{"gene":"PEX14","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1999,"finding":"Human PEX5 binds the N-terminal fragment of PEX14-(1-78) with very high affinity in the low nanomolar range, as measured by surface plasmon resonance. PEX5 possesses multiple binding sites for PEX14 distributed throughout its N-terminal half, and a pentapeptide motif reiterated seven times in PEX5 was identified as the determinant for interaction with PEX14.","method":"Surface plasmon resonance, in vitro binding assay with recombinant proteins, sizing chromatography","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, SPR quantification, multiple orthogonal methods in one study","pmids":["10026185"],"is_preprint":false},{"year":1997,"finding":"H. polymorpha Pex14p is a peroxisomal membrane protein essential for import of matrix proteins; loss of Pex14p results in peroxisomal membrane remnants containing only small amounts of matrix proteins (AO, catalase, DHAS), with the bulk mislocalized to the cytosol. Overproduction of Pex14p causes a peroxisome-deficient phenotype with numerous small vesicles lacking matrix proteins, indicating stoichiometry of Pex14p relative to other biogenesis components is critical.","method":"Genetic complementation, biochemical fractionation, electron microscopy, gene disruption","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation, fractionation, EM, and overexpression phenotype independently replicated in multiple mutant backgrounds","pmids":["9009266"],"is_preprint":false},{"year":2001,"finding":"The seven di-aromatic pentapeptide repeat motifs (WX(E/D/Q/A/S)(E/D/Q)(F/Y)) of human PEX5 each independently bind to the same site in the N-terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis showed the conserved aromatic amino acids at positions 1 and 5 of the motif are essential for high affinity binding, proposed to form hydrophobic anchors via 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 / Strong — mutagenesis plus SPR quantification plus two-hybrid, multiple orthogonal methods confirming the same binding site","pmids":["11438541"],"is_preprint":false},{"year":2009,"finding":"The N-terminal domain of Pex14 (Pex14-N) adopts a three-helical fold. Both PEX5 and PEX19 ligand helices bind competitively to the same surface of Pex14-N, but with opposite directionality. The Pex5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19 mediate this recognition through conserved aromatic side chains. 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, in vitro binding assays, site-directed mutagenesis, in vivo localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with mutagenesis and functional in vivo validation, multiple orthogonal methods in one study","pmids":["19197237"],"is_preprint":false},{"year":2011,"finding":"Human PEX14 interacts directly with tubulin, and this binding is mediated by the conserved N-terminal domain of PEX14. PEX14-deficient cells lose the ability of peroxisomal remnants to move along microtubules, establishing PEX14 as the membrane anchor linking peroxisomes to microtubules for motility.","method":"Affinity purification of native PEX14 complexes, mass spectrometry, size-exclusion chromatography, in vitro binding, live cell imaging, PEX14-deficient cell analysis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-purification, direct binding demonstrated in vitro, loss-of-function phenotype with specific motility readout","pmids":["21525035"],"is_preprint":false},{"year":2011,"finding":"PEX5 binds monomeric catalase and potently inhibits its tetramerization; no such complex was detected with tetrameric catalase. The N-terminal domain of PEX14 disrupts the PEX5-catalase interaction, with one or two of the seven PEX14-binding diaromatic motifs of PEX5 involved, indicating PEX14 participates in the cargo protein release step.","method":"In vitro binding assays, biochemical reconstitution, domain deletion analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined domain mutants, multiple biochemical experiments in a single rigorous study","pmids":["21976670"],"is_preprint":false},{"year":2013,"finding":"A novel PEX5-PEX14 interaction site in PEX5 with the sequence LVAEF (consensus LVXEF) was identified. NMR structure of the Pex5-(57-71)/Pex14-NTD complex showed this motif binds in a similar alpha-helical orientation as the WxxxF/Y motif but with the tryptophan pocket occupied by leucine. The LVXEF motif has 33-fold faster dissociation from PEX14 than WxxxF/Y motifs, and alanine substitution of LVAEF strongly impairs peroxisomal matrix protein import in vivo.","method":"Peptide library ligand blot screening, NMR structure determination, surface plasmon resonance, mutagenesis, in vivo import assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure, SPR kinetics, mutagenesis, and in vivo functional assays in one study","pmids":["24235149"],"is_preprint":false},{"year":2020,"finding":"Pex14 is phosphorylated at Ser232 in response to H2O2-induced oxidative stress in mammalian cells. This phosphorylation suppresses peroxisomal import of catalase specifically (but not canonical PTS1 proteins) by selectively impairing the interaction of catalase with the Pex14-Pex5 complex in vitro. A phosphomimetic Pex14-S232D mutant elevates cytosolic catalase and confers higher cell resistance to H2O2.","method":"Phosphoproteomics, site-directed mutagenesis (S232D phosphomimetic), in vivo import assays, in vitro binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phosphorylation site identified by MS, validated by mutagenesis, in vitro and in vivo functional consequences demonstrated with multiple orthogonal methods","pmids":["32831175"],"is_preprint":false},{"year":2007,"finding":"Hansenula polymorpha Pex14 has a unique dual function: it is required both for matrix protein import into peroxisomes and for selective autophagic degradation of peroxisomes (pexophagy). Other components of the peroxisomal translocon (Pex2, Pex10, Pex12, Pex13, Pex17) are not required for pexophagy, making Pex14 the sole peroxisomal translocon component with this dual function.","method":"Genetic epistasis analysis with multiple pex deletion mutants, pexophagy assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple mutants in a single lab, establishes unique dual role of Pex14 vs. other translocon peroxins","pmids":["17921697"],"is_preprint":false},{"year":2005,"finding":"Pex13 binds Pex14 via two sites: its SH3 domain and a novel intraperoxisomal site. Pex5 also contributes to the association of Pex13 with Pex14. Combining mutations in the novel Pex14-interaction site of Pex13 with a non-Pex13-interacting Pex5(W204A) mutant severely compromised PTS1-dependent import; additionally blocking the SH3-Pex14 interaction completely abrogated PTS2-dependent import and dissociated Pex13 from the docking complex.","method":"In vitro binding assays, genetic epistasis with combined mutants, co-purification, growth assays on oleic acid","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, systematic