Identification and Validation of a PEX5-Dependent Signature for Prognostic Prediction in Glioma.

Gliomas, the most prevalent and lethal form of brain cancer, are known to exhibit metabolic alterations that facilitate tumor growth, invasion, and resistance to therapies. Peroxisomes, essential organelles responsible for fatty acid oxidation and reactive oxygen species (ROS) homeostasis, rely on the receptor PEX5 for the import of metabolic enzymes into their matrix. However, the prognostic significance of peroxisomal enzymes for glioma patients remains unclear. In this study, we elucidate that PEX5 is indispensable for the cell growth, migration, and invasion of glioma cells. We establish a robust prognosis model based on the expression of peroxisomal enzymes, whose localization relies on PEX5. This PEX5-dependent signature not only serves as a robust prognosis model capable of accurately predicting outcomes for glioma patients, but also effectively distinguishes several clinicopathological features, including the grade, isocitrate dehydrogenase (IDH) mutation, and 1p19q codeletion status. Furthermore, we developed a nomogram that integrates the prognostic model with other clinicopathological factors, demonstrating highly accurate performance in estimating patient survival. Patients classified into the high-risk group based on our prognostic model exhibited an immunosuppressive microenvironment. Finally, our validation reveals that the elevated expression of GSTK1, an antioxidant enzyme within the signature, promotes the cell growth and migration of glioma cells, with this effect dependent on the peroxisomal targeting signal recognized by PEX5. These findings identify the PEX5-dependent signature as a promising prognostic tool for gliomas.

Loss of pex5 sensitizes zebrafish to fasting due to deregulated mitochondria, mTOR, and autophagy.

Animal models have been utilized to understand the pathogenesis of Zellweger spectrum disorders (ZSDs); however, the link between clinical manifestations and molecular pathways has not yet been clearly established. We generated peroxin 5 homozygous mutant zebrafish (pex5-/-) to gain insight into the molecular pathogenesis of peroxisome dysfunction. pex5-/- display hallmarks of ZSD in humans and die within one month after birth. Fasting rapidly depletes lipids and glycogen in pex5-/- livers and expedites their mortality. Mechanistically, deregulated mitochondria and mechanistic target of rapamycin (mTOR) signaling act together to induce metabolic alterations that deplete hepatic nutrients and accumulate damaged mitochondria. Accordingly, chemical interventions blocking either the mitochondrial function or mTOR complex 1 (mTORC1) or a combination of both improve the metabolic imbalance shown in the fasted pex5-/- livers and extend the survival of animals. In addition, the suppression of oxidative stress by N-acetyl L-cysteine (NAC) treatment rescued the apoptotic cell death and early mortality observed in pex5-/-. Furthermore, an autophagy activator effectively ameliorated the early mortality of fasted pex5-/-. These results suggest that fasting may be detrimental to patients with peroxisome dysfunction, and that modulating the mitochondria, mTORC1, autophagy activities, or oxidative stress may provide a therapeutic option to alleviate the symptoms of peroxisomal diseases associated with metabolic dysfunction.

Circular RNA Sec61 subunit alpha isoform 1 by competitive absorption of microRNA-513a-5p mediates peroxisomal biogenesis factor 5 expression and promotes the malignant phenotype of non-small cell lung cancer.

Circular RNAs (circRNAs) are functional RNAs in the development and metabolism of non-small cell lung cancer (NSCLC). Therein, this paper particularly elucidated the circRNA SEC61 subunit alpha isoform 1 (circSEC61A1) in NSCLC has not been fully elucidated. Clinical analysis of circSEC61A1 expression was performed on specimens collected from 51 patients with primary NSCLC, together with patients' survival. Cell experiments were performed after interfering with circSEC61A1, microRNA (miR)-513a-5p, and peroxisomal biogenesis factor 5 (PEX5) expression, respectively, and cell malignant phenotypes and aerobic glycolysis were evaluated, as well as epithelial-to-mesenchymal transition (EMT)-related markers and Wnt/ß-catenin pathway. Xenografts experiments studied the performance of circSEC61A1 in vivo. The downstream molecules of circSEC61A1 were searched. Our data demonstrated that circSEC61A1 was upregulated in NSCLC patients, showing an association with poorer survival outcomes. In cell experiments, circSEC61A1 overexpression promoted NSCLC malignant phenotypes, glycolysis, EMT, and Wnt/ß-catenin pathway activation, whereas circSEC61A1 underexpression did the opposite. Knockdown of circSEC61A1 limited tumor growth and metastasis. Furthermore, circSEC61A1 could regulate PEX5 expression through competitive absorption of miR-513a-5p. Generally, circSEC61A1 is a potential biomarker for NSCLC, and circSEC61A1 serves tumor-promoting action in the progression of NSCLC.

