{"gene":"PEX1","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1997,"finding":"Human PEX1 encodes a 147-kDa AAA-family 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 membrane proteins.","method":"Functional complementation in CG1 patient fibroblasts; Western blot analysis of PEX5 levels","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — complementation rescue in patient cells replicated across two independent labs (PMID:9398847 and PMID:9398848) with consistent findings","pmids":["9398847","9398848"],"is_preprint":false},{"year":1998,"finding":"PEX1 and PEX6 physically interact with each other; this interaction is required for peroxisome biogenesis. The common G843D mutation in PEX1 attenuates the PEX1–PEX6 interaction in both yeast two-hybrid and in vitro binding assays, and overexpression of PEX6 can suppress PEX1 G843D phenotypes in an allele-specific manner.","method":"Yeast two-hybrid assay; in vitro binding (pulldown); genetic suppression by overexpression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic suppression plus two orthogonal physical interaction methods (Y2H + in vitro binding), replicated in subsequent work","pmids":["9671729"],"is_preprint":false},{"year":1998,"finding":"The PEX1 G843D missense mutation causes temperature-sensitive peroxisome assembly: peroxisomes form at 30°C but not 37°C in patient fibroblasts and CHO cell mutants transfected with PEX1-G843D, demonstrating the mutation produces a misfolded but partially functional protein.","method":"Immunofluorescence microscopy and biochemical peroxisome assays in patient fibroblasts and transfected CHO mutants at 30°C vs. 37°C","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — demonstrated in both patient fibroblasts and CHO cell model with morphological and biochemical readouts, replicated across multiple studies","pmids":["9817926"],"is_preprint":false},{"year":2001,"finding":"PEX1-G843D protein is largely degraded in vivo at 37°C but present at normal levels at 30°C; ZS-associated PEX1 mutants (L664P, deletion 634–690) are stably expressed at both temperatures but fail to bind PEX6, whereas G843D retains ~50% of wild-type PEX6 binding. PEX1–PEX6 interaction is thus the key determinant of phenotypic severity.","method":"Western blot of patient fibroblasts at permissive/non-permissive temperatures; co-immunoprecipitation of PEX1 mutants with PEX6","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mutant alleles tested by co-IP + protein stability assay, consistent with genetic evidence from PMID:9671729","pmids":["11439091"],"is_preprint":false},{"year":2001,"finding":"Residual PEX1 protein levels correlate with clinical phenotype severity: complete absence of PEX1 protein is associated with severe Zellweger syndrome, whereas NALD/IRD patients retain residual protein (especially those carrying the G843D allele). Growing G843D fibroblasts at 30°C increases PEX1 protein 2–3-fold with recovery of peroxisomal function, indicating G843D generates a misfolded, thermolabile protein.","method":"Western blot quantification of PEX1 protein in patient fibroblasts at 37°C and 30°C; biochemical peroxisome function assays","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic analysis across multiple patient alleles with orthogonal biochemical and protein level measurements","pmids":["11389485"],"is_preprint":false},{"year":2004,"finding":"The crystal structure of the mouse PEX1 N-terminal domain was determined at 2.05-Å resolution, revealing a double-psi-barrel fold structurally similar to the N-terminal domains of p97/VCP and NSF (RMSD ~2.1–2.7 Å), suggesting a common adaptor-binding function in membrane-associated AAA-ATPases.","method":"X-ray crystallography at 2.05-Å resolution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination with structural homology analysis; single study but direct structural data","pmids":["15328346"],"is_preprint":false},{"year":2006,"finding":"The N-terminal domain of PEX1 binds phosphoinositides, preferentially phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate; a conserved arginine residue (surrounded by hydrophobic residues) is essential for this lipid-binding activity, as shown by mutational analysis.","method":"Lipid-binding