combinatorial mutagenesis showing epistatic relationships, co-purification confirming complex assembly","pmids":["15798189"],"is_preprint":false},{"year":2000,"finding":"In H. polymorpha Δpex14 cells, overproduction of the PTS1 receptor Pex5p leads to enhanced import of PTS1 proteins AO and DHAS but not PTS2 protein amine oxidase, and not catalase. Pex5p localizes to the cytosol and outer peroxisomal membrane surface in Δpex14 cells, indicating that Pex5p can bind the peroxisomal membrane and mediate partial import of certain PTS1 proteins in the absence of Pex14p.","method":"Genetic overexpression in pex14 null mutant, subcellular fractionation, immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic null background, specific import assays distinguishing PTS1 vs PTS2 cargo, localization studies","pmids":["10777551"],"is_preprint":false},{"year":2018,"finding":"PEX14 is a bona fide intrinsic membrane protein with Nin-Cout topology (N-terminus facing the peroxisomal lumen, C-terminus facing the cytosol), as determined in proteoliposomes and purified rat liver peroxisomes.","method":"Protease-protection assays on proteoliposomes and native peroxisomes, mass spectrometry, Edman degradation, western blotting with domain-specific antibodies","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — protease protection assays in reconstituted proteoliposomes and native organelles with multiple detection methods in one rigorous study","pmids":["30414318"],"is_preprint":false},{"year":2021,"finding":"The PEX14 N-terminal domain (NTD) binds to microtubular filaments in vitro with nanomolar affinity, interacting with two motifs in the C-terminal region of human β-tubulin. PEX5 competes with β-tubulin for binding to PEX14-NTD, such that PEX5 binding to PEX14 can prevent peroxisome anchoring to microtubules, providing a mechanistic link between peroxisomal protein import and motility.","method":"In vitro binding assays, NMR spectroscopy, competition binding experiments, dissociation constant measurement","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined domains, NMR-based mapping, competition assays identifying specific tubulin motifs","pmids":["33484719"],"is_preprint":false},{"year":2021,"finding":"The PEX14 NTD weakly interacts with membrane-mimicking bicelles via a surface that partially overlaps with the WxxxF/Y binding site. The PEX5-PEX14 NTD interaction is largely unaffected by the presence of membranes, with reduced binding enthalpy compensated by reduced entropy loss, indicating docking of PEX5 to PEX14 at the membrane does not reduce overall 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 in membrane-mimicking systems, single lab, two orthogonal biophysical methods","pmids":["33937250"],"is_preprint":false},{"year":2017,"finding":"Small molecules disrupting the Trypanosoma PEX14-PEX5 protein-protein interaction cause mislocalization of glycosomal enzymes, metabolic catastrophe, and parasite death. High-resolution X-ray crystal structures of inhibitors bound to TbPEX14 were obtained, revealing the structural basis for blocking PEX14-PEX5 interaction as a drug target.","method":"X-ray crystallography of inhibitor-PEX14 complexes, NMR binding studies, cell-based trypanocidal assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — X-ray crystal structures with NMR validation and cell-based functional assays, published in high-impact journal with replication across multiple compound classes","pmids":["28360328"],"is_preprint":false},{"year":2003,"finding":"T. brucei PEX14 is associated with glycosomes and its N-terminal part binds specifically to TbPEX5. RNAi-mediated depletion of TbPEX14 causes mislocalization of glycosomal proteins (PTS1, PTS2, and internal I-PTS-containing) to the cytosol and is essential for survival of both bloodstream-form and procyclic trypanosomes.","method":"Subcellular fractionation, in vitro binding with recombinant proteins, RNA interference, immunofluorescence","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi knockdown with defined import phenotype across multiple cargo classes, direct binding assay, multiple orthogonal methods","pmids":["12709066"],"is_preprint":false},{"year":2004,"finding":"Human PEX14 is required for both PTS1- and PTS2-dependent peroxisomal matrix protein import in human cells. Loss-of-function (nonsense mutation p.Q185X) causes Zellweger syndrome; transfection with wild-type PEX14 rescued import in patient fibroblasts, establishing PEX14 as the 13th complementation group gene for peroxisome biogenesis disorders.","method":"Genetic complementation in patient fibroblasts, immunocytochemistry, mutational analysis","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementation rescue of defined human mutation, import assays for both PTS1 and PTS2, single patient study","pmids":["15146459"],"is_preprint":false},{"year":2009,"finding":"Solution structure of the human Pex5-Pex14-PTS1 complex determined by small angle X-ray scattering reveals a 1:6 stoichiometry for the Pex5-Pex14 complex. The N-terminus of Pex5 remains extended in the presence of cargo and Pex14, 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 model with stoichiometry determination, single lab, limited resolution","pmids":["19584060"],"is_preprint":false},{"year":2025,"finding":"The C-terminal domain (CTD) of human PEX14 undergoes phase separation in vitro, forming condensates that recruit PEX5-cargo complexes carrying PTS1 or PTS2 signals. hPEX14 forms tetramers, and hPEX14 and hPEX13 form immiscible condensates. Replacing hPEX14 CTD with other phase-separating polypeptides partially restores peroxisomal import; electrostatic interactions and the specific CTD sequence are essential for import.","method":"In vitro phase separation assays, structural analysis of PEX14 tetramer, domain replacement experiments, cargo recruitment assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of phase separation, structural analysis, domain swap experiments with functional rescue, multiple orthogonal methods in one study","pmids":["40555785"],"is_preprint":false},{"year":2025,"finding":"PEX14 acts as a docking site for the autophagy receptor optineurin (OPTN) on the peroxisomal membrane, enabling OPTN-mediated pexophagy. PEX14 and OPTN interact through their coiled-coil and ubiquitin-binding domains, respectively. PEX14-OPTN complexes colocalize with LC3, and bafilomycin A1 suppresses OPTN-mediated peroxisome degradation.","method":"Proximity labeling, co-immunoprecipitation, domain mapping, fluorescence microscopy, autophagy inhibitor treatment","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling and biochemical interaction mapping with domain specificity, colocalization with autophagy markers, single lab","pmids":["41071103"],"is_preprint":false},{"year":2026,"finding":"The N-terminal domain of Pex14 undergoes ATP- and ubiquitination-dependent topological remodeling regulated by the AAA+ ATPase Pex6 and its