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.

PEX5 translocation into and out of peroxisomes drives matrix protein import.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes and releases cargo inside the lumen is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in the unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

Insights into the critical role of the PXR in preventing carcinogenesis and chemotherapeutic drug resistance.

Pregnane x receptor (PXR) as a nuclear receptor is well-established in drug metabolism, however, it has pleiotropic functions in regulating inflammatory responses, glucose metabolism, and protects normal cells against carcinogenesis. Most studies focus on its transcriptional regulation, however, PXR can regulate gene expression at the translational level. Emerging evidences have shown that PXR has a broad protein-protein interaction network, by which is implicated in the cross signaling pathways. Furthermore, the interactions between PXR and some critical proteins (e.g., p53, Tip60, p300/CBP-associated factor) in DNA damage pathway highlight its potential roles in this field. A thorough understanding of how PXR maintains genome stability and prevents carcinogenesis will help clinical diagnosis and finally benefit patients. Meanwhile, due to the regulation of CYP450 enzymes CYP3A4 and multidrug resistance protein 1 (MDR1), PXR contributes to chemotherapeutic drug resistance. It is worthy of note that the co-factor of PXR such as RXRα, also has contributions to this process, which makes the PXR-mediated drug resistance more complicated. Although single nucleotide polymorphisms (SNPs) vary between individuals, the amino acid substitution on exon of PXR finally affects PXR transcriptional activity. In this review, we have summarized the updated mechanisms that PXR protects the human body against carcinogenesis, and major contributions of PXR with its co-factors have made on multidrug resistance. Furthermore, we have also reviewed the current promising antagonist and their clinic applications in reversing chemoresistance. We believe our review will bring insight into PXR-targeted cancer therapy, enlighten the future study direction, and provide substantial evidence for the clinic in future.

Stop Codon Context-Specific Induction of Translational Readthrough.

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)-the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.

PEX5 prevents cardiomyocyte hypertrophy via suppressing the redox-sensitive signaling pathways MAPKs and STAT3.

Peroxisomal biogenesis factor 5 (PEX5) is a member of peroxisome biogenesis protein family which serves as a shuttle receptor for the import of peroxisome matrix protein. The function of PEX5 on cardiomyocyte hypertrophy remained to be elucidated. Our study demonstrated that the protein expression level of PEX5 was declined in primary neonatal rat cardiomyocytes treated with phenylephrine (PE) and hearts from cardiac hypertrophic rats induced by abdominal aortic constriction (AAC). Overexpression of PEX5 alleviated cardiomyocyte hypertrophy induced by PE, while silencing of PEX5 exacerbated cardiomyocyte hypertrophy. PEX5 improved redox imbalance by decreasing cellular reactive oxygen species level and preserving peroxisomal catalase. Moreover, PEX5 knockdown aggravated PE-induced activation of redox-sensitive signaling pathways, including mitogen-activated protein kinase (MAPK) pathway and signal transducer and activator of transcription 3 (STAT3); whereas PEX5 overexpression suppressed activation of MAPK and STAT3. But PEX5 did not affect PE-induced phosphorylation of mammalian target of rapamycin (mTOR). In conclusion, the present study suggests that PEX5 protects cardiomyocyte against hypertrophy via regulating redox homeostasis and inhibiting redox-sensitive signaling pathways MAPK and STAT3.

PXR-mediated expression of FABP4 promotes valproate-induced lipid accumulation in HepG2 cells.

