{"gene":"PEX5","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2000,"finding":"Crystal structure of human PEX5 C-terminal fragment containing all seven TPR motifs in complex with a PTS1 pentapeptide revealed that two clusters of three TPRs almost completely surround the peptide, while a hinge region (TPR4) forms a distinct structure enabling the two sets of TPRs to form a single binding site, establishing the molecular basis for PTS1 recognition via a novel TPR-peptide interaction mode.","method":"X-ray crystallography (crystal structure of PEX5 TPR domain in complex with PTS1 peptide)","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with direct functional validation of binding mechanism","pmids":["11101887"],"is_preprint":false},{"year":1999,"finding":"Recombinant human PEX5 forms homotetramers (both PEX5L and PEX5S isoforms) and binds the N-terminal fragment of PEX14 (residues 1-78) with very high affinity in the low nanomolar range. Stable in vitro complexes revealed that PEX5 possesses multiple PEX14-binding sites distributed throughout its N-terminal half, while the C-terminal TPR half binds PTS1 proteins. A pentapeptide motif reiterated seven times in PEX5 was identified as determinant for PEX14 interaction.","method":"Surface plasmon resonance, sizing chromatography, electron microscopy, in vitro binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal biophysical methods (SPR, EM, in vitro reconstitution) in a single study","pmids":["10026185"],"is_preprint":false},{"year":1998,"finding":"PEX5 (PTS1R) is required for import of both PTS1 proteins and PTS2 proteins in CHO cells; the longer isoform PTS1RL is specifically required for PTS2 import, while both isoforms mediate PTS1 import. Missense mutations in TPR1 (G298E) and TPR6 (G485E) of PTS1RS abolished PTS1 import, demonstrating functional importance of TPR domains in protein translocation.","method":"CHO cell mutant complementation, reverse transcription-PCR mutation analysis, import assays in PEX5-defective cell lines","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation with defined mutations, replicated across two CHO mutant cell lines","pmids":["9418886"],"is_preprint":false},{"year":2001,"finding":"The seven di-aromatic pentapeptide repeat motifs (WX(E/D/Q/A/S)(E/D/Q)(F/Y)) in the N-terminal half of human PEX5 each independently bind the same site in the N-terminus of PEX14 with nanomolar affinity. Mutational analysis showed conserved aromatic amino acids at positions 1 and 5 of each motif are essential for high-affinity PEX14 binding, likely forming hydrophobic anchors in an amphipathic alpha-helix.","method":"Two-hybrid analysis in mammalian cells, in vitro binding assays, mutational analysis, surface plasmon resonance","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutational analysis combined with SPR and two orthogonal binding assays","pmids":["11438541"],"is_preprint":false},{"year":2001,"finding":"Human PEX5L (but not PEX5S) physically interacts with PEX7 and is required for PTS2 protein import in mammalian cells. The region amino acids 191-222 of PEX5L is sufficient for PEX7 interaction and amino acids 1-214 are sufficient for peroxisome targeting. A conserved 21-amino-acid motif (aa 209-229) shared with yeast Pex18p/Pex21p is required for both PEX7 interaction and PTS2 import; a serine mutation in this motif abolishes PTS2 import and reduces PEX5L-PEX7 interaction in vitro.","method":"Domain mapping, in vitro binding, complementation in mammalian cells, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple deletion/mutant constructs, in vitro interaction, and in vivo functional rescue","pmids":["11546814"],"is_preprint":false},{"year":1999,"finding":"PEX12 zinc-binding domain interacts with PEX5 and PEX10; a patient missense mutation S320F in PEX12 reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation. PEX12 and PEX10 act downstream of PEX5 docking at the peroxisome surface, not in the docking step itself.","method":"Two-hybrid studies, blot overlay assays, co-immunoprecipitation, genetic suppression by overexpression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods (Y2H, overlay, Co-IP, genetic epistasis) establishing pathway position","pmids":["10562279"],"is_preprint":false},{"year":2000,"finding":"Disruption of the Pex5pL-Pex7p interaction by the S214F missense mutation (adjacent to the PEX5L-specific 37-amino acid insertion) completely abolishes PTS2 protein import in mammals while leaving PTS1 import intact, demonstrating that the Pex5pL-Pex7p interaction is essential specifically for PTS2 import.","method":"CHO cell mutant isolation, complementation with mutant Pex5p constructs, co-immunoprecipitation, import assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — defined missense mutation, reciprocal import and binding assays, confirmed in novel cell mutant","pmids":["10767287"],"is_preprint":false},{"year":2005,"finding":"PEX5 import into peroxisomes is ATP-independent, whereas its export back to the cytosol requires ATP. PEX1 and PEX6 (AAA ATPases) and their recruiter PEX26 are essential for PEX5 export. PEX14 is required for PEX5 docking/import. DTM-embedded PEX5 exists in two distinct complexes of ~500 kDa and ~800 kDa comprising different peroxins (including PEX14 and PEX2), indicating PEX5 transits between subcomplexes during its cycle.","method":"Cell-free translocation system, radiolabeled PEX5 import/export assays with isolated peroxisomes, blue-native PAGE, PEX-defective cell lines","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — cell-free reconstitution with multiple peroxin-defective cell lines and orthogonal biochemical fractionation","pmids":["16314507"],"is_preprint":false},{"year":2009,"finding":"The N-terminal domain of PEX14 adopts a three-helical fold and binds both PEX5 (via its WxxxF/Y motif) and PEX19 competitively at the same surface but with opposite directionality. Mutations of PEX14 residues in the PEX5/PEX19 binding region disrupt binding in vitro and impair peroxisomal membrane localization of PEX14 in vivo.","method":"NMR structure determination, competitive binding assays, mutagenesis, in vivo localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with functional mutagenesis and in vivo validation","pmids":["19197237"],"is_preprint":false},{"year":2012,"finding":"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11 in human) forming a thioester conjugate (Ub-PEX5). USP9X is the primary deubiquitinase acting on Ub-PEX5 in rat liver and HeLa cells, identified by biochemical fractionation; USP9X is an elongated monomeric protein capable of hydrolyzing thioester, isopeptide, and peptide bonds.","method":"Biochemical fractionation, in vitro deubiquitinase assays, identification by mass spectrometry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — unbiased biochemical identification plus enzymatic characterization, replicated in two cell types","pmids":["22371489"],"is_preprint":false},{"year":2013,"finding":"PEX5 binds monomeric catalase (not tetrameric catalase) through domains in both its N- and C-terminal halves, potently inhibiting catalase tetramerization. The PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, with one or two of the seven PEX14-binding diaromatic motifs in PEX5 involved, indicating PEX14 participates in cargo release.","method":"In vitro binding assays, native gel electrophoresis, interaction disruption assays with PEX14 N-terminal domain","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution with multiple complementary assays, single lab","pmids":["21976670"],"is_preprint":false},{"year":2013,"finding":"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11); this modification is mandatory for ATP-dependent extraction of PEX5 from the peroxisomal membrane back to the cytosol. Cargo protein translocation across the peroxisomal membrane occurs upstream of PEX5 ubiquitination (i.e., prior to the first ATP-dependent step).","method":"In vitro import/export system with rat liver peroxisomes, PEGylation assays to monitor cysteine modification, protease protection assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution with defined biochemical intermediates, single lab","pmids":["23963456"],"is_preprint":false},{"year":2013,"finding":"Cys11 of human PEX5 functions as a redox switch: exposure to oxidized glutathione yields a ubiquitination-deficient PEX5, impairing PTS1 import. Substitution of Cys11 by lysine counteracts this effect. Oxidative stress selectively inhibits import of catalase (non-canonical PTS1) more than canonical PTS1 reporters. PEX5 does not oligomerize in cellulo, and oxidative stress does not affect PEX5-substrate binding per se.","method":"PEGylation assays, site-directed mutagenesis (C11K), live-cell import assays in human fibroblasts, redox manipulation","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis combined with functional import assays and redox manipulation, replicated across papers (PMIDs 24118911 and 28760655)","pmids":["24118911","28760655"],"is_preprint":false},{"year":2017,"finding":"TRIM37 localizes to peroxisomal membranes and ubiquitylates PEX5 at K464 via interaction with the C-terminal 51 amino acids (CT51) of PEX5; this monoubiquitination stabilizes PEX5 by preventing its proteasomal degradation. TRIM37 depletion or K464A/ΔCT51 PEX5 mutations reduce PEX5 abundance and impair PTS protein import.","method":"Co-immunoprecipitation, ubiquitylation assays, site-directed mutagenesis, RNAi knockdown, import assays in human cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro ubiquitylation, mutagenesis, and functional import readout in a single study","pmids":["28724525"],"is_preprint":false},{"year":2013,"finding":"In Pichia pastoris, Pex5 functions as a dimer/oligomer whose homo- and hetero-oligomeric interactions (with Pex8 via its N-terminal domain, aa 1-110) regulate cargo binding and release. Cysteine 10 of Pex5 forms redox-sensitive disulfide-linked oligomers with highest PTS1 cargo affinity; DTT reduction causes transition to noncovalent dimer and partial cargo release. Interaction of Pex5 N-terminal domain with a conserved C-terminal motif of Pex8 further facilitates cargo release under reducing conditions.","method":"In vitro binding assays, disulfide crosslinking, dithiothreitol reduction, mutagenesis (C10), import assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods but primarily in yeast (Pichia pastoris), single lab","pmids":["23902771"],"is_preprint":false},{"year":2013,"finding":"A novel PEX5-PEX14 interaction site in PEX5 was identified with the sequence LVAEF (consensus LVXEF). NMR structure of Pex5-(57-71) with PEX14 N-terminal domain showed this motif binds in an alpha-helical orientation similar to WxxxF/Y motifs but with leucine occupying the tryptophan pocket. SPR showed 33-fold faster dissociation than WxxxF/Y motifs; substituting this site with a higher-affinity WxxxF/Y motif impaired protein import, indicating distinct kinetics of this novel site are required for receptor processing.","method":"Peptide library screening, ligand blot analysis, NMR structure determination, SPR, in vivo import assays with alanine substitution mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure, SPR kinetics, and in vivo functional assay in one study","pmids":["24235149"],"is_preprint":false},{"year":2018,"finding":"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, establishing that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 AAA ATPase complex.","method":"Cell-free in vitro system, photoaffinity cross-linking, protein PEGylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution with photoaffinity cross-linking and PEGylation as orthogonal readouts, single lab","pmids":["29884772"],"is_preprint":false},{"year":2022,"finding":"PEX5 accompanies cargo completely into the peroxisomal lumen (not just to the membrane). WxxxF/Y motifs near PEX5's N-terminus bind a lumenal domain of the docking complex inside the peroxisome. An amphipathic helix in PEX5 initiates recycling by binding the lumenal side of the ubiquitin ligase. The N-terminus of PEX5 then emerges in the cytosol for monoubiquitination, and finally PEX5 is extracted from the lumen with unfolding of the receptor and cargo release.","method":"Xenopus egg extract import system, domain deletion/mutation analysis, protease protection and biochemical assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution in Xenopus extract with multiple mechanistic readouts, establishes lumenal translocation mechanism","pmids":["35931083"],"is_preprint":false},{"year":2017,"finding":"The peroxisomal matrix protein translocon (DTM) is a large cavity-forming protein assembly into which cytosolic PEX5 enters to release its cargo. Truncated PEX5 molecules up to residue 197 can be accommodated in excess. PEX5-PEX14 interaction within the DTM is stable at pH 11.5, indicating DTM-bound PEX5 resistance to alkaline extraction does not reflect direct lipid bilayer contact.","method":"Truncated PEX5 molecules as DTM probes, proteinase K accessibility assays, alkaline extraction assays, in vitro binding","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical approaches but mechanistic conclusions are partly inferential, single lab","pmids":["28765278"],"is_preprint":false},{"year":2015,"finding":"PEX5 proteins fused to bulky C-terminal tags trigger peroxisome degradation by autophagy in mammalian cells. This requires monoubiquitination of the N-terminal cysteine of PEX5 (Cys11), which marks PEX5 for recycling. The C-terminal tag does not inhibit monoubiquitination but blocks PEX5 export from the peroxisomal membrane, indicating that prolonged membrane-association of monoubiquitinated PEX5 triggers pexophagy as a quality control mechanism.","method":"Fluorescence microscopy, autophagy inhibitors, mutant PEX5 constructs (C11A abolishes monoubiquitination), quantitative pexophagy assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic dissection with site-directed mutagenesis and pharmacological autophagy inhibition, single lab","pmids":["26086376"],"is_preprint":false},{"year":2011,"finding":"AWP1/ZFAND6 stimulates PEX5 export from peroxisomes. AWP1 interacts with PEX6 AAA ATPase (but not Pex1-Pex6 complexes) and preferentially binds cysteine-ubiquitinated PEX5 over unmodified PEX5 via its A20 zinc-finger domain. AWP1 knockdown reduces PTS1 protein import and destabilizes PEX5, similarly to defects in PEX1/PEX6/PEX26.","method":"Biochemical fractionation, in vitro Pex5 export assay, recombinant protein stimulation assay, antibody inhibition, RNAi knockdown, co-immunoprecipitation","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro reconstitution assay, biochemical identification, RNAi, multiple orthogonal assays","pmids":["21980954"],"is_preprint":false},{"year":2014,"finding":"High-resolution structural analysis revealed that PEX5 adapts its conformation during PTS1 cargo recognition: the receptor binding cavity shrinks to one-third of its original volume upon ligand binding (ligand-induced compaction). A single-residue mutation in cargo protein alanine-glyoxylate aminotransferase that removes steric occlusion increases peroxisomal import efficiency from 34% to 80%.","method":"X-ray crystallography of PEX5-cargo complexes, import efficiency assays in cells","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with quantitative in vivo import assay, single lab","pmids":["25369882"],"is_preprint":false},{"year":2022,"finding":"The PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and serves as the extraction initiator; the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6. Chimeric PEX5 molecules with branched C-terminal polypeptide structures can still be extracted, suggesting simultaneous threading of more than one polypeptide chain. Intra-molecularly cross-linked ubiquitin conjugated to residue 11 of PEX5 blocks extraction, confirming ubiquitin unfolding is required.","method":"Cell-free in vitro extraction system, engineered PEX5 and ubiquitin molecules, cross-linked ubiquitin, PEGylation assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution with chemically defined substrates and mutagenesis controls","pmids":["36442669"],"is_preprint":false},{"year":2024,"finding":"PEX5 ubiquitinated at cysteine 11 (thioester bond) cannot retain a polyubiquitin chain because cysteine-linked ubiquitination is reversible, with E2-mediated deubiquitination being faster than polyubiquitination. A Lys11 PEX5 variant is polyubiquitinated at the peroxisomal membrane, impairing extraction. Thus, cysteine as the ubiquitin acceptor prevents polyubiquitination and proteasomal targeting while enabling reversible monoubiquitination for efficient recycling.","method":"Rat liver cell-free in vitro system, site-directed mutagenesis (C11K), ubiquitin chain analysis, extraction assays","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free reconstitution with defined mutants, mechanistic dissection of ubiquitination reversibility","pmids":["38470934"],"is_preprint":false},{"year":2018,"finding":"Chemically synthesized monoubiquitinated PEX5 (via click chemistry) binds PEX7/PTS2 complex and can restore PTS2 protein import in ΔPEX5 fibroblasts in vivo. In vitro pull-downs showed monoubiquitinated PEX5 interacts with PEX13, PEX14 (independent of ubiquitination status) and with REM components PEX1, PEX6, and PEX26 (interactions enhanced by ubiquitination).","method":"Chemical ubiquitin conjugation (click chemistry), in vitro pull-down assays, complementation in ΔPEX5 fibroblasts","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — chemically defined monoubiquitinated substrate used in both in vitro and in vivo assays","pmids":["30375424"],"is_preprint":false},{"year":2013,"finding":"Absence of PEX5 in CHO cells (ZPEG101 mutant) results in unstable PEX14 due to its inefficient translocation to the peroxisomal membrane, establishing that PEX5 stabilizes PEX14 in addition to its import receptor role. The fifth WXXXF/Y pentapeptide motif in PEX5L is an auxiliary PEX14-binding site required for PEX14 stability. PEX5-PEX13 interaction is essential for PTS1 and catalase import but not PTS2 import.","method":"PEX5-deficient CHO cell mutant (ZPEG101), complementation with Pex5p mutants, western blotting for PEX14 stability, import assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel cell mutant with complementation analysis and defined motif mapping, single lab","pmids":["23009329"],"is_preprint":false},{"year":2020,"finding":"PEX5 mediates fasting-induced lipolysis by escorting adipose triglyceride lipase (ATGL) to contact points between peroxisomes and lipid droplets. During fasting, peroxisomes move toward lipid droplets in a KIFC3-dependent manner, increasing peroxisome-lipid droplet contacts, and PEX5 facilitates ATGL translocation onto lipid droplets. In adipocyte-specific PEX5-knockout mice, ATGL recruitment to lipid droplets was defective and fasting-induced lipolysis was attenuated.","method":"Co-immunoprecipitation, fluorescence microscopy, adipocyte-specific conditional knockout mice, lipolysis assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO mice with quantitative lipolysis phenotype, co-IP, and live imaging of organelle contacts","pmids":["31996685"],"is_preprint":false},{"year":2009,"finding":"Cargo protein translocation across the peroxisomal membrane (including release into the matrix) occurs prior to PEX5 ubiquitination in a cargo-protein-centered in vitro import system, mapping translocation downstream of reversible docking and upstream of the first cytosolic ATP-dependent step.","method":"Cargo-protein-centered in vitro peroxisomal import system, ATP depletion experiments, protease protection assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free import reconstitution with defined biochemical intermediates, consistent with companion paper (PMID 23963456)","pmids":["19632994"],"is_preprint":false},{"year":2021,"finding":"Loss of the PEX5 long isoform (PEX5L) alone causes selective deficiency in PTS2 protein import (not PTS1 import), resulting in rhizomelic chondrodysplasia punctata type 5 (RCDP5) in humans. A frameshift mutation c.722dupA in PEX5L-specific exon 9 abolishes PEX5L expression; reintroduction of PEX5L restores PTS2 import in patient fibroblasts.","method":"Patient mutation analysis, isoform-specific expression studies, PTS1/PTS2 import assays in fibroblasts, complementation with PEX5L","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetics linked to cellular mechanism, complementation confirms isoform-specific PTS2 import function","pmids":["26220973"],"is_preprint":false},{"year":2021,"finding":"PEX5 mutation F218S (missense) allows normal PTS1 import, normal entry into the DTM, normal monoubiquitination and export, but fails to form a stable trimeric complex with PEX7 and a PTS2 cargo protein, thus failing to promote PTS2 protein import, causing congenital cataracts.","method":"In vitro import assays, co-immunoprecipitation, site-directed mutagenesis, patient fibroblast functional studies","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 / Moderate — defined mutation with mechanistic dissection of PTS1 vs PTS2 import steps using in vitro and cell-based assays","pmids":["33389129"],"is_preprint":false},{"year":2022,"finding":"Full-length human PEX5L is monomeric in solution with a compact conformation, spontaneously binds to lipid bilayers (accumulating ~100-fold), and forms ion-conducting membrane channels in artificial horizontal bilayers. The C-terminal cargo-binding domain (residues 336–639) is required for pore formation; truncated PEX5L(1-335) binds membranes but does not form channels, suggesting PEX5L is the pore-forming component of the peroxisomal translocon.","method":"Fluorescence TCSPC (diffusion measurement), electrophysiological single-channel recording in horizontal lipid bilayers, site-specific fluorescent labeling","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology in defined bilayer system with truncation analysis, single lab","pmids":["36260915"],"is_preprint":false},{"year":2021,"finding":"PEX5 NTD harbors multiple membrane interaction sites involving amphipathic alpha-helical regions that include WxxxF/Y motifs; these helical regions are stabilized in the presence of membrane-mimicking bicelles (NMR). PEX14 NTD weakly interacts with bicelles at a surface that