{"gene":"PEX1","run_date":"2026-04-29T11:37:58","timeline":{"discoveries":[{"year":1997,"finding":"Human PEX1 encodes a 147-kDa AAA-ATPase required for peroxisomal matrix protein import; PEX1-deficient cells show severe defects in matrix protein import and destabilization of PEX5 (the PTS1 receptor), even though peroxisomes are present and capable of importing peroxisomal membrane proteins.","method":"Functional complementation of CG1 patient fibroblasts by PEX1 expression; biochemical analysis of PEX5 stability in PEX1-deficient cells","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — independently replicated across two labs in the same journal issue with complementation rescue and biochemical readouts","pmids":["9398847","9398848"],"is_preprint":false},{"year":1998,"finding":"PEX1 and PEX6 physically interact with each other; overexpression of PEX6 can suppress PEX1-deficient phenotypes and vice versa in an allele-specific manner. The common disease mutation G843D attenuates the PEX1–PEX6 interaction both in the yeast two-hybrid system and in vitro pull-down.","method":"Yeast two-hybrid assay, in vitro pull-down, genetic suppression by overexpression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — reciprocal genetic and direct physical interaction evidence with multiple orthogonal methods","pmids":["9671729"],"is_preprint":false},{"year":1998,"finding":"The PEX1-G843D missense mutation causes a temperature-sensitive peroxisome assembly defect: peroxisomes form at 30°C but not at 37°C in IRD patient fibroblasts, indicating the mutation results in a temperature-sensitive protein.","method":"Temperature-shift experiments in patient fibroblasts and transfected CHO mutant cells; morphological and biochemical analysis of peroxisome formation","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — direct cellular experiment with specific phenotypic readout replicated in patient cells and CHO cell model","pmids":["9817926"],"is_preprint":false},{"year":2001,"finding":"PEX1-G843D protein is largely degraded in vivo at 37°C but is stabilized at 30°C; Pex1p-G843D interacts with Pex6p at ~50% of normal efficiency, while ZS-associated PEX1 mutants (L664P; deletion 634–690) are stable but fail to bind Pex6p, linking PEX1–PEX6 interaction failure to severe disease.","method":"Immunoblotting for PEX1 protein levels at permissive/nonpermissive temperature; co-immunoprecipitation of Pex1p mutants with Pex6p","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — direct protein stability and interaction assays with multiple mutants correlating molecular phenotype to disease severity","pmids":["11439091"],"is_preprint":false},{"year":2001,"finding":"Complete absence of PEX1 protein correlates with severe Zellweger syndrome, whereas residual PEX1 protein (especially from the G843D allele) is found in milder NALD/IRD phenotypes. Growing patient fibroblasts at 30°C increases PEX1-G843D protein 2–3-fold and restores peroxisomal function, indicating the G843D mutation produces a misfolded but partially functional protein.","method":"Immunoblotting of patient fibroblasts; peroxisomal function assays at 30°C vs. 37°C","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — multiple patient cell lines with protein level measurement and functional rescue at permissive temperature","pmids":["11389485"],"is_preprint":false},{"year":2004,"finding":"Crystal structure of the mouse PEX1 N-terminal domain (NTD) at 2.05 Å resolution reveals a double-psi-barrel fold homologous to the N-terminal domains of VCP/p97 and NSF, classifying PEX1 as a membrane-related type II AAA-ATPase and suggesting its NTD may serve as an adaptor-binding domain.","method":"X-ray crystallography; computational structure comparison","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure at 2.05 Å with structural validation and functional inference","pmids":["15328346"],"is_preprint":false},{"year":2006,"finding":"The N-terminal domain of PEX1 binds phosphoinositides (preferentially PI3P and PI4P); a conserved arginine residue surrounded by hydrophobic residues is essential for this lipid binding as demonstrated by mutagenesis.","method":"Lipid-binding assay with isolated N-terminal domain; site-directed mutagenesis of conserved arginine","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 1/2 — in vitro lipid-binding assay with mutagenesis, single lab","pmids":["17018057"],"is_preprint":false},{"year":2006,"finding":"Mutation of the Walker A1 motif of Pex1p (G606E in CHO cells, equivalent to a residue in the first AAA cassette) abolishes Pex1p–Pex6p interaction at 37°C but not at 30°C, and prevents Pex1p targeting to peroxisomes at 37°C while allowing peroxisomal localization at 30°C.","method":"Temperature-sensitive CHO pex1 mutant isolation; RT-PCR mutation identification; co-immunoprecipitation; immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — defined mutation with functional and localization readouts, single lab","pmids":["16723118"],"is_preprint":false},{"year":2015,"finding":"Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex (trimer of Pex1/Pex6 dimers) with triangular geometry. The D2 domains of Pex6 constitute the main ATPase activity; both D2 domains harbor essential substrate-binding motifs. ATP hydrolysis produces a pumping motion suggesting substrate translocation through the central channel. Walker B mutation in one D2 domain leads to ATP hydrolysis in the neighboring domain.","method":"Cryo-EM structural analysis; ATPase activity assays; Walker B mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure combined with biochemical ATPase assays and mutagenesis","pmids":["26066397"],"is_preprint":false},{"year":2015,"finding":"Cryo-EM structures of Pex1/Pex6 in different nucleotide states reveal that Pex1 and Pex6 alternate in an unprecedented heterohexameric double ring; N1 of Pex1 is mobile; the D1 ring is catalytically inactive and symmetric while the D2 ring is active and asymmetric. These features are analogous to p97, supporting a role for Pex1/Pex6 in receptor extraction from the peroxisomal membrane analogous to p97 in ERAD.","method":"Cryo-EM with computational domain fitting; nucleotide-state-dependent structural comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structures in multiple nucleotide states with model building","pmids":["26170309"],"is_preprint":false},{"year":2015,"finding":"Induced depletion of Pex1 in yeast blocks import of peroxisomal matrix proteins but does not affect delivery of peroxisomal membrane proteins; pex1 cells contain peroxisomal membrane remnants (ghosts) that lack matrix proteins. Re-introduction of Pex1 into pex1-deficient cells restores matrix protein import into these ghosts, confirming Pex1's direct and essential role in matrix protein import.","method":"Conditional depletion of Pex1; electron microscopy including tomography; immunocytochemistry; complementation by Pex1 re-introduction","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (EM, immuno-EM, functional rescue) in two complementary studies","pmids":["26644516","26644511"],"is_preprint":false},{"year":2018,"finding":"The Pex1/Pex6 complex is a protein translocase that unfolds its substrate Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner by processive threading through the central pore. Pex15 binds the N-terminal domains of Pex6 before its disordered C-terminal region engages pore loops. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to peroxisomal import machinery.","method":"In vitro unfolding assays; cryo-EM of Pex15–Pex1/Pex6 complex; pore-loop mutagenesis; binding assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro translocase activity with structural validation and mutagenesis","pmids":["29321502"],"is_preprint":false},{"year":2018,"finding":"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) interacts directly with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, demonstrating that Ub-PEX5 is a bona fide substrate of the PEX1–PEX6 complex.","method":"Cell-free in vitro system with photoaffinity cross-linking and protein PEGylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro reconstitution with photoaffinity cross-linking demonstrating direct substrate contact and unfolding","pmids":["29884772"],"is_preprint":false},{"year":2010,"finding":"PEX1-G843D is a misfolded protein amenable to chaperone therapy; small-molecule compounds can partially recover peroxisomal matrix protein import in PEX1-G843D patient fibroblasts, as shown by redistribution of a GFP-PTS1 reporter from cytosol to peroxisomes.","method":"High-content screening assay with GFP-PTS1 reporter in patient fibroblasts; confirmatory biochemical assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — cell-based assay with independent confirmatory assays, single lab","pmids":["20212125"],"is_preprint":false},{"year":2014,"finding":"Deficiency in Pex1 (and Pex6/Pex15) leads to accumulation of ubiquitinated receptors at the peroxisomal membrane and enhanced pexophagy mediated by the Atg36 receptor and Atg11; genetic analysis showed that preventing receptor ubiquitin accumulation does not abolish pexophagy in S. cerevisiae.","method":"Genetic epistasis in S. cerevisiae pex1Δ mutants; autophagy assays; immunofluorescence","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with defined pathway components, single lab","pmids":["24657987"],"is_preprint":false},{"year":2020,"finding":"Blocking pexophagy (by deleting ATG genes) in yeast pex1Δ cells does not restore peroxisomal matrix protein import or beta-oxidation function, demonstrating that Pex1 is directly and essentially required for matrix protein import, and that pexophagy is a consequence, not the cause, of import defects.","method":"Genetic epistasis (pex1Δ combined with autophagy gene deletions); peroxisomal import and beta-oxidation functional assays in S. cerevisiae","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with functional readouts, single lab","pmids":["32013259"],"is_preprint":false},{"year":2022,"finding":"An alternatively spliced human PEX1 isoform lacking 321 amino acids of the N-terminal region fails to rescue peroxisomal import defects in PEX1-KO HEK293 cells but does reduce the number of pre-peroxisomal vesicles, suggesting a dual moonlighting function of human PEX1 in both matrix protein import and regulation of pre-peroxisomal vesicles.","method":"CRISPR/Cas9 PEX1 knockout in HEK293 cells; complementation with full-length vs. N-terminal truncated PEX1 isoform; peroxisome import and morphology assays","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — KO complementation with defined truncation mutant, single lab","pmids":["36534601"],"is_preprint":false},{"year":2022,"finding":"Conditional knockout of Pex1 specifically in inner hair cells (IHCs) of the mouse inner ear causes progressive hearing loss with decreased ABR wave I amplitude, reduction in ribbon synapse volume, functional impairment of exocytosis, and decreased peroxisome number—demonstrating a direct role of PEX1 in IHC synapse development and auditory function.","method":"Conditional Pex1 knockout (Gfi1-Cre or VGlut3-Cre crossed to floxed Pex1); ABR recordings; IHC synapse morphology and functional analysis; immunofluorescence","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific KO with defined cellular and functional phenotype, single lab","pmids":["36552747"],"is_preprint":false},{"year":2023,"finding":"CryoEM structures of S. cerevisiae Pex1/Pex6 with endogenous substrate trapped in the D2 pore reveal that pairs of Pex1/Pex6 D2 subdomains engage substrate via a staircase of pore-1 loops; the D1 ring is catalytically inactive but undergoes conformational changes driven by D2 ATP hydrolysis; a 'twin-seam' Pex1/Pex6 D2 heterodimer disengages from the staircase to drive translocation; mechanical forces propagate along unique Pex1/Pex6 interfaces.","method":"CryoEM of substrate-engaged Pex1/Pex6 complex","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM with substrate-engaged complex revealing catalytic mechanism","pmids":["37741838"],"is_preprint":false},{"year":2023,"finding":"The N1 domain of Pex6 binds to both the peroxisomal membrane tether Pex15 and to an extended loop from the D2 ATPase domain of Pex1, influencing Pex1/Pex6 heterohexamer stability; deletion of the Pex6 N1 domain yields an active ATPase in vitro but abolishes Pex1/Pex6 function at the peroxisome in vivo.","method":"X-ray crystallography of Pex6 N1 domain; cryo-EM of Pex1/Pex6; AlphaFold2 predictions; biochemical pull-down assays; in vivo functional complementation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus cryo-EM plus biochemical assays plus in vivo validation, single study","pmids":["38036174"],"is_preprint":false},{"year":2025,"finding":"The HsPEX1-G843D protein is functional as an AAA-ATPase motor but is rapidly degraded by the proteasome due to reduced affinity for PEX6; impaired PEX1–PEX6 assembly is sufficient to trigger PEX1 degradation; fusing a deubiquitinase to