membrane-recruiting partner Pex26. Under normal conditions, the Pex14 N-terminus is oriented toward the peroxisomal lumen; deficiency of Pex6 or Pex26, or pharmacological inhibition of AAA+ ATPases, causes the N-terminus to become exposed to the cytoplasm. Inhibition of ubiquitin activation blocks this reorientation, likely by preventing Pex5 ubiquitination and extraction.","method":"Immunofluorescence microscopy, protease protection assays, pharmacological inhibition of AAA+ ATPases, ubiquitin pathway inhibition","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protease protection assays with genetic and pharmacological perturbations in multiple conditions, single lab study","pmids":["41581879"],"is_preprint":false},{"year":2005,"finding":"In procyclic T. brucei, RNAi knockdown of PEX14 causes mislocalization of glycosomal enzymes and cell death that is triggered by glucose or fructose, but not in the absence of sugars. Double RNAi of PEX14 and hexokinase rescued cells from glucose toxicity even though glycosomal proteins remained mislocalized, establishing that the toxicity of PEX14 depletion in glucose is due to unregulated cytosolic glycolysis.","method":"RNA interference (single and double knockdown), genetic epistasis, cell viability assays with different carbon sources","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with double RNAi, multiple carbon source conditions, establishes pathway position of PEX14 in metabolic toxicity","pmids":["15637070"],"is_preprint":false}],"current_model":"PEX14 is a peroxisomal membrane protein with Nin-Cout topology that serves as the central docking platform of the peroxisomal import machinery: its conserved N-terminal domain binds the WxxxF/Y and LVXEF motifs of the cycling import receptor PEX5 with nanomolar affinity, releases cargo (including catalase, whose release is facilitated by PEX14 displacing PEX5) upon docking, and interacts with PEX13 via two direct sites to organize the translocation complex; the Pex14 N-terminal domain also undergoes ATP- and ubiquitination-dependent topological remodeling driven by Pex6 during Pex5 recycling; the C-terminal domain undergoes phase separation to form condensates that recruit PEX5-cargo complexes; PEX14 additionally binds β-tubulin through its N-terminal domain to anchor peroxisomes to microtubules for motility, a function competitively regulated by PEX5; phosphorylation of PEX14 at Ser232 in response to H2O2 selectively suppresses catalase import to redistribute catalase to the cytosol; and PEX14 serves as the docking site for the autophagy receptor optineurin, enabling selective autophagic degradation of peroxisomes (pexophagy)."},"narrative":{"mechanistic_narrative":"PEX14 is an intrinsic peroxisomal membrane protein that functions as the central docking platform of the matrix protein import machinery, required for import of both PTS1- and PTS2-targeted proteins [PMID:9009266, PMID:15146459]. It adopts an Nin-Cout topology, with the conserved three-helical N-terminal domain (NTD) and the C-terminus exposed to the cytosol [PMID:30414318, PMID:19197237]. The NTD is the principal receptor-binding surface: it engages the cycling import receptor PEX5 with low-nanomolar affinity through PEX5's reiterated di-aromatic WxxxF/Y pentapeptide motifs and a kinetically distinct LVXEF motif, both of which dock as amphipathic helices via conserved aromatic anchors [PMID:10026185, PMID:11438541, PMID:24235149]. The same NTD surface binds the PEX19 F/YFxxxF motif competitively, coupling receptor docking to PEX14 membrane targeting [PMID:19197237]. Beyond receptor capture, PEX14 mediates cargo release, disrupting the PEX5-catalase complex to participate in matrix protein delivery [PMID:21976670], and organizes the translocation complex by binding PEX13 through both an SH3-domain site and a second intraperoxisomal site [PMID:15798189]. The receptor cycle is regulated downstream by AAA+ ATPase-driven, ubiquitination-dependent topological remodeling of the PEX14 N-terminus controlled by PEX6/PEX26 [PMID:41581879], while the cytosolic C-terminal domain undergoes phase separation into condensates that recruit PEX5-cargo complexes to drive import [PMID:40555785]. PEX14 carries additional, separable functions: its NTD binds β-tubulin to anchor peroxisomes to microtubules for motility, a contact competitively displaced by PEX5 to couple import state to organelle movement [PMID:21525035, PMID:33484719]; phosphorylation at Ser232 upon H2O2 stress selectively blocks catalase import to redistribute catalase to the cytosol [PMID:32831175]; and it serves as the membrane docking site for the autophagy receptor optineurin, enabling pexophagy [PMID:41071103, PMID:17921697]. Loss-of-function mutation (p.Q185X) causes Zellweger syndrome, defining PEX14 as a peroxisome biogenesis disorder gene [PMID:15146459].","teleology":[{"year":1997,"claim":"Established that Pex14 is a peroxisomal membrane protein essential for matrix protein import, and that its abundance relative to other biogenesis components must be tightly controlled.","evidence":"Gene disruption, complementation, fractionation and EM in H. polymorpha","pmids":["9009266"],"confidence":"High","gaps":["Molecular partners and mechanism of import not defined","Topology unknown"]},{"year":1999,"claim":"Identified PEX14 as a high-affinity PEX5 receptor docking partner, answering how the cycling receptor engages the membrane.","evidence":"SPR and in vitro binding with recombinant human PEX5 and PEX14-(1-78)","pmids":["10026185"],"confidence":"High","gaps":["Structural basis of binding not resolved","Stoichiometry not determined"]},{"year":2001,"claim":"Defined the molecular determinant of docking, showing seven di-aromatic pentapeptide repeats in PEX5 each bind the PEX14 NTD via conserved aromatic anchors.","evidence":"Two-hybrid, mutagenesis, and SPR in mammalian/in vitro systems","pmids":["11438541"],"confidence":"High","gaps":["Atomic structure of the complex still lacking","Why seven repeats unclear"]},{"year":2005,"claim":"Showed PEX14 organizes the translocon by binding PEX13 through two distinct sites, with PEX5 contributing, defining how the docking complex is assembled and how PTS1 vs PTS2 import depend on these contacts.","evidence":"Combinatorial mutagenesis, co-purification, and oleic-acid growth assays in yeast","pmids":["15798189"],"confidence":"High","gaps":["Quantitative complex architecture not resolved","Dynamics during the import cycle unknown"]},{"year":2009,"claim":"Resolved the three-helical PEX14 NTD fold and revealed competitive, oppositely oriented binding of PEX5 and PEX19 ligand helices, linking receptor docking to PEX14 membrane targeting; SAXS established a 1:6 PEX5:PEX14 stoichiometry.","evidence":"NMR structure, mutagenesis, in vivo localization; SAXS solution model","pmids":["19197237","19584060"],"confidence":"High","gaps":["High-resolution structure of the full complex absent","Functional meaning of 1:6 ratio in vivo