assays (protein–lipid overlay); site-directed mutagenesis of the conserved arginine","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro lipid binding with mutagenesis validation, but physiological significance of this lipid interaction remains uncharacterized","pmids":["17018057"],"is_preprint":false},{"year":2006,"finding":"A mutation in the Walker A1 motif of PEX1 (G606E) abolishes PEX1–PEX6 interaction at 37°C but not 30°C, and causes cytosolic mislocalization of PEX1 at the non-permissive temperature, demonstrating that the Walker A1 motif is essential for both PEX1–PEX6 interaction and peroxisomal targeting of PEX1.","method":"Co-immunoprecipitation; immunofluorescence localization of PEX1 in CHO cell mutants transfected with PEX1-G606E at 30°C vs. 37°C","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP and localization data in a single study without independent replication","pmids":["16723118"],"is_preprint":false},{"year":2015,"finding":"Cryo-EM structures of the Pex1/Pex6 complex reveal an unprecedented heterohexameric double ring in which Pex1 and Pex6 alternate. Each subunit has two N-terminal domains (N1, N2); N1 of Pex1 is mobile. The N-terminal D1 ATPase ring is inactive and symmetric; the C-terminal D2 ring is active and asymmetric with subunits likely in different nucleotide states. This organization is analogous to p97, suggesting a role in protein extraction from the peroxisomal membrane.","method":"Cryo-electron microscopy with computational model building (Monte Carlo placement + energy minimization); nucleotide state variation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure determined in multiple nucleotide states, independently confirmed by parallel cryo-EM study (PMID:26066397)","pmids":["26170309"],"is_preprint":false},{"year":2015,"finding":"Biochemical analysis of yeast Pex1/Pex6 heterohexamer shows that the C-terminal D2 domains of Pex6 constitute the main ATPase activity; ATP hydrolysis drives a pumping motion of the complex consistent with substrate translocation through the central channel. Walker B mutation in one D2 domain promotes ATP hydrolysis in the neighboring domain, revealing inter-domain communication.","method":"ATPase activity assays; cryo-EM structural analysis; Walker B mutagenesis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ATPase reconstitution with mutagenesis plus cryo-EM, independently confirmed by PMID:26170309","pmids":["26066397"],"is_preprint":false},{"year":2015,"finding":"Induced depletion of Pex1 in yeast blocks import of matrix proteins but does not affect peroxisomal membrane protein delivery, confirming that the primary role of the Pex1/Pex6 complex is in matrix protein import rather than peroxisomal membrane biogenesis or vesicle fusion.","method":"Conditional Pex1 depletion; immunofluorescence and biochemical assays for matrix vs. membrane protein import","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean inducible depletion with defined phenotypic readouts separating matrix vs. membrane import, single lab","pmids":["26644516"],"is_preprint":false},{"year":2015,"finding":"Yeast pex1Δ cells contain peroxisomal membrane remnants (ghosts) lacking matrix proteins but retaining major membrane proteins (Pex2, Pex10, Pex11, Pex13, Pex14). Upon re-introduction of Pex1, these ghosts rapidly incorporated peroxisomal matrix proteins and developed into functional peroxisomes.","method":"Electron microscopy (ultrathin sections, electron tomography); immunocytochemistry; Pex1 re-expression rescue","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ultrastructural characterization with functional rescue, multiple orthogonal microscopy methods","pmids":["26644511"],"is_preprint":false},{"year":2014,"finding":"Deficiency of Pex1 (along with Pex6 and Pex15) leads to enhanced pexophagy in S. cerevisiae, with almost all peroxisomal membranes associating with phagophore assembly sites in pex1Δ atg1Δ cells. This pexophagy depends on Atg11 and the pexophagy receptor Atg36. Preventing accumulation of ubiquitinated receptors at the peroxisomal membrane does not abolish pexophagy in yeast.","method":"Genetic analysis (double mutants); fluorescence microscopy; epistasis with atg mutants","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined cellular phenotype, multiple mutant combinations tested","pmids":["24657987"],"is_preprint":false},{"year":2018,"finding":"Pex1/Pex6 is a protein translocase that unfolds Pex15 (its membrane anchor) in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Pex15 binds the N-terminal domains of Pex6, then its C-terminal disordered region engages with the pore loops of the motor, which processively threads Pex15 through the central pore. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to the import machinery.","method":"In vitro unfolding assays; cryo-EM structural analysis of Pex15 alone and in complex with Pex1/Pex6; pore-loop mutagenesis; pulldown assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro unfolding, structural data, and mutagenesis in one study with multiple orthogonal methods","pmids":["29321502"],"is_preprint":false},{"year":2018,"finding":"Peroxisomal monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during ATP-dependent extraction from the peroxisomal membrane, identifying DTM-embedded Ub-PEX5 as a direct substrate of the PEX1–PEX6 complex.","method":"Cell-free in vitro system; photoaffinity cross-linking; protein PEGylation assays (to detect unfolding)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with multiple orthogonal biochemical methods (photoaffinity cross-linking + PEGylation) in a single rigorous study","pmids":["29884772"],"is_preprint":false},{"year":2023,"finding":"CryoEM structures of S. cerevisiae Pex1/Pex6 with an endogenous substrate trapped in the D2 pore reveal that Pex1/Pex6(D2) subdomains engage substrate via a staircase of pore-1 loops with distinct properties. The D1 ring is catalytically inactive but undergoes conformational changes (alternating pore widening/narrowing) fueled by D2 ATP hydrolysis. A 'twin-seam' Pex1/Pex6(D2) heterodimer disengages from the staircase to drive substrate translocation through a unique inter-subunit communication mechanism.","method":"Cryo-EM with endogenous substrate trapped in the pore","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution cryo-EM with substrate-engaged complex, revealing detailed translocation mechanism","pmids":["37741838"],"is_preprint":false},{"year":2023,"finding":"The N1 domain of Pex6 (not Pex1) directly mediates binding to the peroxisomal membrane tether Pex15 and also contacts an extended loop from the D2 ATPase domain of Pex1, influencing Pex1/Pex6 heterohexamer stability. Pex1/ΔN1-Pex6 retains ATPase activity in vitro but fails to support peroxisome function in vivo.","method":"X-ray crystallography of Pex6 N1 domain; cryo-EM of Pex1/Pex6; AlphaFold2 modeling; biochemical binding assays; in vivo complementation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus cryo-EM plus biochemical assays plus in vivo complementation, multiple orthogonal methods","pmids":["38036174"],"is_preprint":false},{"year":2017,"finding":"In Arabidopsis, PEX1 stabilizes PEX6 protein levels and vice versa. A pex1 missense allele (pex1-2) reduces both PEX1 and PEX6 protein levels and causes peroxisome dysfunction that is temperature-sensitive and partially rescued by PEX6 overexpression. A second allele (pex1-3) is semidominant, consistent with PEX1 forming a heterooligomer with PEX6 that is poisoned by mutant subunits. Blocking autophagy partially rescues pex1-3 defects and restores normal peroxisome size.","method":"Genetic analysis of Arabidopsis pex1 alleles; Western blot of PEX1 and PEX6 levels; PEX6 overexpression rescue; autophagy mutant epistasis","journal":"Plant physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and biochemical approaches in plant ortholog, consistent with mammalian/yeast findings","pmids":["28600347"],"is_preprint":false},{"year":2020,"finding":"Blocking pexophagy specifically in pex1Δ yeast (via deletion of pexophagy genes) does not restore peroxisomal matrix protein import or β-oxidation function, demonstrating that PEX1 is directly and essentially involved in peroxisomal matrix protein import and that pex1-induced pexophagy is a consequence rather than the cause of the import defect.","method":"Genetic analysis in S. cerevisiae (pex1Δ combined with pexophagy-null