PEX1-G843D stabilizes the protein; overexpression of PEX1-G843D restores peroxisome import.","method":"In vitro ATPase assays with ScPex1-G700D; HsPEX1-G843D cell line generation; proteasome inhibition; co-immunoprecipitation affinity measurements; deubiquitinase fusion experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro biochemistry plus cell-based assays with multiple orthogonal approaches in single study","pmids":["40158855"],"is_preprint":false},{"year":2021,"finding":"HNRNPA1 controls PEX1 expression post-transcriptionally; depletion of HNRNPA1 downregulates PEX1, leading to increased peroxisomal ROS and pexophagy that is blocked by ATG5 knockout and by NAC (ROS scavenger) treatment.","method":"HNRNPA1 knockdown; PEX1 expression measurement; ATG5-KO epistasis; ROS assays; pexophagy flux assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal approaches linking post-transcriptional regulation of PEX1 to pexophagy, single lab","pmids":["33545634"],"is_preprint":false},{"year":2025,"finding":"In PEX1-depleted HeLa cells, TBK1 becomes phosphorylated and activated (driven by ROS accumulation), which phosphorylates MARCHF7; MARCHF7 ubiquitinates PXMP4 at lysine 20, and ubiquitinated PXMP4 serves as a recognition signal for the pexophagy receptor NBR1.","method":"Functional screening; MARCHF7 depletion; co-immunoprecipitation; site-directed mutagenesis of PXMP4-K20; NBR1 recruitment assays in PEX1-KD cells","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — pathway placement by multiple genetic and biochemical experiments, single lab","pmids":["41267209"],"is_preprint":false}],"current_model":"PEX1 is an AAA+ ATPase that forms a heterohexameric complex with PEX6 (anchored to the peroxisomal membrane via PEX26/Pex15), where the active D2 ATPase ring processively threads and unfolds monoubiquitinated PEX5 through its central pore to extract and recycle this import receptor from the peroxisomal membrane, thereby resetting the peroxisomal matrix protein import cycle; the common disease allele G843D produces a misfolded, proteasome-susceptible protein with reduced PEX6 affinity, and loss of PEX1 function triggers pexophagy via a TBK1–MARCHF7–PXMP4–NBR1 axis."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing that PEX1 encodes an AAA-ATPase required for peroxisomal matrix protein import resolved the molecular basis of complementation group 1 peroxisome biogenesis disorders and showed that peroxisomal membrane assembly proceeds independently of PEX1.","evidence":"Functional complementation of CG1 patient fibroblasts with PEX1 cDNA; PEX5 stability analysis in patient cells","pmids":["9398847","9398848"],"confidence":"High","gaps":["Mechanism by which PEX1 supports import was unknown","Identity of PEX1 substrates was unclear","Subcellular localization of PEX1 was not determined"]},{"year":1998,"claim":"Demonstrating a direct PEX1–PEX6 physical interaction and that the G843D disease mutation weakens this interaction established the functional unit as a PEX1–PEX6 complex and provided the first molecular explanation for the most common PBD allele.","evidence":"Yeast two-hybrid, in vitro pull-down, and genetic suppression between PEX1 and PEX6; temperature-shift experiments in G843D patient fibroblasts","pmids":["9671729","9817926"],"confidence":"High","gaps":["Stoichiometry and architecture of the PEX1–PEX6 complex were unknown","Whether G843D affects ATPase activity or protein stability was unresolved"]},{"year":2001,"claim":"Correlating PEX1 protein levels and PEX6-binding capacity with disease severity across multiple alleles showed that G843D is a misfolded but partially functional protein whose temperature-sensitive degradation underlies the milder IRD phenotype, while null or interaction-dead alleles cause severe Zellweger syndrome.","evidence":"Immunoblotting and co-immunoprecipitation of PEX1 mutants; peroxisomal function assays at permissive vs. non-permissive temperature in patient fibroblasts","pmids":["11439091","11389485"],"confidence":"High","gaps":["Whether G843D retains intrinsic ATPase activity was not tested","Mechanism of PEX1 degradation (proteasomal vs. other) was not defined"]},{"year":2004,"claim":"Solving the crystal structure of the PEX1 N-terminal domain revealed a double-psi-barrel fold shared with p97/NSF, classifying PEX1 as a membrane-associated type II AAA-ATPase and implicating the NTD as an adaptor-binding platform.","evidence":"X-ray crystallography of mouse PEX1 NTD at 2.05 Å resolution","pmids":["15328346"],"confidence":"High","gaps":["No adaptor or substrate bound to the NTD was identified","Full-length PEX1 structure remained unavailable"]},{"year":2006,"claim":"Identifying phosphoinositide binding by the PEX1 NTD and showing that a Walker A1 mutation in the D1 cassette disrupts PEX6 interaction and peroxisomal targeting suggested that both lipid binding and D1-domain integrity contribute to PEX1 recruitment to peroxisomes.","evidence":"In vitro lipid-binding assays with mutagenesis; CHO pex1 temperature-sensitive mutant analysis with co-IP and immunofluorescence","pmids":["17018057","16723118"],"confidence":"Medium","gaps":["Physiological relevance of PI binding in vivo was not demonstrated","Whether D1 ATP binding is structural or regulatory was unclear"]},{"year":2015,"claim":"Cryo-EM structures of the PEX1–PEX6 heterohexamer revealed an alternating double-ring architecture with an active, asymmetric D2 ring and an inactive, symmetric D1 ring, establishing the structural basis for a processive translocation mechanism analogous to p97.","evidence":"Cryo-EM in multiple nucleotide states; ATPase assays; Walker B mutagenesis","pmids":["26066397","26170309"],"confidence":"High","gaps":["No substrate was captured in the pore","How the complex is tethered to the peroxisomal membrane was structurally unresolved"]},{"year":2015,"claim":"Conditional depletion of Pex1 confirmed it is directly required for matrix protein import and not for membrane protein insertion, and showed that re-introduction of Pex1 restores import into pre-existing peroxisomal membrane ghosts.","evidence":"Conditional Pex1 depletion in yeast; electron microscopy/tomography; complementation rescue","pmids":["26644516","26644511"],"confidence":"High","gaps":["Identity of the specific PEX1–PEX6 substrate during import remained unproven"]},{"year":2018,"claim":"Biochemical reconstitution demonstrated that PEX1–PEX6 is a bona fide protein translocase that unfolds substrates through its central pore: Pex15 is threaded via pore-loop engagement, and monoubiquitinated PEX5 is globally unfolded during ATP-dependent extraction, definitively identifying the import receptor as the physiological substrate.","evidence":"In vitro unfolding assays; cryo-EM of Pex15-bound complex; photoaffinity cross-linking and PEGylation of Ub-PEX5","pmids":["29321502","29884772"],"confidence":"High","gaps":["Whether PEX5 unfolding is coupled to cargo release was not shown","Structural view of PEX5 engaged in the pore was lacking"]},{"year":2023,"claim":"Substrate-engaged cryo-EM structures revealed the D2 pore-loop staircase mechanism and a twin-seam heterodimer disengagement cycle that drives processive translocation, while crystallography of the Pex6 N1 domain showed it bridges both the Pex15 membrane anchor and a Pex1 D2 loop to stabilize the functional hexamer at the peroxisome.","evidence":"CryoEM of substrate-trapped Pex1/Pex6; X-ray crystallography of Pex6 N1; in vivo complementation","pmids":["37741838","38036174"],"confidence":"High","gaps":["How the twin-seam mechanism coordinates with ubiquitin recognition is unknown","Structural basis for PEX26 engagement in the human complex not resolved"]},{"year":2025,"claim":"Demonstrating that PEX1-G843D retains full ATPase motor activity but is rapidly degraded by the proteasome due to impaired PEX6 binding resolved the long-standing question of whether the disease allele is catalytically dead or unstable, and showed that stabilization alone is sufficient to restore peroxisome import.","evidence":"In vitro ATPase assays with ScPex1-G700D; proteasome inhibition; deubiquitinase fusion; co-IP affinity measurements in human cells","pmids":["40158855"],"confidence":"High","gaps":["Whether proteasome inhibition is therapeutically viable in patients is untested","Structural basis of G843D misfolding remains unresolved"]},{"year":2014,"claim":"Genetic studies in yeast established that PEX1 deficiency triggers pexophagy via ubiquitinated receptor accumulation at the peroxisomal membrane, and that blocking autophagy does not restore import, separating the import and pexophagy phenotypes.","evidence":"Genetic epistasis in S. cerevisiae pex1Δ with autophagy mutants; autophagy and import assays","pmids":["24657987","32013259"],"confidence":"Medium","gaps":["Mammalian pexophagy signaling downstream of PEX1 loss was not defined","Whether pexophagy contributes to pathology independently of import failure was unclear"]},{"year":2025,"claim":"A mammalian pexophagy signaling cascade was delineated: PEX1 depletion causes ROS-dependent TBK1 activation, which phosphorylates MARCHF7, leading to ubiquitination of PXMP4 at K20 and NBR1-mediated pexophagy, revealing the specific molecular chain linking PEX1 loss to selective peroxisome degradation.","evidence":"Functional screening, co-IP, MARCHF7 depletion, PXMP4-K20 mutagenesis, NBR1 recruitment assays in PEX1-KD HeLa cells","pmids":["41267209"],"confidence":"Medium","gaps":["Whether this pathway operates in vivo in patient tissues is untested","Contribution of pexophagy to Zellweger spectrum pathology versus primary import failure is unresolved"]},{"year":null,"claim":"Key open questions include: the structural basis for ubiquitin recognition by PEX1–PEX6 during PEX5 extraction; how cargo release from PEX5 is coupled to receptor unfolding; the therapeutic potential of PEX1-G843D stabilization strategies in animal models; and whether the PEX1 N-terminal alternatively spliced isoform has a physiologically distinct function in pre-peroxisomal vesicle regulation.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of ubiquitin-engaged PEX1–PEX6 complex exists","Cargo–receptor uncoupling mechanism is undefined","In vivo therapeutic validation of G843D stabilization is lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,8,9,18,20]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[11,12,18]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[7,10,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,10,16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[14,15,21,22]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,10]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,12,18]}],"complexes":["PEX1–PEX6 heterohexamer"],"partners":["PEX6","PEX5","PEX15","PEX26","MARCHF7","NBR1","HNRNPA1"],"other_free_text":[]},"mechanistic_narrative":"PEX1 is a type II AAA+ ATPase essential for peroxisomal matrix protein import, functioning as the motor component of a heterohexameric PEX1–PEX6 complex that extracts monoubiquitinated PEX5 from the peroxisomal membrane to recycle this import receptor [PMID:9398847, PMID:29884772, PMID:26066397]. The PEX1–PEX6 complex adopts a double-ring architecture in which the D1 ring is catalytically inactive and the active D2 ring engages substrate through pore-loop staircases, processively threading and unfolding PEX5 through the central channel in an ATP-hydrolysis-dependent manner [PMID:26170309, PMID:37741838, PMID:29321502]. The complex is anchored to the peroxisomal membrane via the PEX6 N1 domain–Pex15/PEX26 interaction, and loss of PEX1 triggers pexophagy through a TBK1–MARCHF7–PXMP4–NBR1 signaling axis [PMID:38036174, PMID:41267209]. Biallelic PEX1 mutations cause Zellweger spectrum disorders; the common G843D allele produces a motor-competent but misfolded, proteasome-susceptible protein with reduced PEX6 affinity whose function can be partially rescued by lowering temperature or by proteasome inhibition [PMID:9817926, PMID:11439091, PMID:40158855]."},"prefetch_data":{"uniprot":{"accession":"O43933","full_name":"Peroxisomal ATPase PEX1","aliases":["Peroxin-1","Peroxisome biogenesis disorder protein 1","Peroxisome biogenesis factor 1"],"length_aa":1283,"mass_kda":142.9,"function":"Component of the PEX1-PEX6 AAA ATPase complex, a protein dislocase complex that mediates the ATP-dependent extraction of the PEX5 receptor from peroxisomal membranes, an essential step for PEX5 recycling (PubMed:11439091, PubMed:16314507, PubMed:16854980, PubMed:21362118, PubMed:29884772). Specifically recognizes PEX5 monoubiquitinated at 'Cys-11', and pulls it out of the peroxisome lumen through the PEX2-PEX10-PEX12 retrotranslocation channel (PubMed:29884772). Extraction by the PEX1-PEX6 AAA ATPase complex is accompanied by unfolding of the TPR repeats and release of bound cargo from PEX5 (PubMed:29884772)","subcellular_location":"Cytoplasm, cytosol; Peroxisome membrane","url":"https://www.uniprot.org/uniprotkb/O43933/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX1","classification":"Not Classified","n_dependent_lines":306,"n_total_lines":1208,"dependency_fraction":0.2533112582781457},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDOST","stoichiometry":0.2},{"gene":"UBA52","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PEX1","total_profiled":1310},"omim":[{"mim_id":"616617","title":"HEIMLER SYNDROME 2; HMLR2","url":"https://www.omim.org/entry/616617"},{"mim_id":"614863","title":"PEROXISOME BIOGENESIS DISORDER 4B; PBD4B","url":"https://www.omim.org/entry/614863"},{"mim_id":"613749","title":"ZINC FINGER PROTEIN 260; 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The common disease mutation G843D attenuates the PEX1–PEX6 interaction both in the yeast two-hybrid system and in vitro pull-down.