partially overlaps with the WxxxF/Y binding site. The PEX5-PEX14 interaction is largely unaffected by membrane presence (similar binding enthalpies with entropy compensation), indicating membrane docking does not reduce overall PEX5-PEX14 binding affinity.","method":"NMR spectroscopy with bicelles and nanodiscs, isothermal titration calorimetry (ITC)","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR and ITC are rigorous biophysical methods but single lab, functional consequences not directly tested in vivo","pmids":["33937250"],"is_preprint":false},{"year":2025,"finding":"Pex8 in yeast is essential for peroxisomal cargo translocation regardless of receptor/cargo recognition mechanism. Pex8 binds through a 12-fold HEAT repeat array to a short three-helical bundle in the N-terminal domain of Pex5; impairing this interaction abolishes peroxisomal protein translocation. A secondary autonomous Pex8 cargo-like interaction site exists at the C-terminal domain of Pex5, generating a bipartite interaction. The Pex5/Pex8 complex assembly enables association with the Pex2/Pex10/Pex12 E3-ubiquitin ligase complex to initiate receptor recycling.","method":"Cryo-electron microscopy structure of Pex5-Pex8 complex, mutagenesis, functional import assays in yeast","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with functional mutagenesis, but preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.08.30.673231"],"is_preprint":true},{"year":2025,"finding":"Saccharomyces cerevisiae Eci1 can reach peroxisomes and bind Pex5 in the absence of a canonical PTS1 signal. Cryo-EM structure of yeast Pex5-Eci1 complex identified additional binding interfaces beyond the canonical PTS1-TPR interaction, revealing that some cargoes use non-canonical interaction surfaces for Pex5-mediated targeting.","method":"Cryo-electron microscopy, import assays in yeast pex5 mutants lacking PTS1","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with functional import validation, single study","pmids":["40376748"],"is_preprint":false},{"year":2023,"finding":"PEX13 loss causes accumulation of ubiquitinated PEX5 on peroxisomes and increased peroxisome-dependent ROS, both of which induce pexophagy. PEX13 protein levels are downregulated during amino acid starvation to aid pexophagy induction, establishing PEX13 as a regulator that prevents pexophagy of healthy peroxisomes by controlling ubiquitinated PEX5 accumulation.","method":"CRISPR gene editing, quantitative fluorescence microscopy, zebrafish model, autophagy assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells and zebrafish with multiple readouts, single lab","pmids":["36541703"],"is_preprint":false},{"year":2009,"finding":"Solution SAXS analysis of human Pex5-Pex14-PTS1 complexes revealed a 1:6 stoichiometry for Pex5:Pex14 complex. Free full-length Pex5 is monomeric in solution with an elongated, partially unfolded N-terminal domain. In the complex, the Pex5 N-terminus remains extended, with Pex14 significantly intermingled with the Pex5 moiety.","method":"Small-angle X-ray scattering (SAXS), static light scattering, titration studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — SAXS low-resolution structural data, single lab, no mutagenesis validation","pmids":["19584060"],"is_preprint":false},{"year":2003,"finding":"Full-length tetrameric PEX5 binds PTS1 (lissamine-Tyr-Gln-Ser-Lys-Leu-COO-) with Kd of 35 nM. Neither Hsp70 (with or without ATP/ADP) nor the PEX12 zinc RING domain has a detectable effect on PEX5-PTS1 binding kinetics, indicating initial cargo recognition by PEX5 is a relatively simple process not regulated by these factors.","method":"Fluorescence anisotropy binding assay, purified recombinant full-length PEX5, Hsp70 addition experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro binding assay with purified proteins; negative results for Hsp70/PEX12 regulation are well-controlled","pmids":["12456682"],"is_preprint":false}],"current_model":"PEX5 is a shuttling import receptor that recognizes PTS1-containing peroxisomal matrix proteins in the cytosol via its C-terminal TPR domain (crystal structure determined), docks at the peroxisomal membrane through multiple WxxxF/Y motifs in its N-terminal domain that bind PEX14 with nanomolar affinity, and then enters completely into the peroxisomal lumen to release cargo; the receptor is subsequently monoubiquitinated at a conserved N-terminal cysteine (Cys11) by a peroxisomal membrane-embedded E3 ligase complex, and this monoubiquitinated PEX5 is recognized and extracted from the lumen by the AAA ATPase complex PEX1-PEX6 through a threading/unfolding mechanism, with USP9X serving as the primary cytosolic deubiquitinase; the longer isoform PEX5L additionally recruits PEX7 (via a specific 21-aa motif) to co-import PTS2 cargo proteins, TRIM37 stabilizes PEX5 via a separate lysine monoubiquitination, and the redox state of Cys11 acts as a switch coupling oxidative stress to import activity."},"narrative":{"mechanistic_narrative":"PEX5 is the cycling import receptor that delivers folded peroxisomal matrix proteins from the cytosol into the peroxisomal lumen [PMID:9418886, PMID:19632994]. It recognizes PTS1-bearing cargo through its C-terminal TPR domain, where two clusters of three TPRs encircle the PTS1 pentapeptide and the receptor binding cavity compacts upon ligand engagement to enforce selectivity [PMID:11101887, PMID:25369882]. Cargo recognition is a comparatively simple, high-affinity event (Kd ~35 nM) not modulated by Hsp70 or the PEX12 RING domain [PMID:12456682]. The elongated N-terminal half carries seven di-aromatic WxxxF/Y pentapeptide motifs, each binding the three-helical N-terminal domain of the docking peroxin PEX14 with nanomolar affinity, plus a distinct lower-affinity LVxEF site whose fast dissociation kinetics are themselves required for receptor processing [PMID:10026185, PMID:11438541, PMID:19197237, PMID:24235149]. The longer isoform PEX5L additionally recruits PEX7 through a conserved 21-residue motif to co-import PTS2 cargo, a function genetically separable from PTS1 import [PMID:11546814, PMID:10767287]; defects in this PEX5L–PEX7 interaction cause rhizomelic chondrodysplasia punctata type 5 and congenital cataracts [PMID:26220973, PMID:33389129]. Rather than acting only at the membrane, PEX5 enters the lumen of the import translocon (DTM), releases cargo with the participation of the PEX14 N-terminal domain, and is then retrieved [PMID:21976670, PMID:35931083, PMID:28765278]. Recycling is initiated by monoubiquitination of a conserved N-terminal cysteine (Cys11) as a reversible thioester conjugate; this modification is mandatory for the ATP-dependent extraction of PEX5 from the membrane by the PEX1–PEX6 AAA-ATPase, which threads and globally unfolds the receptor together with its unfolded ubiquitin tag [PMID:23963456, PMID:29884772, PMID:36442669, PMID:38470934]. The cysteine acceptor is itself a redox switch coupling glutathione oxidation to import competence, and USP9X is the principal cytosolic deubiquitinase that regenerates free PEX5 [PMID:22371489, PMID:24118911, PMID:28760655]. Beyond matrix import, PEX5 stabilizes PEX14, escorts adipose triglyceride lipase to peroxisome–lipid droplet contacts to drive fasting-induced lipolysis, and—when its export is blocked—accumulates as ubiquitinated PEX5 to trigger pexophagy as an organelle quality-control mechanism [PMID:23009329, PMID:31996685, PMID:26086376].","teleology":[{"year":1998,"claim":"Established PEX5 as the genetic determinant of peroxisomal matrix import and revealed isoform-specific division of labor between PTS1 and PTS2 cargo.","evidence":"CHO mutant complementation and TPR missense mutations in PEX5-defective cells","pmids":["9418886"],"confidence":"High","gaps":["Molecular basis of PTS1 recognition not yet structurally defined","Mechanism by which the long isoform mediates PTS2 import unknown"]},{"year":1999,"claim":"Defined PEX5 architecture as bifunctional — an N-terminal half bearing reiterated PEX14-binding motifs and a C-terminal cargo-binding half — and quantified the nanomolar PEX5–PEX14 docking interaction.","evidence":"SPR, sizing chromatography, EM, and in vitro reconstitution of recombinant PEX5 and PEX14","pmids":["10026185"],"confidence":"High","gaps":["Number and individual contributions of the PEX14 sites not resolved","Oligomeric state in vivo uncertain"]},{"year":2000,"claim":"Solved the structural basis of PTS1 recognition, showing two TPR clusters surround the peptide via a hinge-enabled single binding site.","evidence":"X-ray crystallography of the PEX5 TPR domain with a PTS1 pentapeptide","pmids":["11101887"],"confidence":"High","gaps":["Conformational dynamics of cargo loading/release not addressed","Full-length receptor architecture not captured"]},{"year":2001,"claim":"Defined the molecular grammar of receptor docking and the PEX5L-specific PEX7 recruitment motif that underlies PTS2 import.","evidence":"Two-hybrid, SPR, mutational analysis of WxxxF/Y motifs; domain mapping of the PEX5L 21-aa PEX7-binding motif","pmids":["11438541","11546814"],"confidence":"High","gaps":["Why seven redundant PEX14 sites are needed unresolved","Functional difference between sites not yet defined"]},{"year":2005,"claim":"Separated import (ATP-independent) from export (ATP-dependent), assigning PEX1/PEX6/PEX26 to receptor retrieval and showing PEX5 transits between membrane subcomplexes.","evidence":"Cell-free translocation/export assays with isolated peroxisomes, blue-native PAGE, peroxin-defective cells","pmids":["16314507"],"confidence":"High","gaps":["Molecular trigger linking docking to export not defined","Composition of the 500/800 kDa complexes incomplete"]},{"year":2009,"claim":"Ordered the import cycle by placing cargo translocation across the membrane upstream of the first cytosolic ATP-dependent step, and provided solution architecture of the docking complex.","evidence":"Cargo-centered cell-free import system with ATP depletion and protease protection; SAXS of Pex5–Pex14–PTS1 complexes","pmids":["19632994","19584060"],"confidence":"High","gaps":["Mechanism of translocation across the bilayer unknown","Stoichiometry interpretation limited by SAXS resolution"]},{"year":2009,"claim":"Defined the PEX14 N-terminal fold and showed PEX5 and PEX19 compete for the same surface, integrating receptor docking with membrane-protein biogenesis.","evidence":"NMR structure with competitive binding and in vivo localization","pmids":["19197237"],"confidence":"High","gaps":["Functional consequence of PEX5/PEX19 competition in vivo not fully dissected"]},{"year":2012,"claim":"Identified Cys11 monoubiquitination as a thioester conjugate and USP9X as the cytosolic deubiquitinase regenerating free PEX5.","evidence":"Biochemical fractionation, in vitro DUB assays, mass spectrometry in rat liver and HeLa","pmids":["22371489"],"confidence":"High","gaps":["E3 ligase responsible for Cys11 conjugation not identified here","Regulation of USP9X activity unknown"]},{"year":2013,"claim":"Demonstrated that Cys11 monoubiquitination is mandatory for ATP-dependent extraction and that Cys11 acts as a redox switch coupling oxidative state to import competence, with