unclear"]},{"year":2011,"claim":"Uncovered two distinct PEX14 functions beyond docking: facilitating cargo release by disrupting the PEX5-catalase complex, and anchoring peroxisomes to microtubules via direct tubulin binding through the NTD.","evidence":"In vitro reconstitution and domain deletion; affinity purification, in vitro binding, and live-cell motility assays in PEX14-deficient cells","pmids":["21976670","21525035"],"confidence":"High","gaps":["Mechanism coupling docking surface to two ligand classes not fully resolved","In vivo balance between motility and import unknown"]},{"year":2013,"claim":"Identified the LVXEF motif as a second class of PEX5 docking sequence with faster dissociation kinetics, refining the model of how receptor engagement and release are kinetically tuned.","evidence":"Peptide library screen, NMR structure, SPR kinetics, and in vivo import assays","pmids":["24235149"],"confidence":"High","gaps":["Functional division of labor between WxxxF/Y and LVXEF motifs in vivo unclear"]},{"year":2018,"claim":"Defined PEX14 as an Nin-Cout intrinsic membrane protein, fixing the topological framework for interpreting which domain faces the cytosol versus lumen.","evidence":"Protease-protection assays in proteoliposomes and native rat liver peroxisomes","pmids":["30414318"],"confidence":"High","gaps":["Membrane integration mechanism not addressed","Topological dynamics during import not tested here"]},{"year":2020,"claim":"Showed PEX14 is a regulatory node coupling oxidative stress to selective import: Ser232 phosphorylation specifically suppresses catalase import to redistribute catalase to the cytosol and confer H2O2 resistance.","evidence":"Phosphoproteomics, S232D phosphomimetic mutant, in vivo and in vitro import/binding assays","pmids":["32831175"],"confidence":"High","gaps":["Kinase responsible for Ser232 phosphorylation not identified","Physiological signaling context incompletely mapped"]},{"year":2021,"claim":"Mapped the tubulin-binding interface to two β-tubulin C-terminal motifs and demonstrated PEX5 competes for this site, providing a mechanistic switch linking import state to organelle motility; also showed PEX5 docking is preserved at the membrane.","evidence":"NMR mapping, ITC, and competition binding with bicelles/nanodiscs","pmids":["33484719","33937250"],"confidence":"High","gaps":["In vivo regulation of the motility/import switch not established","Membrane association role of NTD surface unresolved"]},{"year":2025,"claim":"Revealed two new layers of import organization: phase separation of the PEX14 C-terminal domain into condensates that recruit PEX5-cargo, and PEX14 acting as the optineurin docking site for pexophagy, unifying biogenesis and turnover at one protein.","evidence":"In vitro phase separation, tetramer structural analysis, and domain swaps; proximity labeling, Co-IP, domain mapping, and LC3 colocalization","pmids":["40555785","41071103"],"confidence":"High","gaps":["In vivo relevance of condensates not fully established","Optineurin interaction is a single-lab finding without reciprocal cross-validation","Trigger for PEX14-mediated pexophagy unclear"]},{"year":2026,"claim":"Demonstrated that the PEX14 N-terminus undergoes ATP- and ubiquitination-dependent topological reorientation driven by PEX6/PEX26, connecting receptor recycling to dynamic remodeling of the docking platform.","evidence":"Protease protection, immunofluorescence, AAA+ ATPase and ubiquitin pathway inhibition","pmids":["41581879"],"confidence":"Medium","gaps":["Direct demonstration of N-terminal movement at single-molecule level lacking","Functional consequence of reorientation for import cycle not quantified","Single-lab study"]},{"year":null,"claim":"How the docking, cargo-release, condensate, motility, and pexophagy functions of PEX14 are temporally and spatially coordinated within a single import/turnover cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model of the assembled translocon in the membrane","Kinase and upstream signals controlling Ser232 phosphorylation unknown","Interplay between condensate formation and topological remodeling untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,6,9]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,12]},{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[0,18]}],"localization":[{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[1,11,16]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8,19]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,9]}],"complexes":["peroxisomal docking/translocation complex"],"partners":["PEX5","PEX13","PEX19","TUBB","PEX6","PEX26","OPTN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75381","full_name":"Peroxisomal membrane protein PEX14","aliases":["PTS1 receptor-docking protein","Peroxin-14","Peroxisomal membrane anchor protein PEX14"],"length_aa":377,"mass_kda":41.2,"function":"Component of the PEX13-PEX14 docking complex, a translocon channel that specifically mediates the import of peroxisomal cargo proteins bound to PEX5 receptor (PubMed:24235149, PubMed:28765278, PubMed:9653144). The PEX13-PEX14 docking complex forms a large import pore which can be opened to a diameter of about 9 nm (By similarity). Mechanistically, PEX5 receptor along with cargo proteins associates with the PEX14 subunit of the PEX13-PEX14 docking complex in the cytosol, leading to the insertion of the receptor into the organelle membrane with the concomitant translocation of the cargo into the peroxisome matrix (PubMed:24235149, PubMed:28765278). Plays a key role for peroxisome movement through a direct interaction with tubulin (PubMed:21525035)","subcellular_location":"Peroxisome membrane","url":"https://www.uniprot.org/uniprotkb/O75381/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX14","classification":"Not Classified","n_dependent_lines":58,"n_total_lines":1208,"dependency_fraction":0.048013245033112585},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLNS1A","stoichiometry":0.2},{"gene":"EIF3G","stoichiometry":0.2},{"gene":"ENY2","stoichiometry":0.2},{"gene":"STK38L","stoichiometry":0.2},{"gene":"UBA52","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PEX14","total_profiled":1310},"omim":[{"mim_id":"614887","title":"PEROXISOME BIOGENESIS DISORDER 13A (ZELLWEGER); PBD13A","url":"https://www.omim.org/entry/614887"},{"mim_id":"607583","title":"PEROXISOME BIOGENESIS FACTOR 11G; PEX11G","url":"https://www.omim.org/entry/607583"},{"mim_id":"603360","title":"PEROXISOME BIOGENESIS FACTOR 16; PEX16","url":"https://www.omim.org/entry/603360"},{"mim_id":"603164","title":"PEROXISOME BIOGENESIS FACTOR 3; PEX3","url":"https://www.omim.org/entry/603164"},{"mim_id":"601791","title":"PEROXISOME BIOGENESIS FACTOR 14; PEX14","url":"https://www.omim.org/entry/601791"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Peroxisomes","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PEX14"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O75381","domains":[{"cath_id":"1.10.10.10","chopping":"24-73","consensus_level":"medium","plddt":89.2964,"start":24,"end":73}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75381","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75381-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75381-F1-predicted_aligned_error_v6.png","plddt_mean":67.