\",\n      \"method\": \"Yeast two-hybrid assay, in vitro pull-down, genetic suppression by overexpression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal genetic and direct physical interaction evidence with multiple orthogonal methods\",\n      \"pmids\": [\"9671729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The PEX1-G843D missense mutation causes a temperature-sensitive peroxisome assembly defect: peroxisomes form at 30°C but not at 37°C in IRD patient fibroblasts, indicating the mutation results in a temperature-sensitive protein.\",\n      \"method\": \"Temperature-shift experiments in patient fibroblasts and transfected CHO mutant cells; morphological and biochemical analysis of peroxisome formation\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct cellular experiment with specific phenotypic readout replicated in patient cells and CHO cell model\",\n      \"pmids\": [\"9817926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PEX1-G843D protein is largely degraded in vivo at 37°C but is stabilized at 30°C; Pex1p-G843D interacts with Pex6p at ~50% of normal efficiency, while ZS-associated PEX1 mutants (L664P; deletion 634–690) are stable but fail to bind Pex6p, linking PEX1–PEX6 interaction failure to severe disease.\",\n      \"method\": \"Immunoblotting for PEX1 protein levels at permissive/nonpermissive temperature; co-immunoprecipitation of Pex1p mutants with Pex6p\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct protein stability and interaction assays with multiple mutants correlating molecular phenotype to disease severity\",\n      \"pmids\": [\"11439091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Complete absence of PEX1 protein correlates with severe Zellweger syndrome, whereas residual PEX1 protein (especially from the G843D allele) is found in milder NALD/IRD phenotypes. Growing patient fibroblasts at 30°C increases PEX1-G843D protein 2–3-fold and restores peroxisomal function, indicating the G843D mutation produces a misfolded but partially functional protein.\",\n      \"method\": \"Immunoblotting of patient fibroblasts; peroxisomal function assays at 30°C vs. 37°C\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple patient cell lines with protein level measurement and functional rescue at permissive temperature\",\n      \"pmids\": [\"11389485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Crystal structure of the mouse PEX1 N-terminal domain (NTD) at 2.05 Å resolution reveals a double-psi-barrel fold homologous to the N-terminal domains of VCP/p97 and NSF, classifying PEX1 as a membrane-related type II AAA-ATPase and suggesting its NTD may serve as an adaptor-binding domain.\",\n      \"method\": \"X-ray crystallography; computational structure comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure at 2.05 Å with structural validation and functional inference\",\n      \"pmids\": [\"15328346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal domain of PEX1 binds phosphoinositides (preferentially PI3P and PI4P); a conserved arginine residue surrounded by hydrophobic residues is essential for this lipid binding as demonstrated by mutagenesis.\",\n      \"method\": \"Lipid-binding assay with isolated N-terminal domain; site-directed mutagenesis of conserved arginine\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro lipid-binding assay with mutagenesis, single lab\",\n      \"pmids\": [\"17018057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Mutation of the Walker A1 motif of Pex1p (G606E in CHO cells, equivalent to a residue in the first AAA cassette) abolishes Pex1p–Pex6p interaction at 37°C but not at 30°C, and prevents Pex1p targeting to peroxisomes at 37°C while allowing peroxisomal localization at 30°C.\",\n      \"method\": \"Temperature-sensitive CHO pex1 mutant isolation; RT-PCR mutation identification; co-immunoprecipitation; immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined mutation with functional and localization readouts, single lab\",\n      \"pmids\": [\"16723118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex (trimer of Pex1/Pex6 dimers) with triangular geometry. The D2 domains of Pex6 constitute the main ATPase activity; both D2 domains harbor essential substrate-binding motifs. ATP hydrolysis produces a pumping motion suggesting substrate translocation through the central channel. Walker B mutation in one D2 domain leads to ATP hydrolysis in the neighboring domain.\",\n      \"method\": \"Cryo-EM structural analysis; ATPase activity assays; Walker B mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with biochemical ATPase assays and mutagenesis\",\n      \"pmids\": [\"26066397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cryo-EM structures of Pex1/Pex6 in different nucleotide states reveal that Pex1 and Pex6 alternate in an unprecedented heterohexameric double ring; N1 of Pex1 is mobile; the D1 ring is catalytically inactive and symmetric while the D2 ring is active and asymmetric. These features are analogous to p97, supporting a role for Pex1/Pex6 in receptor extraction from the peroxisomal membrane analogous to p97 in ERAD.\",\n      \"method\": \"Cryo-EM with computational domain fitting; nucleotide-state-dependent structural comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structures in multiple nucleotide states with model building\",\n      \"pmids\": [\"26170309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Induced depletion of Pex1 in yeast blocks import of peroxisomal matrix proteins but does not affect delivery of peroxisomal membrane proteins; pex1 cells contain peroxisomal membrane remnants (ghosts) that lack matrix proteins. Re-introduction of Pex1 into pex1-deficient cells restores matrix protein import into these ghosts, confirming Pex1's direct and essential role in matrix protein import.