selective effects on catalase.","evidence":"Cell-free import/export with PEGylation; C11K mutagenesis and redox manipulation in fibroblasts","pmids":["23963456","24118911","28760655"],"confidence":"High","gaps":["How redox state is sensed in vivo not fully resolved","Physiological oxidant relevant to the switch undefined"]},{"year":2013,"claim":"Mechanistically linked PEX5 to cargo release and to a non-WxxxF/Y processing site, and to PEX14 stabilization.","evidence":"In vitro catalase tetramerization assays; NMR/SPR of the LVxEF site; CHO mutant complementation for PEX14 stability","pmids":["21976670","24235149","23009329"],"confidence":"High","gaps":["Catalase work is single-lab in vitro","PEX14-stabilization findings Medium-confidence and single-lab"]},{"year":2014,"claim":"Showed ligand-induced compaction of the TPR cavity and that cargo steric features tune import efficiency.","evidence":"X-ray crystallography of PEX5–cargo complexes with in-cell import efficiency assays","pmids":["25369882"],"confidence":"High","gaps":["Generality of compaction across diverse cargoes untested","Single-lab"]},{"year":2015,"claim":"Connected receptor recycling failure to organelle quality control, showing stalled monoubiquitinated PEX5 triggers pexophagy.","evidence":"Fluorescence microscopy, autophagy inhibitors, C11A mutants in mammalian cells","pmids":["26086376"],"confidence":"High","gaps":["Autophagy receptor recognizing stalled Ub-PEX5 not identified","Single-lab"]},{"year":2017,"claim":"Characterized the import translocon as a large cavity (DTM) into which PEX5 enters, and identified TRIM37-mediated K464 monoubiquitination as a PEX5-stabilizing modification distinct from Cys11.","evidence":"Truncated PEX5 DTM probes with proteinase K/alkaline extraction; Co-IP, in vitro ubiquitylation, RNAi, import assays","pmids":["28765278","28724525"],"confidence":"High","gaps":["DTM mechanistic conclusions partly inferential","Interplay between TRIM37 K464 and Cys11 ubiquitination unresolved"]},{"year":2018,"claim":"Established DTM-embedded Ub-PEX5 as a direct PEX1–PEX6 substrate that is globally unfolded during extraction, and defined the interaction map of chemically defined monoubiquitinated PEX5 with docking and export machinery.","evidence":"Cell-free system with photoaffinity cross-linking/PEGylation; click-chemistry monoubiquitinated PEX5 pull-downs and complementation","pmids":["29884772","30375424"],"confidence":"High","gaps":["Force-generation and threading directionality mechanistically incomplete","Single-lab reconstitutions"]},{"year":2020,"claim":"Extended PEX5 function beyond matrix import to fasting-induced lipolysis by escorting ATGL to peroxisome–lipid droplet contacts in vivo.","evidence":"Co-IP, live imaging, adipocyte-specific PEX5-knockout mice with lipolysis assays","pmids":["31996685"],"confidence":"High","gaps":["Whether this uses the canonical TPR cargo pathway unclear","Molecular basis of ATGL recognition undefined"]},{"year":2021,"claim":"Linked PEX5L-specific PTS2 import defects to human disease and dissected step-specific mutations that uncouple PTS1 from PTS2 import.","evidence":"Patient mutation analysis (c.722dupA; F218S), import assays, Co-IP, complementation in fibroblasts","pmids":["26220973","33389129"],"confidence":"High","gaps":["Tissue-specific consequences of PTS2 loss not mechanistically explained"]},{"year":2022,"claim":"Resolved the complete lumenal import-and-recycling itinerary and the ubiquitin-unfolding requirement that initiates threading-based extraction.","evidence":"Xenopus egg extract import system; cell-free extraction with engineered/cross-linked ubiquitin and chimeric PEX5","pmids":["35931083","36442669"],"confidence":"High","gaps":["Identity of the lumenal docking/ligase contacts incompletely mapped","How multiple chains are threaded simultaneously unresolved"]},{"year":2023,"claim":"Placed PEX13 as a regulator preventing pexophagy of healthy peroxisomes by limiting accumulation of ubiquitinated PEX5.","evidence":"CRISPR KO, quantitative microscopy, zebrafish, autophagy assays","pmids":["36541703"],"confidence":"Medium","gaps":["Single-lab","Direct link between PEX13 levels and Ub-PEX5 turnover correlative"]},{"year":2024,"claim":"Explained why cysteine, not lysine, is the ubiquitin acceptor: reversible thioester ubiquitination prevents polyubiquitination and proteasomal targeting, enabling efficient recycling.","evidence":"Rat liver cell-free system, C11K mutagenesis, ubiquitin chain and extraction analysis","pmids":["38470934"],"confidence":"High","gaps":["In vivo balance of E2-mediated deubiquitination versus chain extension not quantified"]},{"year":2025,"claim":"Provided structural basis for the Pex5–Pex8 interaction in yeast that couples cargo translocation to recruitment of the Pex2/Pex10/Pex12 ligase, and revealed non-canonical cargo interaction surfaces.","evidence":"Cryo-EM of Pex5–Pex8 (preprint) and Pex5–Eci1 complexes with functional import assays in yeast","pmids":["bio_10.1101_2025.08.30.673231","40376748"],"confidence":"Medium","gaps":["One source is a preprint","Whether the Pex8-dependent step generalizes to mammals (which lack a clear Pex8) unknown"]},{"year":null,"claim":"The physical nature of the translocation pore and how the receptor and cargo cross the bilayer remain incompletely defined.","evidence":"PEX5L membrane channel activity is reported but the in vivo pore identity is unresolved","pmids":[],"confidence":"Medium","gaps":["Whether PEX5L itself forms the physiological translocon pore is not established in vivo","The complete subunit composition and gating of the DTM channel are unknown","How cargo of varying size and fold is accommodated remains undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[0,2,21,36]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,6,26]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[30]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,9,11]},{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[7,17,18]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[26]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[2,7,17,27]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,16,22,23]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[19,34]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[28,29]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[26]}],"complexes":["DTM (peroxisomal matrix protein translocon)","PEX1-PEX6 AAA-ATPase complex (substrate)","PEX5L-PEX7 receptor complex"],"partners":["PEX14","PEX7","PEX13","PEX12","PEX1","PEX6","USP9X","TRIM37"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P50542","full_name":"Peroxisomal targeting signal 1 receptor","aliases":["PTS1-BP","Peroxin-5","Peroxisomal C-terminal targeting signal import receptor","Peroxisome receptor 1"],"length_aa":639,"mass_kda":70.9,"function":"Receptor that mediates peroxisomal import of proteins containing a C-terminal PTS1-type tripeptide peroxisomal targeting signal (SKL-type) (PubMed:11101887, PubMed:11336669, PubMed:12456682, PubMed:16314507, PubMed:17157249, PubMed:17428317, PubMed:21976670, PubMed:26344566, PubMed:7706321, PubMed:7719337, PubMed:7790377). Binds to cargo proteins containing a PTS1 peroxisomal targeting signal in the cytosol, and translocates them into the peroxisome matrix by passing through the PEX13-PEX14 docking complex along with cargo proteins (PubMed:12456682, PubMed:17157249, PubMed:21976670, PubMed:26344566). PEX5 receptor is then retrotranslocated into the cytosol, leading to release of bound cargo in the peroxisome matrix, and reset for a subsequent peroxisome import cycle (PubMed:11336669, PubMed:24662292) In addition to promoting peroxisomal translocation of proteins containing a PTS1 peroxisomal targeting signal, mediates peroxisomal import of proteins containing a C-terminal PTS2-type peroxisomal targeting signal via its interaction with PEX7 (PubMed:11336669, PubMed:11546814, PubMed:25538232, PubMed:33389129, PubMed:9668159). Interaction with PEX7 only takes place when PEX7 is associated with cargo proteins containing a PTS2 peroxisomal targeting signal (PubMed:25538232). PEX7 along with PTS2-containing cargo proteins are then translocated through the PEX13-PEX14 docking complex together with PEX5 (PubMed:25538232) Does not mediate translocation of peroxisomal import of proteins containing a C-terminal PTS2-type peroxisomal targeting signal","subcellular_location":"Cytoplasm, cytosol; Peroxisome matrix","url":"https://www.uniprot.org/uniprotkb/P50542/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX5","classification":"Not Classified","n_dependent_lines":57,"n_total_lines":1208,"dependency_fraction":0.04718543046357616},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PEX5","total_profiled":1310},"omim":[{"mim_id":"621410","title":"PEROXISOME BIOGENESIS FACTOR 39; PEX39","url":"https://www.omim.org/entry/621410"},{"mim_id":"616716","title":"RHIZOMELIC CHONDRODYSPLASIA PUNCTATA, TYPE 5; RCDP5","url":"https://www.omim.org/entry/616716"},{"mim_id":"616154","title":"PEROXISOMAL FATTY ACYL-CoA REDUCTASE 1 DISORDER; PFCRD","url":"https://www.omim.org/entry/616154"},{"mim_id":"611058","title":"PEROXISOME BIOGENESIS FACTOR 5-LIKE; PEX5L","url":"https://www.omim.org/entry/611058"},{"mim_id":"607622","title":"PHOSPHOMEVALONATE KINASE; PMVK","url":"https://www.omim.org/entry/607622"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PEX5"},"hgnc":{"alias_symbol":["PTS1R"],"prev_symbol":["PXR1"]},"alphafold":{"accession":"P50542","domains":[{"cath_id":"1.25.40.10","chopping":"330-399","consensus_level":"medium","plddt":95.7083,"start":330,"end":399},{"cath_id":"1.25.40.10","chopping":"417-441_459-518","consensus_level":"medium","plddt":91.6894,"start":417,"end":518},{"cath_id":"1.25.40.10","chopping":"523-589_598-639","consensus_level":"medium","plddt":94.1368,"start":523,"end":639}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P50542","model_url":"https://alphafold.ebi.ac.uk/files/AF-P50542-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P50542-F1-predicted_aligned_error_v6.png","plddt_mean":69.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PEX5","jax_strain_url":"https://www.jax.org/strain/search?query=PEX5"},"sequence":{"accession":"P50542","fasta_url":"https://rest.uniprot.org/uniprotkb/P50542.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P50542/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P50542"}},"corpus_meta":[{"pmid":"11101887","id":"PMC_11101887","title":"Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.","date":"2000","source":"Nature structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/11101887","citation_count":313,"is_preprint":false},{"pmid":"11583975","id":"PMC_11583975","title":"Mitochondrial alterations caused by defective peroxisomal biogenesis in a mouse model for Zellweger syndrome (PEX5 knockout mouse).","date":"2001","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/11583975","citation_count":178,"is_preprint":false},{"pmid":"9418886","id":"PMC_9418886","title":"Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.","date":"1998","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9418886","citation_count":177,"is_preprint":false},{"pmid":"16314507","id":"PMC_16314507","title":"Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16314507","citation_count":175,"is_preprint":false},{"pmid":"10026185","id":"PMC_10026185","title":"Recombinant human peroxisomal targeting signal receptor PEX5. 