mutants); β-oxidation functional assays; matrix protein import assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis analysis with defined functional readouts, single lab","pmids":["32013259"],"is_preprint":false},{"year":2021,"finding":"HNRNPA1 controls PEX1 expression at the post-transcriptional level; depletion of HNRNPA1 reduces PEX1 protein levels, increases peroxisomal ROS, and induces pexophagy (blocked in ATG5-KO cells). Inhibiting ROS with NAC suppresses pexophagy in HNRNPA1-deficient cells.","method":"siRNA knockdown of HNRNPA1; Western blot of PEX1; ROS measurement; pexophagy assays; ATG5-KO epistasis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — knockdown with functional readouts and genetic epistasis, but mechanism of HNRNPA1 regulation of PEX1 mRNA not fully characterized","pmids":["33545634"],"is_preprint":false},{"year":2022,"finding":"In PEX1-KO HEK293 cells, complete absence of PEX1 significantly increases the number of peroxisomal ghosts (import-incompetent membrane vesicles). Re-expression of full-length PEX1 restores peroxisome import competence and abundance. An alternatively spliced PEX1 isoform lacking 321 N-terminal amino acids fails to rescue peroxisomal import defects but reduces the number of peroxisomal vesicles, suggesting a 'moonlighting' role for the N-terminal region of PEX1 in regulating pre-peroxisomal vesicles.","method":"CRISPR/Cas9 PEX1-KO in HEK293 cells; re-expression of full-length and truncated PEX1 isoforms; immunofluorescence quantification of peroxisomal ghosts; matrix protein import assays","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean CRISPR KO with defined rescue experiments using domain-truncated isoforms, single lab","pmids":["36534601"],"is_preprint":false},{"year":2025,"finding":"PEX1-G843D (and its yeast homolog ScPex1-G700D) destabilizes Pex1's active D2 ATPase domain and impairs assembly with Pex6, but retains residual AAA-ATPase motor activity in vitro. In human cells, PEX1-G843D is rapidly degraded by the proteasome; impaired Pex1/Pex6 assembly is itself sufficient to trigger proteasomal degradation of PEX1-WT. Fusing a deubiquitinase to PEX1-G843D substantially stabilizes the protein. Induced overexpression of PEX1-G843D restores peroxisome import.","method":"In vitro ATPase assays; co-IP for PEX1–PEX6 assembly; proteasome inhibitor treatment; deubiquitinase fusion stabilization; peroxisome import assays in human cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution plus cell-based degradation assays with multiple orthogonal methods in a single rigorous study","pmids":["40158855"],"is_preprint":false},{"year":2025,"finding":"PEX1 depletion in HeLa cells activates a TBK1-MARCHF7-PXMP4-NBR1 pexophagy axis: PEX1 deficiency induces ROS accumulation that activates TBK1 (phospho-S172), which phosphorylates MARCHF7 (an E3 ligase); MARCHF7 ubiquitinates PXMP4 at K20, and ubiquitinated PXMP4 recruits NBR1 as a pexophagy receptor.","method":"siRNA knockdown; functional screening; co-IP; ubiquitination assays; phosphorylation analysis; pexophagy flux assays; PXMP4 K20R mutant rescue","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical and genetic approaches in a single study defining a pathway downstream of PEX1 depletion","pmids":["41267209"],"is_preprint":false},{"year":2022,"finding":"Conditional deletion of Pex1 specifically in inner hair cells (IHCs) of the mouse inner ear causes progressive hearing loss, decreased ABR wave I amplitude (indicative of synaptic defects), decreased ribbon synapse volume, altered IHC exocytosis, and reduced peroxisome number in IHCs, establishing a direct role for PEX1 in IHC synapse development and auditory function.","method":"Conditional knockout mice (Gfi1-Cre or VGlut3-Cre × floxed Pex1); ABR electrophysiology; IHC synapse immunofluorescence; exocytosis assays; peroxisome counting","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific KO with defined electrophysiological and synaptic phenotypes, single lab","pmids":["36552747"],"is_preprint":false}],"current_model":"PEX1 is an AAA-ATPase that forms a heterohexameric complex with PEX6 (alternating subunits in a double ring), anchored to the peroxisomal membrane via PEX15/PEX26; this complex uses ATP hydrolysis in its active D2 ring to processively thread and unfold substrates through its central pore—most critically monoubiquitinated PEX5—thereby extracting and recycling the peroxisomal matrix protein import receptor from the membrane; PEX1–PEX6 assembly is mutually stabilizing (reduced affinity triggers proteasomal degradation of PEX1), and loss of PEX1 function causes peroxisomal matrix protein import failure, accumulation of import-incompetent peroxisomal ghosts, and secondary pexophagy."