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PEX14","jax_strain_url":"https://www.jax.org/strain/search?query=PEX14"},"sequence":{"accession":"O75381","fasta_url":"https://rest.uniprot.org/uniprotkb/O75381.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75381/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75381"}},"corpus_meta":[{"pmid":"10026185","id":"PMC_10026185","title":"Recombinant human peroxisomal targeting signal receptor PEX5. Structural basis for interaction of PEX5 with PEX14.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10026185","citation_count":154,"is_preprint":false},{"pmid":"9009266","id":"PMC_9009266","title":"The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.","date":"1997","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9009266","citation_count":116,"is_preprint":false},{"pmid":"11438541","id":"PMC_11438541","title":"The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11438541","citation_count":101,"is_preprint":false},{"pmid":"21976670","id":"PMC_21976670","title":"PEX5 protein binds monomeric catalase blocking its tetramerization and releases 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PEX5 possesses multiple binding sites for PEX14 distributed throughout its N-terminal half, and a pentapeptide motif reiterated seven times in PEX5 was identified as the determinant for interaction with PEX14.\",\n      \"method\": \"Surface plasmon resonance, in vitro binding assay with recombinant proteins, sizing chromatography\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, SPR quantification, multiple orthogonal methods in one study\",\n      \"pmids\": [\"10026185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"H. polymorpha Pex14p is a peroxisomal membrane protein essential for import of matrix proteins; loss of Pex14p results in peroxisomal membrane remnants containing only small amounts of matrix proteins (AO, catalase, DHAS), with the bulk mislocalized to the cytosol. Overproduction of Pex14p causes a peroxisome-deficient phenotype with numerous small vesicles lacking matrix proteins, indicating stoichiometry of Pex14p relative to other biogenesis components is critical.\",\n      \"method\": \"Genetic complementation, biochemical fractionation, electron microscopy, gene disruption\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation, fractionation, EM, and overexpression phenotype independently replicated in multiple mutant backgrounds\",\n      \"pmids\": [\"9009266\"],\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)) of human PEX5 each independently bind to the same site in the N-terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis showed the conserved aromatic amino acids at positions 1 and 5 of the motif are essential for high affinity binding, proposed to form hydrophobic anchors via 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 / Strong — mutagenesis plus SPR quantification plus two-hybrid, multiple orthogonal methods confirming the same binding site\",\n      \"pmids\": [\"11438541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The N-terminal domain of Pex14 (Pex14-N) adopts a three-helical fold. Both PEX5 and PEX19 ligand helices bind competitively to the same surface of Pex14-N, but with opposite directionality. The Pex5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19 mediate this recognition through conserved aromatic side chains. 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, in vitro binding assays, site-directed mutagenesis, in vivo localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with mutagenesis and functional in vivo validation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"19197237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human PEX14 interacts directly with tubulin, and this binding is mediated by the conserved N-terminal domain of PEX14. PEX14-deficient cells lose the ability of peroxisomal remnants to move along microtubules, establishing PEX14 as the membrane anchor linking peroxisomes to microtubules for motility.\",\n      \"method\": \"Affinity purification of native PEX14 complexes, mass spectrometry, size-exclusion chromatography, in vitro binding, live cell imaging, PEX14-deficient cell analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-purification, direct binding demonstrated in vitro, loss-of-function phenotype with specific motility readout\",\n      \"pmids\": [\"21525035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PEX5 binds monomeric catalase and potently inhibits its tetramerization; no such complex was detected with tetrameric catalase. The N-terminal domain of PEX14 disrupts the PEX5-catalase interaction, with one or two of the seven PEX14-binding diaromatic motifs of PEX5 involved, indicating PEX14 participates in the cargo protein release step.\",\n      \"method\": \"In vitro binding assays, biochemical reconstitution, domain deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined domain mutants, multiple biochemical experiments in a single rigorous study\",\n      \"pmids\": [\"21976670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A novel PEX5-PEX14 interaction site in PEX5 with the sequence LVAEF (consensus LVXEF) was identified. NMR structure of the Pex5-(57-71)/Pex14-NTD complex showed this motif binds in a similar alpha-helical orientation as the WxxxF/Y motif but with the tryptophan pocket occupied by leucine. The LVXEF motif has 33-fold faster dissociation from PEX14 than WxxxF/Y motifs, and alanine substitution of LVAEF strongly impairs peroxisomal matrix protein import in vivo.\",\n      \"method\": \"Peptide library ligand blot screening, NMR structure determination, surface plasmon resonance, mutagenesis, in vivo import assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure, SPR kinetics, mutagenesis, and in vivo functional assays in one study\",\n      \"pmids\": [\"24235149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Pex14 is phosphorylated at Ser232 in response to H2O2-induced oxidative stress in mammalian cells. This phosphorylation suppresses peroxisomal import of catalase specifically (but not canonical PTS1 proteins) by selectively impairing the interaction of catalase with the Pex14-Pex5 complex in vitro. A phosphomimetic Pex14-S232D mutant elevates cytosolic catalase and confers higher cell resistance to H2O2.