO4. Cd uptake and translocation, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) levels, and reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels and catalase activity were analyzed in pex7-1 mutant primary and lateral roots in comparison with the wild type (wt). The peroxisomal defect due to PEX7 mutation did not reduce Cd-uptake but reduced its translocation to the shoot and the root cell peroxisomal signal detected by 8-(4-Nitrophenyl) Bodipy (N-BODIPY) probe. The trend of nitric oxide (NO) and peroxynitrite in pex7-1 roots, exposed/not exposed to Cd, was as in wt, with the higher Cd-concentration inducing higher levels of these RNS. By contrast, PEX7 mutation caused changes in Cd-induced hydrogen peroxide (H2O2) and superoxide anion (O2●−) levels in the roots, delaying ROS-scavenging. Results show that PEX7 is involved in counteracting Cd toxicity in Arabidopsis root system by controlling ROS metabolism and affecting auxin levels. These results add further information to the important role of peroxisomes in plant responses to Cd.

Recombinant Production of a Novel Fusion Protein: Listeriolysin O Fragment Fused to S1 Subunit of Pertussis Toxin.

Background:Some resources have suggested that genetically inactivated PTs bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive PTS1 in a fusion form with N-terminal half of the LLO pore-forming toxin. Methods:Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results:The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion:The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future.

Evolutionary Maintenance of the PTS2 Protein Import Pathway in the Stramenopile Alga Nannochloropsis.

The stramenopile alga Nannochloropsis evolved by secondary endosymbiosis of a red alga by a heterotrophic host cell and emerged as a promising organism for biotechnological applications, such as the production of polyunsaturated fatty acids and biodiesel. Peroxisomes play major roles in fatty acid metabolism but experimental analyses of peroxisome biogenesis and metabolism in Nannochloropsis are not reported yet. In fungi, animals, and land plants, soluble proteins of peroxisomes are targeted to the matrix by one of two peroxisome targeting signals (type 1, PTS1, or type 2, PTS2), which are generally conserved across kingdoms and allow the prediction of peroxisomal matrix proteins from nuclear genome sequences. Because diatoms lost the PTS2 pathway secondarily, we investigated its presence in the stramenopile sister group of diatoms, the Eustigmatophyceae, represented by Nannochloropsis. We detected a full-length gene of a putative PEX7 ortholog coding for the cytosolic receptor of PTS2 proteins and demonstrated its expression in Nannochloropsis gaditana. The search for predicted PTS2 cargo proteins in N. gaditana yielded several candidates. In vivo subcellular targeting analyses of representative fusion proteins in different plant expression systems demonstrated that two predicted PTS2 domains were indeed functional and sufficient to direct a reporter protein to peroxisomes. Peroxisome targeting of the predicted PTS2 cargo proteins was further confirmed in Nannochloropsis oceanica by confocal and transmission electron microscopy. Taken together, the results demonstrate for the first time that one group of stramenopile algae maintained the import pathway for PTS2 cargo proteins. To comprehensively map and model the metabolic capabilities of Nannochloropsis peroxisomes, in silico predictions needs to encompass both the PTS1 and the PTS2 matrix proteome.

Membrane Processing and Steady-State Regulation of the Alternative Peroxisomal Import Receptor Pex9p.

Import of peroxisomal matrix proteins with a type 1 peroxisomal targeting signal (PTS1) in Saccharomyces cerevisiae is facilitated by cytosolic import receptors Pex5p and Pex9p. While Pex5p has a broad specificity for all PTS1 proteins independent of the growth conditions, Pex9p is only expressed in fatty-acid containing media to mediate peroxisomal import of the two malate synthases, Mls1p and Mls2p, as well as the glutathione transferase Gto1p. Pex5p-cargo complexes dock at the peroxisomal membrane, translocate their cargo-protein via a transient pore and are recycled into the cytosol for a further round of import. The processing of Pex5p has been shown to require a complex network of interactions with other membrane-bound peroxins, as well as decoration with ubiquitin as signal for its ATP-dependent release and recycling. Here, we show that the alternative receptor Pex9p requires the same set of interacting peroxins to mediate peroxisomal import of Mls1p. However, while Pex5p is rather stable, Pex9p is rapidly degraded during its normal life cycle. The steady-state regulation of Pex9p, combining oleate-induced expression with high turnover rates resembles that of Pex18p, one of the two co-receptors of the PTS2-dependent targeting pathway into peroxisomes. Both Pex9p- and Pex18p-dependent import routes serve the fast metabolic adaptation to changes of carbon sources in baker’s yeast. By sequence similarities, we identified another Pex9p homolog in the human pathogenic fungus Candida glabrata, in which similar metabolic reprogramming strategies are crucial for survival of the pathogen.