Valproate (valproic acid, VPA) is widely used in the therapy of epilepsy. However, adverse effect like hepatic steatosis has been reported in patients receiving VPA treatment. But whether nuclear receptor pregnane X receptor (PXR) and fatty acid binding protein 4 (FABP4) are involved in the regulation of VPA-induced steatosis or not is still unknown. In this study, the roles of PXR and FABP4 in VPA-induced lipid accumulation in HepG2 cells were investigated. We found that the expression of PXR and FABP4 were increased by VPA in a dose-dependent manner. Knockdown of PXR not only reduced lipid accumulation but also impaired the induction of FABP4 by VPA. While overexpression of PXR enhanced both lipid accumulation and FABP4 expression. Moreover, exogenous expression of FABP4 increased triglyceride levels and enhanced lipid accumulation caused by VPA. Taken together, these results suggest that PXR-mediated expression of FABP4 is responsible for lipid accumulation caused by VPA.

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility.

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

A missense allele of PEX5 is responsible for the defective import of PTS2 cargo proteins into peroxisomes.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery.

Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.

PEX5, a novel target of microRNA-31-5p, increases radioresistance in hepatocellular carcinoma by activating Wnt/ß-catenin signaling and homologous recombination.

Rationale: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with high recurrence and metastasis rates. Although radiation is an effective treatment for tumors, it is often limited by intrinsic radioresistance in HCC. The contributions of dysregulated microRNAs, including miR-31-5p, to HCC progression have been recently reported. However, the role of miR-31-5p in the radiation response of HCC is unknown. In this study, we aimed to investigate the impact of miR-31-5p on HCC radiosensitivity. Methods: miR-31-5p expression in HCC tissues, paired adjacent tissues, and HCC cell lines was measured using quantitative real-time polymerase chain reaction and in situ hybridization. Bioinformatic analyses, gain- and loss-of-function experiments, and luciferase reporter assays were performed to validate peroxisomal biogenesis factor 5 (PEX5) as a direct target of miR-31-5p. The biofunctions of PEX5 and miR-31-5p in HCC were determined by Transwell, wound-healing, and Cell Counting Kit-8 (CCK8) assays. A colony formation assay was used to evaluate the radiosensitivity of HCC cells. The interaction among PEX5, ß-catenin, Rac1, and JNK-2 was confirmed by coimmunoprecipitation. A xenograft tumor model was established to validate the effects of miR-31-5p and PEX5 on HCC progression and radiosensitivity in vivo.Results: Low expression of miR-31-5p in HCC specimens, as observed in this study, predicted a poor clinical outcome. However, the expression pattern of PEX5, as a direct target of miR-31-5p, was opposite that of miR-31-5p, and high PEX5 expression indicated poor prognosis in HCC patients. Ectopic expression of PEX5 increased the proliferation, migration, and invasion abilities and enhanced the radioresistance of HCC cells in vitro and in vivo; however, these phenotypes were inhibited by miR-31-5p. Mechanistically, PEX5 stabilized cytoplasmic ß-catenin and facilitated ß-catenin nuclear translocation to activate Wnt/ß-catenin signaling. Moreover, upon radiation exposure, PEX5 reduced excessive reactive oxygen species (ROS) accumulation and activated the homologous recombination (HR) pathway, which protected HCC cells from radiation-induced damage. Conclusions: Our findings demonstrated a novel role for PEX5 as a miR-31-5p target and a mediator of the Wnt/ß-catenin signaling and HR pathways, providing new insights into studying HCC radiation responses and implicating PEX5 and miR-31-5p as potential therapeutic targets in HCC.

Nobiletin, sinensetin, and tangeretin are the main perpetrators in clementines provoking food-drug interactions in vitro.

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.

Spatiotemporal contact between peroxisomes and lipid droplets regulates fasting-induced lipolysis via PEX5.

Lipid droplets (LDs) are key subcellular organelles for regulating lipid metabolism. Although several subcellular organelles participate in lipid metabolism, it remains elusive whether physical contacts between subcellular organelles and LDs might be involved in lipolysis upon nutritional deprivation. Here, we demonstrate that peroxisomes and peroxisomal protein PEX5 mediate fasting-induced lipolysis by stimulating adipose triglyceride lipase (ATGL) translocation onto LDs. During fasting, physical contacts between peroxisomes and LDs are increased by KIFC3-dependent movement of peroxisomes toward LDs, which facilitates spatial translocations of ATGL onto LDs. In addition, PEX5 could escort ATGL to contact points between peroxisomes and LDs in the presence of fasting cues. Moreover, in adipocyte-specific PEX5-knockout mice, the recruitment of ATGL onto LDs was defective and fasting-induced lipolysis is attenuated. Collectively, these data suggest that physical contacts between peroxisomes and LDs are required for spatiotemporal translocation of ATGL, which is escorted by PEX5 upon fasting, to maintain energy homeostasis.