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Study with a novel Pex5-impaired Chinese hamster ovary cell mutant.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10767287","citation_count":91,"is_preprint":false},{"pmid":"17317787","id":"PMC_17317787","title":"Glaucoma-causing myocilin mutants require the Peroxisomal targeting signal-1 receptor (PTS1R) to elevate intraocular pressure.","date":"2007","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17317787","citation_count":90,"is_preprint":false},{"pmid":"22371489","id":"PMC_22371489","title":"Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22371489","citation_count":84,"is_preprint":false},{"pmid":"31996685","id":"PMC_31996685","title":"Spatiotemporal contact between peroxisomes and lipid droplets regulates fasting-induced lipolysis via PEX5.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31996685","citation_count":80,"is_preprint":false},{"pmid":"26086376","id":"PMC_26086376","title":"Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.","date":"2015","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/26086376","citation_count":80,"is_preprint":false},{"pmid":"21976670","id":"PMC_21976670","title":"PEX5 protein binds monomeric catalase blocking its tetramerization and releases it upon binding the N-terminal domain of PEX14.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21976670","citation_count":74,"is_preprint":false},{"pmid":"19197237","id":"PMC_19197237","title":"Structural basis for competitive interactions of Pex14 with the import receptors Pex5 and Pex19.","date":"2009","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/19197237","citation_count":73,"is_preprint":false},{"pmid":"24118911","id":"PMC_24118911","title":"PEX5, the shuttling import receptor for peroxisomal matrix proteins, is a redox-sensitive protein.","date":"2013","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/24118911","citation_count":71,"is_preprint":false},{"pmid":"28724525","id":"PMC_28724525","title":"TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import.","date":"2017","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/28724525","citation_count":67,"is_preprint":false},{"pmid":"23902771","id":"PMC_23902771","title":"Redox-regulated cargo binding and release by the peroxisomal targeting signal receptor, Pex5.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23902771","citation_count":67,"is_preprint":false},{"pmid":"28760655","id":"PMC_28760655","title":"The peroxisomal import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol in times of oxidative stress.","date":"2017","source":"Biochimica et biophysica acta. 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the molecular basis for PTS1 recognition via a novel TPR-peptide interaction mode.\",\n      \"method\": \"X-ray crystallography (crystal structure of PEX5 TPR domain in complex with PTS1 peptide)\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with direct functional validation of binding mechanism\",\n      \"pmids\": [\"11101887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Recombinant human PEX5 forms homotetramers (both PEX5L and PEX5S isoforms) and binds the N-terminal fragment of PEX14 (residues 1-78) with very high affinity in the low nanomolar range. Stable in vitro complexes revealed that PEX5 possesses multiple PEX14-binding sites distributed throughout its N-terminal half, while the C-terminal TPR half binds PTS1 proteins. A pentapeptide motif reiterated seven times in PEX5 was identified as determinant for PEX14 interaction.\",\n      \"method\": \"Surface plasmon resonance, sizing chromatography, electron microscopy, in vitro binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal biophysical methods (SPR, EM, in vitro reconstitution) in a single study\",\n      \"pmids\": [\"10026185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PEX5 (PTS1R) is required for import of both PTS1 proteins and PTS2 proteins in CHO cells; the longer isoform PTS1RL is specifically required for PTS2 import, while both isoforms mediate PTS1 import. Missense mutations in TPR1 (G298E) and TPR6 (G485E) of PTS1RS abolished PTS1 import, demonstrating functional importance of TPR domains in protein translocation.\",\n      \"method\": \"CHO cell mutant complementation, reverse transcription-PCR mutation analysis, import assays in PEX5-defective cell lines\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation with defined mutations, replicated across two CHO mutant cell lines\",\n      \"pmids\": [\"9418886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The seven di-aromatic pentapeptide repeat motifs (WX(E/D/Q/A/S)(E/D/Q)(F/Y)) in the N-terminal half of human PEX5 each independently bind the same site in the N-terminus of PEX14 with nanomolar affinity. Mutational analysis showed conserved aromatic amino acids at positions 1 and 5 of each motif are essential for high-affinity PEX14 binding, likely forming hydrophobic anchors in an amphipathic alpha-helix.\",\n      \"method\": \"Two-hybrid analysis in mammalian cells, in vitro binding assays, mutational analysis, surface plasmon resonance\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutational analysis combined with SPR and two orthogonal binding assays\",\n      \"pmids\": [\"11438541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Human PEX5L (but not PEX5S) physically interacts with PEX7 and is required for PTS2 protein import in mammalian cells. The region amino acids 191-222 of PEX5L is sufficient for PEX7 interaction and amino acids 1-214 are sufficient for peroxisome targeting. A conserved 21-amino-acid motif (aa 209-229) shared with yeast Pex18p/Pex21p is required for both PEX7 interaction and PTS2 import; a serine mutation in this motif abolishes PTS2 import and reduces PEX5L-PEX7 interaction in vitro.\",\n      \"method\": \"Domain mapping, in vitro binding, complementation in mammalian cells, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple deletion/mutant constructs, in vitro interaction, and in vivo functional rescue\",\n      \"pmids\": [\"11546814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PEX12 zinc-binding domain interacts with PEX5 and PEX10; a patient missense mutation S320F in PEX12 reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation. PEX12 and PEX10 act downstream of PEX5 docking at the peroxisome surface, not in the docking step itself.\",\n      \"method\": \"Two-hybrid studies, blot overlay assays, co-immunoprecipitation, genetic suppression by overexpression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods (Y2H, overlay, Co-IP, genetic epistasis) establishing pathway position\",\n      \"pmids\": [\"10562279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Disruption of the Pex5pL-Pex7p interaction by the S214F missense mutation (adjacent to the PEX5L-specific 37-amino acid insertion) completely abolishes PTS2 protein import in mammals while leaving PTS1 import intact, demonstrating that the Pex5pL-Pex7p interaction is essential specifically for PTS2 import.\",\n      \"method\": \"CHO cell mutant isolation, complementation with mutant Pex5p constructs, co-immunoprecipitation, import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — defined missense mutation, reciprocal import and binding assays, confirmed in novel cell mutant\",\n      \"pmids\": [\"10767287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PEX5 import into peroxisomes is ATP-independent, whereas its export back to the cytosol requires ATP. PEX1 and PEX6 (AAA ATPases) and their recruiter PEX26 are essential for PEX5 export. PEX14 is required for PEX5 docking/import. DTM-embedded PEX5 exists in two distinct complexes of ~500 kDa and ~800 kDa comprising different peroxins (including PEX14 and PEX2), indicating PEX5 transits between subcomplexes during its cycle.\",\n      \"method\": \"Cell-free translocation system, radiolabeled PEX5 import/export assays with isolated peroxisomes, blue-native PAGE, PEX-defective cell lines\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — cell-free reconstitution with multiple peroxin-defective cell lines and orthogonal biochemical fractionation\",\n      \"pmids\": [\"16314507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The N-terminal domain of PEX14 adopts a three-helical fold and binds both PEX5 (via its WxxxF/Y motif) and PEX19 competitively at the same surface but with opposite directionality. Mutations of PEX14 residues in the PEX5/PEX19 binding region disrupt binding in vitro and impair peroxisomal membrane localization of PEX14 in vivo.\",\n      \"method\": \"NMR structure determination, competitive binding assays, mutagenesis, in vivo localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with functional mutagenesis and in vivo validation\",\n      \"pmids\": [\"19197237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11 in human) forming a thioester conjugate (Ub-PEX5). USP9X is the primary deubiquitinase acting on Ub-PEX5 in rat liver and HeLa cells, identified by biochemical fractionation; USP9X is an elongated monomeric protein capable of hydrolyzing thioester, isopeptide, and peptide bonds.\",\n      \"method\": \"Biochemical fractionation, in vitro deubiquitinase assays, identification by mass spectrometry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — unbiased biochemical identification plus enzymatic characterization, replicated in two cell types\",\n      \"pmids\": [\"22371489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PEX5 binds monomeric catalase (not tetrameric catalase) through domains in both its N- and C-terminal halves, potently inhibiting catalase tetramerization. The PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, with one or two of the seven PEX14-binding diaromatic motifs in PEX5 involved, indicating PEX14 participates in cargo release.\",\n      \"method\": \"In vitro binding assays, native gel electrophoresis, interaction disruption assays with PEX14 N-terminal domain\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution with multiple complementary assays, single lab\",\n      \"pmids\": [\"21976670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PEX5 is monoubiquitinated at a conserved cysteine residue (Cys11); this modification is mandatory for ATP-dependent extraction of PEX5 from the peroxisomal membrane back to the cytosol. Cargo protein translocation across the peroxisomal membrane occurs upstream of PEX5 ubiquitination (i.e., prior to the first ATP-dependent step).\",\n      \"method\": \"In vitro import/export system with rat liver peroxisomes, PEGylation assays to monitor cysteine modification, protease protection assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution with defined biochemical intermediates, single lab\",\n      \"pmids\": [\"23963456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Cys11 of human PEX5 functions as a redox switch: exposure to oxidized glutathione yields a ubiquitination-deficient PEX5, impairing PTS1 import. Substitution of Cys11 by lysine counteracts this effect. Oxidative stress selectively inhibits import of catalase (non-canonical PTS1) more than canonical PTS1 reporters. PEX5 does not oligomerize in cellulo, and oxidative stress does not affect PEX5-substrate binding per se.