\",\n      \"method\": \"Conditional depletion of Pex1; electron microscopy including tomography; immunocytochemistry; complementation by Pex1 re-introduction\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (EM, immuno-EM, functional rescue) in two complementary studies\",\n      \"pmids\": [\"26644516\", \"26644511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The Pex1/Pex6 complex is a protein translocase that unfolds its substrate Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner by processive threading through the central pore. Pex15 binds the N-terminal domains of Pex6 before its disordered C-terminal region engages pore loops. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to peroxisomal import machinery.\",\n      \"method\": \"In vitro unfolding assays; cryo-EM of Pex15–Pex1/Pex6 complex; pore-loop mutagenesis; binding assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro translocase activity with structural validation and mutagenesis\",\n      \"pmids\": [\"29321502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) interacts directly with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event, demonstrating that Ub-PEX5 is a bona fide substrate of the PEX1–PEX6 complex.\",\n      \"method\": \"Cell-free in vitro system with photoaffinity cross-linking and protein PEGylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro reconstitution with photoaffinity cross-linking demonstrating direct substrate contact and unfolding\",\n      \"pmids\": [\"29884772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PEX1-G843D is a misfolded protein amenable to chaperone therapy; small-molecule compounds can partially recover peroxisomal matrix protein import in PEX1-G843D patient fibroblasts, as shown by redistribution of a GFP-PTS1 reporter from cytosol to peroxisomes.\",\n      \"method\": \"High-content screening assay with GFP-PTS1 reporter in patient fibroblasts; confirmatory biochemical assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-based assay with independent confirmatory assays, single lab\",\n      \"pmids\": [\"20212125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deficiency in Pex1 (and Pex6/Pex15) leads to accumulation of ubiquitinated receptors at the peroxisomal membrane and enhanced pexophagy mediated by the Atg36 receptor and Atg11; genetic analysis showed that preventing receptor ubiquitin accumulation does not abolish pexophagy in S. cerevisiae.\",\n      \"method\": \"Genetic epistasis in S. cerevisiae pex1Δ mutants; autophagy assays; immunofluorescence\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with defined pathway components, single lab\",\n      \"pmids\": [\"24657987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Blocking pexophagy (by deleting ATG genes) in yeast pex1Δ cells does not restore peroxisomal matrix protein import or beta-oxidation function, demonstrating that Pex1 is directly and essentially required for matrix protein import, and that pexophagy is a consequence, not the cause, of import defects.\",\n      \"method\": \"Genetic epistasis (pex1Δ combined with autophagy gene deletions); peroxisomal import and beta-oxidation functional assays in S. cerevisiae\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with functional readouts, single lab\",\n      \"pmids\": [\"32013259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"An alternatively spliced human PEX1 isoform lacking 321 amino acids of the N-terminal region fails to rescue peroxisomal import defects in PEX1-KO HEK293 cells but does reduce the number of pre-peroxisomal vesicles, suggesting a dual moonlighting function of human PEX1 in both matrix protein import and regulation of pre-peroxisomal vesicles.\",\n      \"method\": \"CRISPR/Cas9 PEX1 knockout in HEK293 cells; complementation with full-length vs. N-terminal truncated PEX1 isoform; peroxisome import and morphology assays\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO complementation with defined truncation mutant, single lab\",\n      \"pmids\": [\"36534601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Conditional knockout of Pex1 specifically in inner hair cells (IHCs) of the mouse inner ear causes progressive hearing loss with decreased ABR wave I amplitude, reduction in ribbon synapse volume, functional impairment of exocytosis, and decreased peroxisome number—demonstrating a direct role of PEX1 in IHC synapse development and auditory function.\",\n      \"method\": \"Conditional Pex1 knockout (Gfi1-Cre or VGlut3-Cre crossed to floxed Pex1); ABR recordings; IHC synapse morphology and functional analysis; immunofluorescence\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with defined cellular and functional phenotype, single lab\",\n      \"pmids\": [\"36552747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CryoEM structures of S. cerevisiae Pex1/Pex6 with endogenous substrate trapped in the D2 pore reveal that pairs of Pex1/Pex6 D2 subdomains engage substrate via a staircase of pore-1 loops; the D1 ring is catalytically inactive but undergoes conformational changes driven by D2 ATP hydrolysis; a 'twin-seam' Pex1/Pex6 D2 heterodimer disengages from the staircase to drive translocation; mechanical forces propagate along unique Pex1/Pex6 interfaces.\",\n      \"method\": \"CryoEM of substrate-engaged Pex1/Pex6 complex\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM with substrate-engaged complex revealing catalytic mechanism\",\n      \"pmids\": [\"37741838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The N1 domain of Pex6 binds to both the peroxisomal membrane tether Pex15 and to an extended loop from the D2 ATPase domain of Pex1, influencing Pex1/Pex6 heterohexamer stability; deletion of the Pex6 N1 domain yields an active ATPase in vitro but abolishes Pex1/Pex6 function at the peroxisome in vivo.\",\n      \"method\": \"X-ray crystallography of Pex6 N1 domain; cryo-EM of Pex1/Pex6; AlphaFold2 predictions; biochemical pull-down assays; in vivo functional complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus cryo-EM plus biochemical assays plus in vivo validation, single study\",\n      \"pmids\": [\"38036174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The HsPEX1-G843D protein is functional as an AAA-ATPase motor but is rapidly degraded by the proteasome due to reduced affinity for PEX6; impaired PEX1–PEX6 assembly is sufficient to trigger PEX1 degradation; fusing a deubiquitinase to PEX1-G843D stabilizes the protein; overexpression of PEX1-G843D restores peroxisome import.\",\n      \"method\": \"In vitro ATPase assays with ScPex1-G700D; HsPEX1-G843D cell line generation; proteasome inhibition; co-immunoprecipitation affinity measurements; deubiquitinase fusion experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro biochemistry plus cell-based assays with multiple orthogonal approaches in single study\",\n      \"pmids\": [\"40158855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HNRNPA1 controls PEX1 expression post-transcriptionally; depletion of HNRNPA1 downregulates PEX1, leading to increased peroxisomal ROS and pexophagy that is blocked by ATG5 knockout and by NAC (ROS scavenger) treatment.\",\n      \"method\": \"HNRNPA1 knockdown; PEX1 expression measurement; ATG5-KO epistasis; ROS assays; pexophagy flux assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches linking post-transcriptional regulation of PEX1 to pexophagy, single lab\",\n      \"pmids\": [\"33545634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In PEX1-depleted HeLa cells, TBK1 becomes phosphorylated and activated (driven by ROS accumulation), which phosphorylates MARCHF7; MARCHF7 ubiquitinates PXMP4 at lysine 20, and ubiquitinated PXMP4 serves as a recognition signal for the pexophagy receptor NBR1.\",\n      \"method\": \"Functional screening; MARCHF7 depletion; co-immunoprecipitation; site-directed mutagenesis of PXMP4-K20; NBR1 recruitment assays in PEX1-KD cells\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway placement by multiple genetic and biochemical experiments, single lab\",\n      \"pmids\": [\"41267209\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX1 is an AAA+ ATPase that forms a heterohexameric complex with PEX6 (anchored to the peroxisomal membrane via PEX26/Pex15), where the active D2 ATPase ring processively threads and unfolds monoubiquitinated PEX5 through its central pore to extract and recycle this import receptor from the peroxisomal membrane, thereby resetting the peroxisomal matrix protein import cycle; the common disease allele G843D produces a misfolded, proteasome-susceptible protein with reduced PEX6 affinity, and loss of PEX1 function triggers pexophagy via a TBK1–MARCHF7–PXMP4–NBR1 axis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PEX1 is a type II AAA+ ATPase essential for peroxisomal matrix protein import, functioning as the motor component of a heterohexameric PEX1–PEX6 complex that extracts monoubiquitinated PEX5 from the peroxisomal membrane to recycle this import receptor [PMID:9398847, PMID:29884772, PMID:26066397]. The PEX1–PEX6 complex adopts a double-ring architecture in which the D1 ring is catalytically inactive and the active D2 ring engages substrate through pore-loop staircases, processively threading and unfolding PEX5 through the central channel in an ATP-hydrolysis-dependent manner [PMID:26170309, PMID:37741838, PMID:29321502]. The complex is anchored to the peroxisomal membrane via the PEX6 N1 domain–Pex15/PEX26 interaction, and loss of PEX1 triggers pexophagy through a TBK1–MARCHF7–PXMP4–NBR1 signaling axis [PMID:38036174, PMID:41267209]. Biallelic PEX1 mutations cause Zellweger spectrum disorders; the common G843D allele produces a motor-competent but misfolded, proteasome-susceptible protein with reduced PEX6 affinity whose function can be partially rescued by lowering temperature or by proteasome inhibition [PMID:9817926, PMID:11439091, PMID:40158855].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that PEX1 encodes an AAA-ATPase required for peroxisomal matrix protein import resolved the molecular basis of complementation group 1 peroxisome biogenesis disorders and showed that peroxisomal membrane assembly proceeds independently of PEX1.\",\n      \"evidence\": \"Functional complementation of CG1 patient fibroblasts with PEX1 cDNA; PEX5 stability analysis in patient cells\",\n      \"pmids\": [\"9398847\", \"9398848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PEX1 supports import was unknown\", \"Identity of PEX1 substrates was unclear\", \"Subcellular localization of PEX1 was not determined\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrating a direct PEX1–PEX6 physical interaction and that the G843D disease mutation weakens this interaction established the functional unit as a PEX1–PEX6 complex and provided the first molecular explanation for the most common PBD allele.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pull-down, and genetic suppression between PEX1 and PEX6; temperature-shift experiments in G843D patient fibroblasts\",\n      \"pmids\": [\"9671729\", \"9817926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the PEX1–PEX6 complex were unknown\", \"Whether G843D affects ATPase activity or protein stability was unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Correlating PEX1 protein levels and PEX6-binding capacity with disease severity across multiple alleles showed that G843D is a misfolded but partially functional protein whose temperature-sensitive degradation underlies the milder IRD phenotype, while null or interaction-dead alleles cause severe Zellweger syndrome.\",\n      \"evidence\": \"Immunoblotting and co-immunoprecipitation of PEX1 mutants; peroxisomal function assays at permissive vs. non-permissive temperature in patient fibroblasts\",\n      \"pmids\": [\"11439091\", \"11389485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether G843D retains intrinsic ATPase activity was not tested\", \"Mechanism of PEX1 degradation (proteasomal vs. other) was not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Solving the crystal structure of the PEX1 N-terminal domain revealed a double-psi-barrel fold shared with p97/NSF, classifying PEX1 as a membrane-associated type II AAA-ATPase and implicating the NTD as an adaptor-binding platform.\",\n      \"evidence\": \"X-ray crystallography of mouse PEX1 NTD at 2.05 Å resolution\",\n      \"pmids\": [\"15328346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No adaptor or substrate bound to the NTD was identified\", \"Full-length PEX1 structure remained unavailable\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying phosphoinositide binding by the PEX1 NTD and showing that a Walker A1 mutation in the D1 cassette disrupts PEX6 interaction and peroxisomal targeting suggested that both lipid binding and D1-domain integrity contribute to PEX1 recruitment to peroxisomes.\",\n      \"evidence\": \"In vitro lipid-binding assays with mutagenesis; CHO pex1 temperature-sensitive mutant analysis with co-IP and immunofluorescence\",\n      \"pmids\": [\"17018057\", \"16723118\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of PI binding in vivo was not demonstrated\", \"Whether D1 ATP binding is structural or regulatory was unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Cryo-EM structures of the PEX1–PEX6 heterohexamer revealed an alternating double-ring architecture with an active, asymmetric D2 ring and an inactive, symmetric D1 ring, establishing the structural basis for a processive translocation mechanism analogous to p97.\",\n      \"evidence\": \"Cryo-EM in multiple nucleotide states; ATPase assays; Walker B mutagenesis\",\n      \"pmids\": [\"26066397\", \"26170309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrate was captured in the pore\", \"How the complex is tethered to the peroxisomal membrane was structurally unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Conditional depletion of Pex1 confirmed it is directly required for matrix protein import and not for membrane protein insertion, and showed that re-introduction of Pex1 restores import into pre-existing peroxisomal membrane ghosts.\",\n      \"evidence\": \"Conditional Pex1 depletion in yeast; electron microscopy/tomography; complementation rescue\",\n      \"pmids\": [\"26644516\", \"26644511\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the specific PEX1–PEX6 substrate during import remained unproven\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Biochemical reconstitution demonstrated that PEX1–PEX6 is a bona fide protein translocase that unfolds substrates through its central pore: Pex15 is threaded via pore-loop engagement, and monoubiquitinated PEX5 is globally unfolded during ATP-dependent extraction, definitively identifying the import receptor as the physiological substrate.\",\n      \"evidence\": \"In vitro unfolding assays; cryo-EM of Pex15-bound complex; photoaffinity cross-linking and PEGylation of Ub-PEX5\",\n      \"pmids\": [\"29321502\", \"29884772\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PEX5 unfolding is coupled to cargo release was not shown\", \"Structural view of PEX5 engaged in the pore was lacking\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Substrate-engaged cryo-EM structures revealed the D2 pore-loop staircase mechanism and a twin-seam heterodimer disengagement cycle that drives processive translocation, while crystallography of the Pex6 N1 domain showed it bridges both the Pex15 membrane anchor and a Pex1 D2 loop to stabilize the functional hexamer at the peroxisome.\",\n      \"evidence\": \"CryoEM of substrate-trapped Pex1/Pex6; X-ray crystallography of Pex6 N1; in vivo complementation\",\n      \"pmids\": [\"37741838\", \"38036174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the twin-seam mechanism coordinates with ubiquitin recognition is unknown\", \"Structural basis for PEX26 engagement in the human complex not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that PEX1-G843D retains full ATPase motor activity but is rapidly degraded by the proteasome due to impaired PEX6 binding resolved the long-standing question of whether the disease allele is catalytically dead or unstable, and showed that stabilization alone is sufficient to restore peroxisome import.\",\n      \"evidence\": \"In vitro ATPase assays with ScPex1-G700D; proteasome inhibition; deubiquitinase fusion; co-IP affinity measurements in human cells\",\n      \"pmids\": [\"40158855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether proteasome inhibition is therapeutically viable in patients is untested\", \"Structural basis of G843D misfolding remains unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Genetic studies in yeast established that PEX1 deficiency triggers pexophagy via ubiquitinated receptor accumulation at the peroxisomal membrane, and that blocking autophagy does not restore import, separating the import and pexophagy phenotypes.\",\n      \"evidence\": \"Genetic epistasis in S. cerevisiae pex1Δ with autophagy mutants; autophagy and import assays\",\n      \"pmids\": [\"24657987\", \"32013259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian pexophagy signaling downstream of PEX1 loss was not defined\", \"Whether pexophagy contributes to pathology independently of import failure was unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A mammalian pexophagy signaling cascade was delineated: PEX1 depletion causes ROS-dependent TBK1 activation, which phosphorylates MARCHF7, leading to ubiquitination of PXMP4 at K20 and NBR1-mediated pexophagy, revealing the specific molecular chain linking PEX1 loss to selective peroxisome degradation.\",\n      \"evidence\": \"Functional screening, co-IP, MARCHF7 depletion, PXMP4-K20 mutagenesis, NBR1 recruitment assays in PEX1-KD HeLa cells\",\n      \"pmids\": [\"41267209\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this pathway operates in vivo in patient tissues is untested\", \"Contribution of pexophagy to Zellweger spectrum pathology versus primary import failure is unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: the structural basis for ubiquitin recognition by PEX1–PEX6 during PEX5 extraction; how cargo release from PEX5 is coupled to receptor unfolding; the therapeutic potential of PEX1-G843D stabilization strategies in animal models; and whether the PEX1 N-terminal alternatively spliced isoform has a physiologically distinct function in pre-peroxisomal vesicle regulation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of ubiquitin-engaged PEX1–PEX6 complex exists\", \"Cargo–receptor uncoupling mechanism is undefined\", \"In vivo therapeutic validation of G843D stabilization is lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 8, 9, 18, 20]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [11, 12, 18]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [7, 10, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 10, 16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [14, 15, 21, 22]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 12, 18]}\n    ],\n    \"complexes\": [\n      \"PEX1–PEX6 heterohexamer\"\n    ],\n    \"partners\": [\n      \"PEX6\",\n      \"PEX5\",\n      \"PEX15\",\n      \"PEX26\",\n      \"MARCHF7\",\n      \"NBR1\",\n      \"HNRNPA1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}