OsPEX5 regulates rice spikelet development through modulating jasmonic acid biosynthesis.

Spikelet is the primary reproductive structure and a critical determinant of grain yield in rice. The molecular mechanisms regulating rice spikelet development still remain largely unclear. Here, we report that mutations in OsPEX5, which encodes a peroxisomal targeting sequence 1 (PTS1) receptor protein, cause abnormal spikelet morphology. We show that OsPEX5 can physically interact with OsOPR7, an enzyme involved in jasmonic acid (JA) biosynthesis and is required for its import into peroxisome. Similar to Ospex5 mutant, the knockout mutant of OsOPR7 generated via CRISPR-Cas9 technology has reduced levels of endogenous JA and also displays an abnormal spikelet phenotype. Application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7. Furthermore, we show that OsMYC2 directly binds to the promoters of OsMADS1, OsMADS7 and OsMADS14 to activate their expression, and subsequently regulate spikelet development. Our results suggest that OsPEX5 plays a critical role in regulating spikelet development through mediating peroxisomal import of OsOPR7, therefore providing new insights into regulation of JA biosynthesis in plants and expanding our understanding of the biological role of JA in regulating rice reproduction.

Peroxisomes control mitochondrial dynamics and the mitochondrion-dependent apoptosis pathway.

Peroxisomes cooperate with mitochondria in the performance of cellular metabolic functions, such as fatty acid oxidation and the maintenance of redox homeostasis. However, whether peroxisomes also regulate mitochondrial fission-fusion dynamics or mitochondrion-dependent apoptosis remained unclear. We now show that genetic ablation of the peroxins Pex3 or Pex5, which are essential for peroxisome biogenesis, results in mitochondrial fragmentation in mouse embryonic fibroblasts (MEFs) in a manner dependent on Drp1 (also known as DNM1L). Conversely, treatment with 4-PBA, which results in peroxisome proliferation, resulted in mitochondrial elongation in wild-type MEFs, but not in Pex3-knockout MEFs. We further found that peroxisome deficiency increased the levels of cytosolic cytochrome c and caspase activity under basal conditions without inducing apoptosis. It also greatly enhanced etoposide-induced caspase activation and apoptosis, which is indicative of an enhanced cellular sensitivity to death signals. Taken together, our data unveil a previously unrecognized role for peroxisomes in the regulation of mitochondrial dynamics and mitochondrion-dependent apoptosis. Effects of peroxin gene mutations on mitochondrion-dependent apoptosis may contribute to pathogenesis of peroxisome biogenesis disorders.This article has an associated First Person interview with the first author of the paper.

Peroxisome protein import recapitulated in Xenopus egg extracts.

Peroxisomes import their luminal proteins from the cytosol. Most substrates contain a C-terminal Ser-Lys-Leu (SKL) sequence that is recognized by the receptor Pex5. Pex5 binds to peroxisomes via a docking complex containing Pex14, and recycles back into the cytosol following its mono-ubiquitination at a conserved Cys residue. The mechanism of peroxisome protein import remains incompletely understood. Here, we developed an in vitro import system based on Xenopus egg extracts. Import is dependent on the SKL motif in the substrate and on the presence of Pex5 and Pex14, and is sustained by ATP hydrolysis. A protein lacking an SKL sequence can be coimported, providing strong evidence for import of a folded protein. The conserved cysteine in Pex5 is not essential for import or to clear import sites for subsequent rounds of translocation. This new in vitro assay will be useful for further dissecting the mechanism of peroxisome protein import.

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import.

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed. In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.

Distinct Roles for Peroxisomal Targeting Signal Receptors Pex5 and Pex7 in Drosophila.

Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX, or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. We characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle, similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts.