FpPEX5 and FpPEX7 are involved in the growth, reproduction, DON toxin production, and pathogenicity in Fusarium pseudograminearum.

Fusarium crown rot, caused by Fusarium pseudograminearum, is a devastating disease affecting the yield and quality of cereal crops. Peroxisomes are single-membrane organelles that play a critical role in various biological processes in eukaryotic cells. To functionally characterise peroxisome biosynthetic receptor proteins FpPEX5 and FpPEX7 in F. pseudograminearum, we constructed deletion mutants, ΔFpPEX5 and ΔFpPEX7, and complementary strains, ΔFpPEX5-C and ΔFpPEX7-C, and analysed the functions of FpPEX5 and FpPEX7 proteins using various phenotypic observations. The deletion of FpPEX5 and FpPEX7 resulted in a significant deficiency in mycelial growth and conidiation and blocked the peroxisomal targeting signal 1 and peroxisomal targeting signal 2 pathways, which are involved in peroxisomal matrix protein transport, increasing the accumulation of lipid droplets and reactive oxygen species. The deletion of FpPEX5 and FpPEX7 may reduce the formation of toxigenic bodies and decrease the pathogenicity of F. pseudograminearum. These results indicate that FpPEX5 and FpPEX7 play vital roles in the growth, asexual reproduction, virulence, and fatty acid utilisation of F. pseudograminearum. This study provides a theoretical basis for controlling stem rot in wheat.

Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Identification of Leishmania donovani PEX5-PTS1 Interaction Inhibitors through Fluorescence Polarization-Based High-Throughput Screening.

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 µM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds.

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.

Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.

Peroxisome biogenesis initiated by protein phase separation.

Peroxisomes are organelles that carry out ß-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction1. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts2, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes3. Current models postulate a large pore formed by transmembrane proteins4; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS5,6. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels7.

Estimating the Interaction Strength Between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytometer-Based FRET (flowFRET) Measurements.

The import of many peroxisomal matrix proteins is initiated by the interaction of type-1 peroxisomal targeting signals (PTS1) residing at the extreme C-terminus of cargo proteins and their receptor protein PEX5. This interaction has been amply investigated by biophysical methods using isolated proteins and peptides or heterologous systems such as two-hybrid assays. However, a recently developed novel application of Fluorescence resonance energy transfer (FRET) allows a quantifying measurement of this interaction in living cells. This method combines the systematic measurement of FRET-efficiency in a high number of cells with a well-suited normalization protocol and a fitting algorithm, which together allow the estimation of numerical values for the apparent interaction strength that correlates with other measures of binding strength but can be obtained under rather physiological conditions.

Cell-free reconstitution of peroxisomal matrix protein import using Xenopus egg extract.

Peroxisomes are vital metabolic organelles whose matrix enzymes are imported from the cytosol in a folded state by the soluble receptor PEX5. The import mechanism has been challenging to decipher because of the lack of suitable in vitro systems. Here, we present a protocol for reconstituting matrix protein import using Xenopus egg extract. We describe how extract is prepared, how to replace endogenous PEX5 with recombinant versions, and how to perform and interpret a peroxisomal import reaction using a fluorescent cargo. For complete details on the use and execution of this protocol, please refer to Skowyra and Rapoport (2022).1.

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.

The Extraction Mechanism of Monoubiquitinated PEX5 from the Peroxisomal Membrane.

The AAA ATPases PEX1•PEX6 extract PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane so that a new protein transport cycle can start. Extraction requires ubiquitination of PEX5 at residue 11 and involves a threading mechanism, but how exactly this occurs is unclear. We used a cell-free in vitro system and a variety of engineered PEX5 and ubiquitin molecules to challenge the extraction machinery. We show that PEX5 modified with a single ubiquitin is a substrate for extraction and extend previous findings proposing that neither the N- nor the C-terminus of PEX5 are required for extraction. Chimeric PEX5 molecules possessing a branched polypeptide structure at their C-terminal domains can still be extracted from the peroxisomal membrane thus suggesting that the extraction machinery can thread more than one polypeptide chain simultaneously. Importantly, we found that the PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and, accordingly, an intra-molecularly cross-linked ubiquitin blocked extraction when conjugated to residue 11 of PEX5. Collectively, our data suggest that the PEX5-linked monoubiquitin is the extraction initiator and that the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6.

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.

Normal plasmalogen levels are maintained in tissues from mice with hepatocyte-specific deletion in peroxin 5.

On the basis of findings that cultured rat hepatocytes secrete lipoprotein with a high plasmalogen content and the occurrence of this lipid in human serum, it has been suggested that hepatocytes play a role in the supply of plasmalogens to tissues. We tested this hypothesis in a mouse with a hepatocyte-specific defect in peroxisomes, an organelle essentially required for plasmalogen biosynthesis. We analyzed plasmalogens in lipid extracts of forebrain, liver and five further tissues and in plasma by reaction with dansylhydrazine in hydrochloric acid, which cleaves the vinyl ether of plasmalogens and forms a fluorescent dansylhydrazone, which we quantified by reversed phase high performance liquid chromatography. Reaction with dansylhydrazine in acetic acid was used to quantify free aldehydes as a control. Our results show normal levels of plasmalogens in plasma and in all tissues examined, including forebrain and the liver, irrespective of the inactivation of hepatic peroxisomes. None of the selected ether lipids analyzed by mass spectrometry in plasma and liver was decreased in the mice deficient in liver peroxisomes. In contrast, we found three plasmenylcholine species which were even significantly increased in the livers of these animals. Quantification of mRNA expression of plasmalogen biosynthetic enzymes revealed particularly low expression of fatty acyl-CoA reductase, the key regulatory enzyme of plasmalogen biosynthesis, in liver, with and without hepatic peroxisome deficiency. Our results do not support the suggested role of hepatocytes in supplying plasmalogens to tissues.

Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer.

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1-335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1-335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3-4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1-335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9.

Accurate and regulated protein targeting is crucial for cellular function and proteostasis. In the yeast Saccharomyces cerevisiae, peroxisomal matrix proteins, which harboring a Peroxisomal Targeting Signal 1 (PTS1), can utilize two paralog targeting factors, Pex5 and Pex9, to target correctly. While both proteins are similar and recognize PTS1 signals, Pex9 targets only a subset of Pex5 cargo proteins. However, what defines this substrate selectivity remains uncovered. Here, we used unbiased screens alongside directed experiments to identify the properties underlying Pex9 targeting specificity. We find that the specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is explained by structural modeling of the PTS1-binding cavities of the two factors showing differences in their surface hydrophobicity. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes.

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

Dynamics of the translocation pore of the human peroxisomal protein import machinery.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported in a posttranslational manner. Intricate protein import machineries have evolved that catalyze the different stages of translocation. In humans, PEX5L was found to be an essential component of the peroxisomal translocon. PEX5L is the main receptor for substrate proteins carrying a peroxisomal targeting signal (PTS). Substrates are bound by soluble PEX5L in the cytosol after which the cargo-receptor complex is recruited to peroxisomal membranes. Here, PEX5L interacts with the docking protein PEX14 and becomes part of an integral membrane protein complex that facilitates substrate translocation into the peroxisomal lumen in a still unknown process. In this study, we show that PEX5L containing complexes purified from human peroxisomal membranes constitute water-filled pores when reconstituted into planar-lipid membranes. Channel characteristics were highly dynamic in terms of conductance states, selectivity and voltage- and substrate-sensitivity. Our results show that a PEX5L associated pore exists in human peroxisomes, which can be activated by receptor-cargo complexes.

Structure and function of the peroxisomal ubiquitin ligase complex.

Peroxisomes are membrane-bounded organelles that exist in most eukaryotic cells and are involved in the oxidation of fatty acids and the destruction of reactive oxygen species. Depending on the organism, they house additional metabolic reactions that range from glycolysis in parasitic protozoa to the production of ether lipids in animals and antibiotics in fungi. The importance of peroxisomes for human health is revealed by various disorders - notably the Zellweger spectrum - that are caused by defects in peroxisome biogenesis and are often fatal. Most peroxisomal metabolic enzymes reside in the lumen, but are synthesized in the cytosol and imported into the organelle by mobile receptors. The receptors accompany cargo all the way into the lumen and must return to the cytosol to start a new import cycle. Recycling requires receptor monoubiquitination by a membrane-embedded ubiquitin ligase complex composed of three RING finger (RF) domain-containing proteins: PEX2, PEX10, and PEX12. A recent cryo-electron microscopy (cryo-EM) structure of the complex reveals its function as a retro-translocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that assemble into an open channel. The N terminus of a receptor likely inserts into the pore from the lumenal side, and is then monoubiquitinated by one of the RFs to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitinated by the concerted action of the other two RFs and ultimately degraded. The new data provide mechanistic insight into a crucial step of peroxisomal protein import.

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.

Small molecule mediated inhibition of protein cargo recognition by peroxisomal transport receptor PEX5 is toxic to Trypanosoma.

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5-PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5-PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.

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.