\",\n      \"method\": \"Phosphoproteomics, site-directed mutagenesis (S232D phosphomimetic), in vivo import assays, in vitro binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphorylation site identified by MS, validated by mutagenesis, in vitro and in vivo functional consequences demonstrated with multiple orthogonal methods\",\n      \"pmids\": [\"32831175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Hansenula polymorpha Pex14 has a unique dual function: it is required both for matrix protein import into peroxisomes and for selective autophagic degradation of peroxisomes (pexophagy). Other components of the peroxisomal translocon (Pex2, Pex10, Pex12, Pex13, Pex17) are not required for pexophagy, making Pex14 the sole peroxisomal translocon component with this dual function.\",\n      \"method\": \"Genetic epistasis analysis with multiple pex deletion mutants, pexophagy assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple mutants in a single lab, establishes unique dual role of Pex14 vs. other translocon peroxins\",\n      \"pmids\": [\"17921697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Pex13 binds Pex14 via two sites: its SH3 domain and a novel intraperoxisomal site. Pex5 also contributes to the association of Pex13 with Pex14. Combining mutations in the novel Pex14-interaction site of Pex13 with a non-Pex13-interacting Pex5(W204A) mutant severely compromised PTS1-dependent import; additionally blocking the SH3-Pex14 interaction completely abrogated PTS2-dependent import and dissociated Pex13 from the docking complex.\",\n      \"method\": \"In vitro binding assays, genetic epistasis with combined mutants, co-purification, growth assays on oleic acid\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, systematic combinatorial mutagenesis showing epistatic relationships, co-purification confirming complex assembly\",\n      \"pmids\": [\"15798189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In H. polymorpha Δpex14 cells, overproduction of the PTS1 receptor Pex5p leads to enhanced import of PTS1 proteins AO and DHAS but not PTS2 protein amine oxidase, and not catalase. Pex5p localizes to the cytosol and outer peroxisomal membrane surface in Δpex14 cells, indicating that Pex5p can bind the peroxisomal membrane and mediate partial import of certain PTS1 proteins in the absence of Pex14p.\",\n      \"method\": \"Genetic overexpression in pex14 null mutant, subcellular fractionation, immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic null background, specific import assays distinguishing PTS1 vs PTS2 cargo, localization studies\",\n      \"pmids\": [\"10777551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PEX14 is a bona fide intrinsic membrane protein with Nin-Cout topology (N-terminus facing the peroxisomal lumen, C-terminus facing the cytosol), as determined in proteoliposomes and purified rat liver peroxisomes.\",\n      \"method\": \"Protease-protection assays on proteoliposomes and native peroxisomes, mass spectrometry, Edman degradation, western blotting with domain-specific antibodies\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — protease protection assays in reconstituted proteoliposomes and native organelles with multiple detection methods in one rigorous study\",\n      \"pmids\": [\"30414318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The PEX14 N-terminal domain (NTD) binds to microtubular filaments in vitro with nanomolar affinity, interacting with two motifs in the C-terminal region of human β-tubulin. PEX5 competes with β-tubulin for binding to PEX14-NTD, such that PEX5 binding to PEX14 can prevent peroxisome anchoring to microtubules, providing a mechanistic link between peroxisomal protein import and motility.\",\n      \"method\": \"In vitro binding assays, NMR spectroscopy, competition binding experiments, dissociation constant measurement\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined domains, NMR-based mapping, competition assays identifying specific tubulin motifs\",\n      \"pmids\": [\"33484719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The PEX14 NTD weakly interacts with membrane-mimicking bicelles via a surface that partially overlaps with the WxxxF/Y binding site. The PEX5-PEX14 NTD interaction is largely unaffected by the presence of membranes, with reduced binding enthalpy compensated by reduced entropy loss, indicating docking of PEX5 to PEX14 at the membrane does not reduce overall 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 in membrane-mimicking systems, single lab, two orthogonal biophysical methods\",\n      \"pmids\": [\"33937250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Small molecules disrupting the Trypanosoma PEX14-PEX5 protein-protein interaction cause mislocalization of glycosomal enzymes, metabolic catastrophe, and parasite death. High-resolution X-ray crystal structures of inhibitors bound to TbPEX14 were obtained, revealing the structural basis for blocking PEX14-PEX5 interaction as a drug target.\",\n      \"method\": \"X-ray crystallography of inhibitor-PEX14 complexes, NMR binding studies, cell-based trypanocidal assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — X-ray crystal structures with NMR validation and cell-based functional assays, published in high-impact journal with replication across multiple compound classes\",\n      \"pmids\": [\"28360328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"T. brucei PEX14 is associated with glycosomes and its N-terminal part binds specifically to TbPEX5. RNAi-mediated depletion of TbPEX14 causes mislocalization of glycosomal proteins (PTS1, PTS2, and internal I-PTS-containing) to the cytosol and is essential for survival of both bloodstream-form and procyclic trypanosomes.\",\n      \"method\": \"Subcellular fractionation, in vitro binding with recombinant proteins, RNA interference, immunofluorescence\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi knockdown with defined import phenotype across multiple cargo classes, direct binding assay, multiple orthogonal methods\",\n      \"pmids\": [\"12709066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human PEX14 is required for both PTS1- and PTS2-dependent peroxisomal matrix protein import in human cells. Loss-of-function (nonsense mutation p.Q185X) causes Zellweger syndrome; transfection with wild-type PEX14 rescued import in patient fibroblasts, establishing PEX14 as the 13th complementation group gene for peroxisome biogenesis disorders.\",\n      \"method\": \"Genetic complementation in patient fibroblasts, immunocytochemistry, mutational analysis\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementation rescue of defined human mutation, import assays for both PTS1 and PTS2, single patient study\",\n      \"pmids\": [\"15146459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Solution structure of the human Pex5-Pex14-PTS1 complex determined by small angle X-ray scattering reveals a 1:6 stoichiometry for the Pex5-Pex14 complex. The N-terminus of Pex5 remains extended in the presence of cargo and Pex14, 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 model with stoichiometry determination, single lab, limited resolution\",\n      \"pmids\": [\"19584060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The C-terminal domain (CTD) of human PEX14 undergoes phase separation in vitro, forming condensates that recruit PEX5-cargo complexes carrying PTS1 or PTS2 signals. hPEX14 forms tetramers, and hPEX14 and hPEX13 form immiscible condensates. Replacing hPEX14 CTD with other phase-separating polypeptides partially restores peroxisomal import; electrostatic interactions and the specific CTD sequence are essential for import.