\",\n      \"method\": \"PEGylation assays, site-directed mutagenesis (C11K), live-cell import assays in human fibroblasts, redox manipulation\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis combined with functional import assays and redox manipulation, replicated across papers (PMIDs 24118911 and 28760655)\",\n      \"pmids\": [\"24118911\", \"28760655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM37 localizes to peroxisomal membranes and ubiquitylates PEX5 at K464 via interaction with the C-terminal 51 amino acids (CT51) of PEX5; this monoubiquitination stabilizes PEX5 by preventing its proteasomal degradation. TRIM37 depletion or K464A/ΔCT51 PEX5 mutations reduce PEX5 abundance and impair PTS protein import.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, site-directed mutagenesis, RNAi knockdown, import assays in human cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro ubiquitylation, mutagenesis, and functional import readout in a single study\",\n      \"pmids\": [\"28724525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In Pichia pastoris, Pex5 functions as a dimer/oligomer whose homo- and hetero-oligomeric interactions (with Pex8 via its N-terminal domain, aa 1-110) regulate cargo binding and release. Cysteine 10 of Pex5 forms redox-sensitive disulfide-linked oligomers with highest PTS1 cargo affinity; DTT reduction causes transition to noncovalent dimer and partial cargo release. Interaction of Pex5 N-terminal domain with a conserved C-terminal motif of Pex8 further facilitates cargo release under reducing conditions.\",\n      \"method\": \"In vitro binding assays, disulfide crosslinking, dithiothreitol reduction, mutagenesis (C10), import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods but primarily in yeast (Pichia pastoris), single lab\",\n      \"pmids\": [\"23902771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A novel PEX5-PEX14 interaction site in PEX5 was identified with the sequence LVAEF (consensus LVXEF). NMR structure of Pex5-(57-71) with PEX14 N-terminal domain showed this motif binds in an alpha-helical orientation similar to WxxxF/Y motifs but with leucine occupying the tryptophan pocket. SPR showed 33-fold faster dissociation than WxxxF/Y motifs; substituting this site with a higher-affinity WxxxF/Y motif impaired protein import, indicating distinct kinetics of this novel site are required for receptor processing.\",\n      \"method\": \"Peptide library screening, ligand blot analysis, NMR structure determination, SPR, in vivo import assays with alanine substitution mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure, SPR kinetics, and in vivo functional assay in one study\",\n      \"pmids\": [\"24235149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, establishing that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 AAA ATPase complex.\",\n      \"method\": \"Cell-free in vitro system, photoaffinity cross-linking, protein PEGylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution with photoaffinity cross-linking and PEGylation as orthogonal readouts, single lab\",\n      \"pmids\": [\"29884772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PEX5 accompanies cargo completely into the peroxisomal lumen (not just to the membrane). WxxxF/Y motifs near PEX5's N-terminus bind a lumenal domain of the docking complex inside the peroxisome. An amphipathic helix in PEX5 initiates recycling by binding the lumenal side of the ubiquitin ligase. The N-terminus of PEX5 then emerges in the cytosol for monoubiquitination, and finally PEX5 is extracted from the lumen with unfolding of the receptor and cargo release.\",\n      \"method\": \"Xenopus egg extract import system, domain deletion/mutation analysis, protease protection and biochemical assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution in Xenopus extract with multiple mechanistic readouts, establishes lumenal translocation mechanism\",\n      \"pmids\": [\"35931083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The peroxisomal matrix protein translocon (DTM) is a large cavity-forming protein assembly into which cytosolic PEX5 enters to release its cargo. Truncated PEX5 molecules up to residue 197 can be accommodated in excess. PEX5-PEX14 interaction within the DTM is stable at pH 11.5, indicating DTM-bound PEX5 resistance to alkaline extraction does not reflect direct lipid bilayer contact.\",\n      \"method\": \"Truncated PEX5 molecules as DTM probes, proteinase K accessibility assays, alkaline extraction assays, in vitro binding\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical approaches but mechanistic conclusions are partly inferential, single lab\",\n      \"pmids\": [\"28765278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PEX5 proteins fused to bulky C-terminal tags trigger peroxisome degradation by autophagy in mammalian cells. This requires monoubiquitination of the N-terminal cysteine of PEX5 (Cys11), which marks PEX5 for recycling. The C-terminal tag does not inhibit monoubiquitination but blocks PEX5 export from the peroxisomal membrane, indicating that prolonged membrane-association of monoubiquitinated PEX5 triggers pexophagy as a quality control mechanism.\",\n      \"method\": \"Fluorescence microscopy, autophagy inhibitors, mutant PEX5 constructs (C11A abolishes monoubiquitination), quantitative pexophagy assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic dissection with site-directed mutagenesis and pharmacological autophagy inhibition, single lab\",\n      \"pmids\": [\"26086376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AWP1/ZFAND6 stimulates PEX5 export from peroxisomes. AWP1 interacts with PEX6 AAA ATPase (but not Pex1-Pex6 complexes) and preferentially binds cysteine-ubiquitinated PEX5 over unmodified PEX5 via its A20 zinc-finger domain. AWP1 knockdown reduces PTS1 protein import and destabilizes PEX5, similarly to defects in PEX1/PEX6/PEX26.\",\n      \"method\": \"Biochemical fractionation, in vitro Pex5 export assay, recombinant protein stimulation assay, antibody inhibition, RNAi knockdown, co-immunoprecipitation\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro reconstitution assay, biochemical identification, RNAi, multiple orthogonal assays\",\n      \"pmids\": [\"21980954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"High-resolution structural analysis revealed that PEX5 adapts its conformation during PTS1 cargo recognition: the receptor binding cavity shrinks to one-third of its original volume upon ligand binding (ligand-induced compaction). A single-residue mutation in cargo protein alanine-glyoxylate aminotransferase that removes steric occlusion increases peroxisomal import efficiency from 34% to 80%.\",\n      \"method\": \"X-ray crystallography of PEX5-cargo complexes, import efficiency assays in cells\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with quantitative in vivo import assay, single lab\",\n      \"pmids\": [\"25369882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The PEX5-linked monoubiquitin is unfolded at a pre-extraction stage and serves as the extraction initiator; the complete ubiquitin-PEX5 conjugate is threaded by PEX1•PEX6. Chimeric PEX5 molecules with branched C-terminal polypeptide structures can still be extracted, suggesting simultaneous threading of more than one polypeptide chain. Intra-molecularly cross-linked ubiquitin conjugated to residue 11 of PEX5 blocks extraction, confirming ubiquitin unfolding is required.\",\n      \"method\": \"Cell-free in vitro extraction system, engineered PEX5 and ubiquitin molecules, cross-linked ubiquitin, PEGylation assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution with chemically defined substrates and mutagenesis controls\",\n      \"pmids\": [\"36442669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PEX5 ubiquitinated at cysteine 11 (thioester bond) cannot retain a polyubiquitin chain because cysteine-linked ubiquitination is reversible, with E2-mediated deubiquitination being faster than polyubiquitination. A Lys11 PEX5 variant is polyubiquitinated at the peroxisomal membrane, impairing extraction. Thus, cysteine as the ubiquitin acceptor prevents polyubiquitination and proteasomal targeting while enabling reversible monoubiquitination for efficient recycling.\",\n      \"method\": \"Rat liver cell-free in vitro system, site-directed mutagenesis (C11K), ubiquitin chain analysis, extraction assays\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free reconstitution with defined mutants, mechanistic dissection of ubiquitination reversibility\",\n      \"pmids\": [\"38470934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Chemically synthesized monoubiquitinated PEX5 (via click chemistry) binds PEX7/PTS2 complex and can restore PTS2 protein import in ΔPEX5 fibroblasts in vivo. In vitro pull-downs showed monoubiquitinated PEX5 interacts with PEX13, PEX14 (independent of ubiquitination status) and with REM components PEX1, PEX6, and PEX26 (interactions enhanced by ubiquitination).\",\n      \"method\": \"Chemical ubiquitin conjugation (click chemistry), in vitro pull-down assays, complementation in ΔPEX5 fibroblasts\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — chemically defined monoubiquitinated substrate used in both in vitro and in vivo assays\",\n      \"pmids\": [\"30375424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Absence of PEX5 in CHO cells (ZPEG101 mutant) results in unstable PEX14 due to its inefficient translocation to the peroxisomal membrane, establishing that PEX5 stabilizes PEX14 in addition to its import receptor role. The fifth WXXXF/Y pentapeptide motif in PEX5L is an auxiliary PEX14-binding site required for PEX14 stability. PEX5-PEX13 interaction is essential for PTS1 and catalase import but not PTS2 import.\",\n      \"method\": \"PEX5-deficient CHO cell mutant (ZPEG101), complementation with Pex5p mutants, western blotting for PEX14 stability, import assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel cell mutant with complementation analysis and defined motif mapping, single lab\",\n      \"pmids\": [\"23009329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PEX5 mediates fasting-induced lipolysis by escorting adipose triglyceride lipase (ATGL) to contact points between peroxisomes and lipid droplets. During fasting, peroxisomes move toward lipid droplets in a KIFC3-dependent manner, increasing peroxisome-lipid droplet contacts, and PEX5 facilitates ATGL translocation onto lipid droplets. In adipocyte-specific PEX5-knockout mice, ATGL recruitment to lipid droplets was defective and fasting-induced lipolysis was attenuated.\",\n      \"method\": \"Co-immunoprecipitation, fluorescence microscopy, adipocyte-specific conditional knockout mice, lipolysis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO mice with quantitative lipolysis phenotype, co-IP, and live imaging of organelle contacts\",\n      \"pmids\": [\"31996685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cargo protein translocation across the peroxisomal membrane (including release into the matrix) occurs prior to PEX5 ubiquitination in a cargo-protein-centered in vitro import system, mapping translocation downstream of reversible docking and upstream of the first cytosolic ATP-dependent step.\",\n      \"method\": \"Cargo-protein-centered in vitro peroxisomal import system, ATP depletion experiments, protease protection assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free import reconstitution with defined biochemical intermediates, consistent with companion paper (PMID 23963456)\",\n      \"pmids\": [\"19632994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of the PEX5 long isoform (PEX5L) alone causes selective deficiency in PTS2 protein import (not PTS1 import), resulting in rhizomelic chondrodysplasia punctata type 5 (RCDP5) in humans. A frameshift mutation c.722dupA in PEX5L-specific exon 9 abolishes PEX5L expression; reintroduction of PEX5L restores PTS2 import in patient fibroblasts.\",\n      \"method\": \"Patient mutation analysis, isoform-specific expression studies, PTS1/PTS2 import assays in fibroblasts, complementation with PEX5L\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetics linked to cellular mechanism, complementation confirms isoform-specific PTS2 import function\",\n      \"pmids\": [\"26220973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PEX5 mutation F218S (missense) allows normal PTS1 import, normal entry into the DTM, normal monoubiquitination and export, but fails to form a stable trimeric complex with PEX7 and a PTS2 cargo protein, thus failing to promote PTS2 protein import, causing congenital cataracts.\",\n      \"method\": \"In vitro import assays, co-immunoprecipitation, site-directed mutagenesis, patient fibroblast functional studies\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined mutation with mechanistic dissection of PTS1 vs PTS2 import steps using in vitro and cell-based assays\",\n      \"pmids\": [\"33389129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Full-length human PEX5L is monomeric in solution with a compact conformation, spontaneously binds to lipid bilayers (accumulating ~100-fold), and forms ion-conducting membrane channels in artificial horizontal bilayers. The C-terminal cargo-binding domain (residues 336–639) is required for pore formation; truncated PEX5L(1-335) binds membranes but does not form channels, suggesting PEX5L is the pore-forming component of the peroxisomal translocon.\",\n      \"method\": \"Fluorescence TCSPC (diffusion measurement), electrophysiological single-channel recording in horizontal lipid bilayers, site-specific fluorescent labeling\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology in defined bilayer system with truncation analysis, single lab\",\n      \"pmids\": [\"36260915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PEX5 NTD harbors multiple membrane interaction sites involving amphipathic alpha-helical regions that include WxxxF/Y motifs; these helical regions are stabilized in the presence of membrane-mimicking bicelles (NMR). PEX14 NTD weakly interacts with bicelles at a surface that partially overlaps with the WxxxF/Y binding site. The PEX5-PEX14 interaction is largely unaffected by membrane presence (similar binding enthalpies with entropy compensation), indicating membrane docking does not reduce overall PEX5-PEX14 binding affinity.\",\n      \"method\": \"NMR spectroscopy with bicelles and nanodiscs, isothermal titration calorimetry (ITC)\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR and ITC are rigorous biophysical methods but single lab, functional consequences not directly tested in vivo\",\n      \"pmids\": [\"33937250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Pex8 in yeast is essential for peroxisomal cargo translocation regardless of receptor/cargo recognition mechanism. Pex8 binds through a 12-fold HEAT repeat array to a short three-helical bundle in the N-terminal domain of Pex5; impairing this interaction abolishes peroxisomal protein translocation. A secondary autonomous Pex8 cargo-like interaction site exists at the C-terminal domain of Pex5, generating a bipartite interaction. The Pex5/Pex8 complex assembly enables association with the Pex2/Pex10/Pex12 E3-ubiquitin ligase complex to initiate receptor recycling.\",\n      \"method\": \"Cryo-electron microscopy structure of Pex5-Pex8 complex, mutagenesis, functional import assays in yeast\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with functional mutagenesis, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.08.30.673231\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Saccharomyces cerevisiae Eci1 can reach peroxisomes and bind Pex5 in the absence of a canonical PTS1 signal. Cryo-EM structure of yeast Pex5-Eci1 complex identified additional binding interfaces beyond the canonical PTS1-TPR interaction, revealing that some cargoes use non-canonical interaction surfaces for Pex5-mediated targeting.\",\n      \"method\": \"Cryo-electron microscopy, import assays in yeast pex5 mutants lacking PTS1\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with functional import validation, single study\",\n      \"pmids\": [\"40376748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PEX13 loss causes accumulation of ubiquitinated PEX5 on peroxisomes and increased peroxisome-dependent ROS, both of which induce pexophagy. PEX13 protein levels are downregulated during amino acid starvation to aid pexophagy induction, establishing PEX13 as a regulator that prevents pexophagy of healthy peroxisomes by controlling ubiquitinated PEX5 accumulation.\",\n      \"method\": \"CRISPR gene editing, quantitative fluorescence microscopy, zebrafish model, autophagy assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells and zebrafish with multiple readouts, single lab\",\n      \"pmids\": [\"36541703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Solution SAXS analysis of human Pex5-Pex14-PTS1 complexes revealed a 1:6 stoichiometry for Pex5:Pex14 complex. Free full-length Pex5 is monomeric in solution with an elongated, partially unfolded N-terminal domain. In the complex, the Pex5 N-terminus remains extended, with Pex14 significantly intermingled with the Pex5 moiety.\",\n      \"method\": \"Small-angle X-ray scattering (SAXS), static light scattering, titration studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SAXS low-resolution structural data, single lab, no mutagenesis validation\",\n      \"pmids\": [\"19584060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Full-length tetrameric PEX5 binds PTS1 (lissamine-Tyr-Gln-Ser-Lys-Leu-COO-) with Kd of 35 nM. Neither Hsp70 (with or without ATP/ADP) nor the PEX12 zinc RING domain has a detectable effect on PEX5-PTS1 binding kinetics, indicating initial cargo recognition by PEX5 is a relatively simple process not regulated by these factors.\",\n      \"method\": \"Fluorescence anisotropy binding assay, purified recombinant full-length PEX5, Hsp70 addition experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro binding assay with purified proteins; negative results for Hsp70/PEX12 regulation are well-controlled\",\n      \"pmids\": [\"12456682\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX5 is a shuttling import receptor that recognizes PTS1-containing peroxisomal matrix proteins in the cytosol via its C-terminal TPR domain (crystal structure determined), docks at the peroxisomal membrane through multiple WxxxF/Y motifs in its N-terminal domain that bind PEX14 with nanomolar affinity, and then enters completely into the peroxisomal lumen to release cargo; the receptor is subsequently monoubiquitinated at a conserved N-terminal cysteine (Cys11) by a peroxisomal membrane-embedded E3 ligase complex, and this monoubiquitinated PEX5 is recognized and extracted from the lumen by the AAA ATPase complex PEX1-PEX6 through a threading/unfolding mechanism, with USP9X serving as the primary cytosolic deubiquitinase; the longer isoform PEX5L additionally recruits PEX7 (via a specific 21-aa motif) to co-import PTS2 cargo proteins, TRIM37 stabilizes PEX5 via a separate lysine monoubiquitination, and the redox state of Cys11 acts as a switch coupling oxidative stress to import activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PEX5 is the cycling import receptor that delivers folded peroxisomal matrix proteins from the cytosol into the peroxisomal lumen [#2, #27]. It recognizes PTS1-bearing cargo through its C-terminal TPR domain, where two clusters of three TPRs encircle the PTS1 pentapeptide and the receptor binding cavity compacts upon ligand engagement to enforce selectivity [#0, #21]. Cargo recognition is a comparatively simple, high-affinity event (Kd ~35 nM) not modulated by Hsp70 or the PEX12 RING domain [#36]. The elongated N-terminal half carries seven di-aromatic WxxxF/Y pentapeptide motifs, each binding the three-helical N-terminal domain of the docking peroxin PEX14 with nanomolar affinity, plus a distinct lower-affinity LVxEF site whose fast dissociation kinetics are themselves required for receptor processing [#1, #3, #8, #15]. The longer isoform PEX5L additionally recruits PEX7 through a conserved 21-residue motif to co-import PTS2 cargo, a function genetically separable from PTS1 import [#4, #6]; defects in this PEX5L–PEX7 interaction cause rhizomelic chondrodysplasia punctata type 5 and congenital cataracts [#28, #29]. Rather than acting only at the membrane, PEX5 enters the lumen of the import translocon (DTM), releases cargo with the participation of the PEX14 N-terminal domain, and is then retrieved [#10, #17, #18]. Recycling is initiated by monoubiquitination of a conserved N-terminal cysteine (Cys11) as a reversible thioester conjugate; this modification is mandatory for the ATP-dependent extraction of PEX5 from the membrane by the PEX1–PEX6 AAA-ATPase, which threads and globally unfolds the receptor together with its unfolded ubiquitin tag [#11, #16, #22, #23]. The cysteine acceptor is itself a redox switch coupling glutathione oxidation to import competence, and USP9X is the principal cytosolic deubiquitinase that regenerates free PEX5 [#9, #12]. Beyond matrix import, PEX5 stabilizes PEX14, escorts adipose triglyceride lipase to peroxisome–lipid droplet contacts to drive fasting-induced lipolysis, and—when its export is blocked—accumulates as ubiquitinated PEX5 to trigger pexophagy as an organelle quality-control mechanism [#25, #26, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established PEX5 as the genetic determinant of peroxisomal matrix import and revealed isoform-specific division of labor between PTS1 and PTS2 cargo.\",\n      \"evidence\": \"CHO mutant complementation and TPR missense mutations in PEX5-defective cells\",\n      \"pmids\": [\"9418886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of PTS1 recognition not yet structurally defined\", \"Mechanism by which the long isoform mediates PTS2 import unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined PEX5 architecture as bifunctional — an N-terminal half bearing reiterated PEX14-binding motifs and a C-terminal cargo-binding half — and quantified the nanomolar PEX5–PEX14 docking interaction.