A novel dynein-type AAA+ protein with peroxisomal targeting signal type 2.

Peroxisomal matrix proteins are imported into peroxisomes in a process mediated by peroxisomal targeting signal (PTS) type 1 and 2. The PTS2 proteins are imported into peroxisomes after binding with Pex7p. Niwa et al. (A newly isolated Pex7-binding, atypical PTS2 protein P7BP2 is a novel dynein-type AAA+ protein. J Biochem 2018;164:437-447) identified a novel Pex7p-binding protein in CHO cells and characterized the subcellular distribution and molecular properties of the human homologue, 'P7BP2'. Interestingly, P7BP2 possesses PTS2 at the NH2 terminal and six putative AAA+ domains. Another group has suggested that the protein also possesses mitochondrial targeting signal at the NH2 terminal. In fact, the P7BP2 expressed in mammalian cells is targeted to both peroxisomes and mitochondria. The purified protein from Sf9 cells is a monomer and has a disc-like ring structure, suggesting that P7BP2 is a novel dynein-type AAA+ family protein. The protein expressed in insect cells exhibits ATPase activity. P7BP2 localizes to peroxisomes and mitochondria, and has a common function related to dynein-type ATPases in both organelles.

Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.

Prediction of Peroxisomal Matrix Proteins in Plants.

Our knowledge of the proteome of plant peroxisomes is far from being complete, and the functional complexity and plasticity of this cell organelle are amazingly high particularly in plants, as exemplified by the model species Arabidopsis thaliana. Plant-specific peroxisome functions that have been uncovered only recently include, for instance, the participation of peroxisomes in phylloquinone and biotin biosynthesis. Experimental proteome studies have been proved very successful in defining the proteome of Arabidopsis peroxisomes but this approach also faces significant challenges and limitations. Complementary to experimental approaches, computational methods have emerged as important powerful tools to define the proteome of soluble matrix proteins of plant peroxisomes. Compared to other cell organelles such as mitochondria, plastids and the ER, the simultaneous operation of two major import pathways for soluble proteins in peroxisomes is rather atypical. Novel machine learning prediction approaches have been developed for peroxisome targeting signals type 1 (PTS1) and revealed high sensitivity and specificity, as validated by in vivo subcellular targeting analyses in diverse transient plant expression systems. Accordingly, the algorithms allow the correct prediction of many novel peroxisome-targeted proteins from plant genome sequences and the discovery of additional organelle functions. In contrast, the prediction of PTS2 proteins largely remains restricted to genome searches by conserved patterns contrary to more advanced machine learning methods. Here, we summarize and discuss the capabilities and accuracies of available prediction algorithms for PTS1 and PTS2 carrying proteins.

Identification of d-amino acid oxidase and propiverine interaction partners and their potential role in the propiverine-mediated nephropathy

Propiverine, a frequently-prescribed pharmaceutical for the treatment of symptoms associated with overactive bladder syndrome, provoked massive intranuclear and cytosolic protein inclusions in rat proximal tubule epithelium, primarily consisting of the peroxisomal targeting signal 1 (PTS1) containing protein d-amino acid oxidase (DAAO). As this type of nephropathy was also observed for other drugs, the aim was to determine whether propiverine interferes with trafficking and/or import of peroxisomal proteins. To elucidate this, DAAO- and propiverine-specific interaction partners from human HEK293 and rat WKPT cell lines and rat kidney and liver homogenate were determined using co-immunoprecipitation with subsequent nano-ESI-LC-MS/MS analyses. Corroboration of the role of DAAO- and/or propiverine-specific interaction partners in the drug-induced DAAO accumulation was sought via specific immunofluorescence staining of rat kidney sections from control and propiverine-treated rats. Above analyses demonstrated the interaction of propiverine with several protein classes, foremost peroxisomal proteins (DAAO, MFE2, HAOX2) and proteins of the protein quality control system, i.e. chaperones (HSP70 and DnaJ co-chaperones), proteases and proteasomal proteins (regulatory subunits of the 26S proteasome; Rpn1/2). The immunofluorescence analysis revealed mislocalization of many PTS1-proteins (DAAO, CAT, MFE2, ACOX1, EHHADH) in rat renal sections, strongly suggesting that propiverine primarily binds to PTS1 proteins resulting in the formation of PTS1 but not PTS2 or peroxisomal membrane protein (PMP) accumulations. Moreover, chaperones involved in peroxisomal trafficking (HSC70, DnaJB1) and peroxisomal biogenesis factor proteins (PEX3, PEX5, PEX7), also presented with distinct mislocalization patterns. Concomitantly, an increased number of peroxisomes was observed, suggestive of a compensatory mechanism for the presumably suboptimally functioning peroxisomes.Overall, the data presented suggested that propiverine interacts exclusively with DAAO or with a selected number of PTS1 proteins. The consequence of this interaction is the abrogated trafficking and peroxisomal import of PTS1 proteins concomitant with their nuclear and cytosolic accumulation due to inhibited degradation and imbalanced protein homeostasis.

PPero, a Computational Model for Plant PTS1 Type Peroxisomal Protein Prediction

Well-defined motifs often make it easy to investigate protein function and localization. In plants, peroxisomal proteins are guided to peroxisomes mainly by a conserved type 1 (PTS1) or type 2 (PTS2) targeting signal, and the PTS1 motif is commonly used for peroxisome targeting protein prediction. Currently computational prediction of peroxisome targeted PTS1-type proteins are mostly based on the 3 amino acids PTS1 motif and the adjacent sequence which is less than 14 amino acid residue in length. The potential contribution of the adjacent sequences beyond this short region has never been well investigated in plants. In this work, we develop a bi-profile Bayesian SVM method to extract and learn position-based amino acid features for both PTS1 motifs and their extended adjacent sequences in plants. Our proposed model outperformed other implementations with similar applications and achieved the highest accuracy of 93.6% and 92.6% for Arabidosis and other plant species respectively. A large scale analysis for Arabidopsis, Rice, Maize, Potato, Wheat, and Soybean proteome was conducted using the proposed model and a batch of candidate PTS1 proteins were predicted. The DNA segments corresponding to the C-terminal sequences of 9 selected candidates were cloned and transformed into Arabidopsis for experimental validation, and 5 of them demonstrated peroxisome targeting.

Distinct Pores for Peroxisomal Import of PTS1 and PTS2 Proteins.

Two peroxisomal targeting signals, PTS1 and PTS2, recognized by cytosolic receptors Pex5 and cooperating Pex7/Pex18, direct folded proteins to the peroxisomal matrix. A pore consisting of the PTS1 receptor Pex5 and the docking protein Pex14 imports PTS1 proteins. We identified a distinct PTS2-specific pore, which contains the PTS2 co-receptor Pex18 and the Pex14/Pex17-docking complex as major constituents. The estimated maximal pore size of ∼ 4.7 nm is large enough to allow import of folded PTS2 proteins. PTS2 cargo proteins modulate complex gating, open probability, and subconductance states of the pore. While the PTS1 channel is transiently activated by arriving receptor-cargo complexes, the reconstituted PTS2 channel is constitutively present in an open state. However, the cargo-loaded PTS2 channel is largely impermeable to solutes and ions. Our results demonstrate that import of PTS1 and PTS2 proteins does not converge at the peroxisomal membrane as previously anticipated but is performed by distinct pores.

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

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.

Revisiting the intraperoxisomal pathway of mammalian PEX7.

Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix.

Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis

Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins, Is a Redox‐Sensitive Protein

Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5

In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1-110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.

Identification of a novel missense mutation of PEX7 gene in an Iranian patient with rhizomelic chondrodysplasia punctata type 1

Deficiency in the PTS2 protein import pathway due to mutations in PEX7 gene results in the rhizomelic chondrodysplasia punctata (RCDP) type 1. In the present study, we have reported a novel missense mutation, W75R, in the PEX7 gene in an Iranian patient with the RCDP type 1. The inability of PEX7 protein to transport PTS2 containing proteins including peroxisomal 3-ketoacyl-CoA thiolase and PTS2-EGFP protein to the surface of the peroxisomes showed that the W75R mutation in PEX7 gene severely impaired the function of PEX7 protein and was responsible for RCDP type 1 in this patient.