\",\n      \"method\": \"In vitro phase separation assays, structural analysis of PEX14 tetramer, domain replacement experiments, cargo recruitment assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of phase separation, structural analysis, domain swap experiments with functional rescue, multiple orthogonal methods in one study\",\n      \"pmids\": [\"40555785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PEX14 acts as a docking site for the autophagy receptor optineurin (OPTN) on the peroxisomal membrane, enabling OPTN-mediated pexophagy. PEX14 and OPTN interact through their coiled-coil and ubiquitin-binding domains, respectively. PEX14-OPTN complexes colocalize with LC3, and bafilomycin A1 suppresses OPTN-mediated peroxisome degradation.\",\n      \"method\": \"Proximity labeling, co-immunoprecipitation, domain mapping, fluorescence microscopy, autophagy inhibitor treatment\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling and biochemical interaction mapping with domain specificity, colocalization with autophagy markers, single lab\",\n      \"pmids\": [\"41071103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The N-terminal domain of Pex14 undergoes ATP- and ubiquitination-dependent topological remodeling regulated by the AAA+ ATPase Pex6 and its membrane-recruiting partner Pex26. Under normal conditions, the Pex14 N-terminus is oriented toward the peroxisomal lumen; deficiency of Pex6 or Pex26, or pharmacological inhibition of AAA+ ATPases, causes the N-terminus to become exposed to the cytoplasm. Inhibition of ubiquitin activation blocks this reorientation, likely by preventing Pex5 ubiquitination and extraction.\",\n      \"method\": \"Immunofluorescence microscopy, protease protection assays, pharmacological inhibition of AAA+ ATPases, ubiquitin pathway inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protease protection assays with genetic and pharmacological perturbations in multiple conditions, single lab study\",\n      \"pmids\": [\"41581879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In procyclic T. brucei, RNAi knockdown of PEX14 causes mislocalization of glycosomal enzymes and cell death that is triggered by glucose or fructose, but not in the absence of sugars. Double RNAi of PEX14 and hexokinase rescued cells from glucose toxicity even though glycosomal proteins remained mislocalized, establishing that the toxicity of PEX14 depletion in glucose is due to unregulated cytosolic glycolysis.\",\n      \"method\": \"RNA interference (single and double knockdown), genetic epistasis, cell viability assays with different carbon sources\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with double RNAi, multiple carbon source conditions, establishes pathway position of PEX14 in metabolic toxicity\",\n      \"pmids\": [\"15637070\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX14 is a peroxisomal membrane protein with Nin-Cout topology that serves as the central docking platform of the peroxisomal import machinery: its conserved N-terminal domain binds the WxxxF/Y and LVXEF motifs of the cycling import receptor PEX5 with nanomolar affinity, releases cargo (including catalase, whose release is facilitated by PEX14 displacing PEX5) upon docking, and interacts with PEX13 via two direct sites to organize the translocation complex; the Pex14 N-terminal domain also undergoes ATP- and ubiquitination-dependent topological remodeling driven by Pex6 during Pex5 recycling; the C-terminal domain undergoes phase separation to form condensates that recruit PEX5-cargo complexes; PEX14 additionally binds β-tubulin through its N-terminal domain to anchor peroxisomes to microtubules for motility, a function competitively regulated by PEX5; phosphorylation of PEX14 at Ser232 in response to H2O2 selectively suppresses catalase import to redistribute catalase to the cytosol; and PEX14 serves as the docking site for the autophagy receptor optineurin, enabling selective autophagic degradation of peroxisomes (pexophagy).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PEX14 is an intrinsic peroxisomal membrane protein that functions as the central docking platform of the matrix protein import machinery, required for import of both PTS1- and PTS2-targeted proteins [#1, #16]. It adopts an Nin-Cout topology, with the conserved three-helical N-terminal domain (NTD) and the C-terminus exposed to the cytosol [#11, #3]. The NTD is the principal receptor-binding surface: it engages the cycling import receptor PEX5 with low-nanomolar affinity through PEX5's reiterated di-aromatic WxxxF/Y pentapeptide motifs and a kinetically distinct LVXEF motif, both of which dock as amphipathic helices via conserved aromatic anchors [#0, #2, #6]. The same NTD surface binds the PEX19 F/YFxxxF motif competitively, coupling receptor docking to PEX14 membrane targeting [#3]. Beyond receptor capture, PEX14 mediates cargo release, disrupting the PEX5-catalase complex to participate in matrix protein delivery [#5], and organizes the translocation complex by binding PEX13 through both an SH3-domain site and a second intraperoxisomal site [#9]. The receptor cycle is regulated downstream by AAA+ ATPase-driven, ubiquitination-dependent topological remodeling of the PEX14 N-terminus controlled by PEX6/PEX26 [#20], while the cytosolic C-terminal domain undergoes phase separation into condensates that recruit PEX5-cargo complexes to drive import [#18]. PEX14 carries additional, separable functions: its NTD binds β-tubulin to anchor peroxisomes to microtubules for motility, a contact competitively displaced by PEX5 to couple import state to organelle movement [#4, #12]; phosphorylation at Ser232 upon H2O2 stress selectively blocks catalase import to redistribute catalase to the cytosol [#7]; and it serves as the membrane docking site for the autophagy receptor optineurin, enabling pexophagy [#19, #8]. Loss-of-function mutation (p.Q185X) causes Zellweger syndrome, defining PEX14 as a peroxisome biogenesis disorder gene [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that Pex14 is a peroxisomal membrane protein essential for matrix protein import, and that its abundance relative to other biogenesis components must be tightly controlled.\",\n      \"evidence\": \"Gene disruption, complementation, fractionation and EM in H. polymorpha\",\n      \"pmids\": [\"9009266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners and mechanism of import not defined\", \"Topology unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified PEX14 as a high-affinity PEX5 receptor docking partner, answering how the cycling receptor engages the membrane.\",\n      \"evidence\": \"SPR and in vitro binding with recombinant human PEX5 and PEX14-(1-78)\",\n      \"pmids\": [\"10026185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of binding not resolved\", \"Stoichiometry not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined the molecular determinant of docking, showing seven di-aromatic pentapeptide repeats in PEX5 each bind the PEX14 NTD via conserved aromatic anchors.\",\n      \"evidence\": \"Two-hybrid, mutagenesis, and SPR in mammalian/in vitro systems\",\n      \"pmids\": [\"11438541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the complex still lacking\", \"Why seven repeats unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed PEX14 organizes the translocon by binding PEX13 through two distinct sites, with PEX5 contributing, defining how the docking complex is assembled and how PTS1 vs PTS2 import depend on these contacts.\",\n      \"evidence\": \"Combinatorial mutagenesis, co-purification, and oleic-acid growth assays in yeast\",\n      \"pmids\": [\"15798189\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative complex architecture not resolved\", \"Dynamics during the import cycle unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved the three-helical PEX14 NTD fold and revealed competitive, oppositely oriented binding of PEX5 and PEX19 ligand helices, linking receptor docking to PEX14 membrane targeting; SAXS established a 1:6 PEX5:PEX14 stoichiometry.\",\n      \"evidence\": \"NMR structure, mutagenesis, in vivo localization; SAXS solution model\",\n      \"pmids\": [\"19197237\", \"19584060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the full complex absent\", \"Functional meaning of 1:6 ratio in vivo unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Uncovered two distinct PEX14 functions beyond docking: facilitating cargo release by disrupting the PEX5-catalase complex, and anchoring peroxisomes to microtubules via direct tubulin binding through the NTD.\",\n      \"evidence\": \"In vitro reconstitution and domain deletion; affinity purification, in vitro binding, and live-cell motility assays in PEX14-deficient cells\",\n      \"pmids\": [\"21976670\", \"21525035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling docking surface to two ligand classes not fully resolved\", \"In vivo balance between motility and import unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified the LVXEF motif as a second class of PEX5 docking sequence with faster dissociation kinetics, refining the model of how receptor engagement and release are kinetically tuned.\",\n      \"evidence\": \"Peptide library screen, NMR structure, SPR kinetics, and in vivo import assays\",\n      \"pmids\": [\"24235149\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional division of labor between WxxxF/Y and LVXEF motifs in vivo unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined PEX14 as an Nin-Cout intrinsic membrane protein, fixing the topological framework for interpreting which domain faces the cytosol versus lumen.\",\n      \"evidence\": \"Protease-protection assays in proteoliposomes and native rat liver peroxisomes\",\n      \"pmids\": [\"30414318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane integration mechanism not addressed\", \"Topological dynamics during import not tested here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed PEX14 is a regulatory node coupling oxidative stress to selective import: Ser232 phosphorylation specifically suppresses catalase import to redistribute catalase to the cytosol and confer H2O2 resistance.\",\n      \"evidence\": \"Phosphoproteomics, S232D phosphomimetic mutant, in vivo and in vitro import/binding assays\",\n      \"pmids\": [\"32831175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Ser232 phosphorylation not identified\", \"Physiological signaling context incompletely mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped the tubulin-binding interface to two β-tubulin C-terminal motifs and demonstrated PEX5 competes for this site, providing a mechanistic switch linking import state to organelle motility; also showed PEX5 docking is preserved at the membrane.\",\n      \"evidence\": \"NMR mapping, ITC, and competition binding with bicelles/nanodiscs\",\n      \"pmids\": [\"33484719\", \"33937250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulation of the motility/import switch not established\", \"Membrane association role of NTD surface unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed two new layers of import organization: phase separation of the PEX14 C-terminal domain into condensates that recruit PEX5-cargo, and PEX14 acting as the optineurin docking site for pexophagy, unifying biogenesis and turnover at one protein.\",\n      \"evidence\": \"In vitro phase separation, tetramer structural analysis, and domain swaps; proximity labeling, Co-IP, domain mapping, and LC3 colocalization\",\n      \"pmids\": [\"40555785\", \"41071103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of condensates not fully established\", \"Optineurin interaction is a single-lab finding without reciprocal cross-validation\", \"Trigger for PEX14-mediated pexophagy unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated that the PEX14 N-terminus undergoes ATP- and ubiquitination-dependent topological reorientation driven by PEX6/PEX26, connecting receptor recycling to dynamic remodeling of the docking platform.\",\n      \"evidence\": \"Protease protection, immunofluorescence, AAA+ ATPase and ubiquitin pathway inhibition\",\n      \"pmids\": [\"41581879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration of N-terminal movement at single-molecule level lacking\", \"Functional consequence of reorientation for import cycle not quantified\", \"Single-lab study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the docking, cargo-release, condensate, motility, and pexophagy functions of PEX14 are temporally and spatially coordinated within a single import/turnover cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model of the assembled translocon in the membrane\", \"Kinase and upstream signals controlling Ser232 phosphorylation unknown\", \"Interplay between condensate formation and topological remodeling untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 6, 9]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 12]},\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [0, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005778\", \"supporting_discovery_ids\": [1, 11]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [1, 11, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8, 19]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 9]}\n    ],\n    \"complexes\": [\"peroxisomal docking/translocation complex\"],\n    \"partners\": [\"PEX5\", \"PEX13\", \"PEX19\", \"TUBB\", \"PEX6\", \"PEX26\", \"OPTN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}