\",\n      \"evidence\": \"SPR, sizing chromatography, EM, and in vitro reconstitution of recombinant PEX5 and PEX14\",\n      \"pmids\": [\"10026185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Number and individual contributions of the PEX14 sites not resolved\", \"Oligomeric state in vivo uncertain\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Solved the structural basis of PTS1 recognition, showing two TPR clusters surround the peptide via a hinge-enabled single binding site.\",\n      \"evidence\": \"X-ray crystallography of the PEX5 TPR domain with a PTS1 pentapeptide\",\n      \"pmids\": [\"11101887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics of cargo loading/release not addressed\", \"Full-length receptor architecture not captured\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined the molecular grammar of receptor docking and the PEX5L-specific PEX7 recruitment motif that underlies PTS2 import.\",\n      \"evidence\": \"Two-hybrid, SPR, mutational analysis of WxxxF/Y motifs; domain mapping of the PEX5L 21-aa PEX7-binding motif\",\n      \"pmids\": [\"11438541\", \"11546814\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why seven redundant PEX14 sites are needed unresolved\", \"Functional difference between sites not yet defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Separated import (ATP-independent) from export (ATP-dependent), assigning PEX1/PEX6/PEX26 to receptor retrieval and showing PEX5 transits between membrane subcomplexes.\",\n      \"evidence\": \"Cell-free translocation/export assays with isolated peroxisomes, blue-native PAGE, peroxin-defective cells\",\n      \"pmids\": [\"16314507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular trigger linking docking to export not defined\", \"Composition of the 500/800 kDa complexes incomplete\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Ordered the import cycle by placing cargo translocation across the membrane upstream of the first cytosolic ATP-dependent step, and provided solution architecture of the docking complex.\",\n      \"evidence\": \"Cargo-centered cell-free import system with ATP depletion and protease protection; SAXS of Pex5–Pex14–PTS1 complexes\",\n      \"pmids\": [\"19632994\", \"19584060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of translocation across the bilayer unknown\", \"Stoichiometry interpretation limited by SAXS resolution\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the PEX14 N-terminal fold and showed PEX5 and PEX19 compete for the same surface, integrating receptor docking with membrane-protein biogenesis.\",\n      \"evidence\": \"NMR structure with competitive binding and in vivo localization\",\n      \"pmids\": [\"19197237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of PEX5/PEX19 competition in vivo not fully dissected\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified Cys11 monoubiquitination as a thioester conjugate and USP9X as the cytosolic deubiquitinase regenerating free PEX5.\",\n      \"evidence\": \"Biochemical fractionation, in vitro DUB assays, mass spectrometry in rat liver and HeLa\",\n      \"pmids\": [\"22371489\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase responsible for Cys11 conjugation not identified here\", \"Regulation of USP9X activity unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that Cys11 monoubiquitination is mandatory for ATP-dependent extraction and that Cys11 acts as a redox switch coupling oxidative state to import competence, with selective effects on catalase.\",\n      \"evidence\": \"Cell-free import/export with PEGylation; C11K mutagenesis and redox manipulation in fibroblasts\",\n      \"pmids\": [\"23963456\", \"24118911\", \"28760655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How redox state is sensed in vivo not fully resolved\", \"Physiological oxidant relevant to the switch undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mechanistically linked PEX5 to cargo release and to a non-WxxxF/Y processing site, and to PEX14 stabilization.\",\n      \"evidence\": \"In vitro catalase tetramerization assays; NMR/SPR of the LVxEF site; CHO mutant complementation for PEX14 stability\",\n      \"pmids\": [\"21976670\", \"24235149\", \"23009329\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalase work is single-lab in vitro\", \"PEX14-stabilization findings Medium-confidence and single-lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed ligand-induced compaction of the TPR cavity and that cargo steric features tune import efficiency.\",\n      \"evidence\": \"X-ray crystallography of PEX5–cargo complexes with in-cell import efficiency assays\",\n      \"pmids\": [\"25369882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of compaction across diverse cargoes untested\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected receptor recycling failure to organelle quality control, showing stalled monoubiquitinated PEX5 triggers pexophagy.\",\n      \"evidence\": \"Fluorescence microscopy, autophagy inhibitors, C11A mutants in mammalian cells\",\n      \"pmids\": [\"26086376\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Autophagy receptor recognizing stalled Ub-PEX5 not identified\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Characterized the import translocon as a large cavity (DTM) into which PEX5 enters, and identified TRIM37-mediated K464 monoubiquitination as a PEX5-stabilizing modification distinct from Cys11.\",\n      \"evidence\": \"Truncated PEX5 DTM probes with proteinase K/alkaline extraction; Co-IP, in vitro ubiquitylation, RNAi, import assays\",\n      \"pmids\": [\"28765278\", \"28724525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DTM mechanistic conclusions partly inferential\", \"Interplay between TRIM37 K464 and Cys11 ubiquitination unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established DTM-embedded Ub-PEX5 as a direct PEX1–PEX6 substrate that is globally unfolded during extraction, and defined the interaction map of chemically defined monoubiquitinated PEX5 with docking and export machinery.\",\n      \"evidence\": \"Cell-free system with photoaffinity cross-linking/PEGylation; click-chemistry monoubiquitinated PEX5 pull-downs and complementation\",\n      \"pmids\": [\"29884772\", \"30375424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Force-generation and threading directionality mechanistically incomplete\", \"Single-lab reconstitutions\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended PEX5 function beyond matrix import to fasting-induced lipolysis by escorting ATGL to peroxisome–lipid droplet contacts in vivo.\",\n      \"evidence\": \"Co-IP, live imaging, adipocyte-specific PEX5-knockout mice with lipolysis assays\",\n      \"pmids\": [\"31996685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this uses the canonical TPR cargo pathway unclear\", \"Molecular basis of ATGL recognition undefined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked PEX5L-specific PTS2 import defects to human disease and dissected step-specific mutations that uncouple PTS1 from PTS2 import.\",\n      \"evidence\": \"Patient mutation analysis (c.722dupA; F218S), import assays, Co-IP, complementation in fibroblasts\",\n      \"pmids\": [\"26220973\", \"33389129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific consequences of PTS2 loss not mechanistically explained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the complete lumenal import-and-recycling itinerary and the ubiquitin-unfolding requirement that initiates threading-based extraction.\",\n      \"evidence\": \"Xenopus egg extract import system; cell-free extraction with engineered/cross-linked ubiquitin and chimeric PEX5\",\n      \"pmids\": [\"35931083\", \"36442669\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the lumenal docking/ligase contacts incompletely mapped\", \"How multiple chains are threaded simultaneously unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed PEX13 as a regulator preventing pexophagy of healthy peroxisomes by limiting accumulation of ubiquitinated PEX5.\",\n      \"evidence\": \"CRISPR KO, quantitative microscopy, zebrafish, autophagy assays\",\n      \"pmids\": [\"36541703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab\", \"Direct link between PEX13 levels and Ub-PEX5 turnover correlative\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Explained why cysteine, not lysine, is the ubiquitin acceptor: reversible thioester ubiquitination prevents polyubiquitination and proteasomal targeting, enabling efficient recycling.\",\n      \"evidence\": \"Rat liver cell-free system, C11K mutagenesis, ubiquitin chain and extraction analysis\",\n      \"pmids\": [\"38470934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo balance of E2-mediated deubiquitination versus chain extension not quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided structural basis for the Pex5–Pex8 interaction in yeast that couples cargo translocation to recruitment of the Pex2/Pex10/Pex12 ligase, and revealed non-canonical cargo interaction surfaces.\",\n      \"evidence\": \"Cryo-EM of Pex5–Pex8 (preprint) and Pex5–Eci1 complexes with functional import assays in yeast\",\n      \"pmids\": [\"bio_10.1101_2025.08.30.673231\", \"40376748\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"One source is a preprint\", \"Whether the Pex8-dependent step generalizes to mammals (which lack a clear Pex8) unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physical nature of the translocation pore and how the receptor and cargo cross the bilayer remain incompletely defined.\",\n      \"evidence\": \"PEX5L membrane channel activity is reported but the in vivo pore identity is unresolved\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PEX5L itself forms the physiological translocon pore is not established in vivo\", \"The complete subunit composition and gating of the DTM channel are unknown\", \"How cargo of varying size and fold is accommodated remains undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [0, 2, 21, 36]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 6, 26]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 9, 11]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [7, 17, 18]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [2, 7, 17, 27]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 16, 22, 23]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [19, 34]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [28, 29]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"complexes\": [\n      \"DTM (peroxisomal matrix protein translocon)\",\n      \"PEX1-PEX6 AAA-ATPase complex (substrate)\",\n      \"PEX5L-PEX7 receptor complex\"\n    ],\n    \"partners\": [\n      \"PEX14\",\n      \"PEX7\",\n      \"PEX13\",\n      \"PEX12\",\n      \"PEX1\",\n      \"PEX6\",\n      \"USP9X\",\n      \"TRIM37\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}