},"narrative":{"mechanistic_narrative":"PEX1 is an AAA-family ATPase essential for peroxisomal matrix protein import; its loss produces import-incompetent peroxisomal membrane remnants (\"ghosts\") that retain membrane proteins but lack matrix content and can be rescued by PEX1 re-expression [PMID:9398847, PMID:9398848, PMID:26644511, PMID:36534601]. PEX1 functions as an obligate partner of PEX6, with which it forms a heterohexameric double-ring complex in which the two subunits alternate; the N-terminal D1 ring is catalytically inactive and the C-terminal D2 ring carries out the ATP hydrolysis that powers the motor [PMID:26170309, PMID:26066397]. This complex acts as a protein translocase that processively threads and unfolds substrates through its central pore via a pore-loop staircase, driven by D2 ATP hydrolysis and inter-subunit communication [PMID:29321502, PMID:37741838]. Its critical substrate is monoubiquitinated PEX5, the recycled PTS1 import receptor, which engages the complex through its ubiquitin moiety and is globally unfolded during ATP-dependent extraction from the peroxisomal membrane [PMID:29884772]; the complex similarly unfolds and is anchored to the membrane via the tether Pex15/PEX26, bound by the Pex6 N1 domain [PMID:29321502, PMID:38036174]. PEX1 and PEX6 are mutually stabilizing, and disrupted assembly triggers proteasomal degradation of PEX1, a mechanism that underlies the common disease allele G843D, which destabilizes the D2 domain and impairs assembly while retaining residual motor activity [PMID:11439091, PMID:40158855]. Mutations spanning loss of PEX1 protein to attenuated PEX1–PEX6 interaction cause the peroxisome biogenesis disorder spectrum from severe Zellweger syndrome to milder NALD/IRD, with residual protein levels and PEX6-binding capacity determining phenotypic severity [PMID:9671729, PMID:11439091, PMID:11389485]. Loss of PEX1 secondarily activates ROS-driven pexophagy, which is a consequence rather than the cause of the import defect [PMID:32013259, PMID:41267209].","teleology":[{"year":1997,"claim":"Established PEX1 as an AAA-ATPase whose loss specifically abolishes matrix protein import while leaving peroxisomal membranes intact, distinguishing import failure from organelle absence.","evidence":"Functional complementation in CG1 patient fibroblasts with PEX5 Western blot","pmids":["9398847","9398848"],"confidence":"High","gaps":["Mechanism by which PEX1 acts on PEX5 not yet defined","No partner protein identified at this stage"]},{"year":1998,"claim":"Identified PEX6 as the direct physical partner of PEX1 and showed the interaction is required for biogenesis, framing PEX1 function as dependent on heterocomplex formation.","evidence":"Yeast two-hybrid, in vitro pulldown, and allele-specific genetic suppression by PEX6 overexpression","pmids":["9671729"],"confidence":"High","gaps":["Stoichiometry and architecture of the complex unknown","How the interaction enables import not addressed"]},{"year":1998,"claim":"Demonstrated that the common G843D allele is temperature-sensitive, defining it as a misfolded but partially functional protein rather than a null.","evidence":"Immunofluorescence and biochemical peroxisome assays at 30°C vs 37°C in patient fibroblasts and CHO mutants","pmids":["9817926"],"confidence":"High","gaps":["Molecular basis of thermolability unresolved","Fate of misfolded protein not determined"]},{"year":2001,"claim":"Linked PEX1–PEX6 binding capacity and residual protein level to clinical severity, establishing the genotype-phenotype logic of the disease spectrum.","evidence":"Co-IP of PEX1 mutants with PEX6 and protein stability quantification across multiple alleles in patient fibroblasts","pmids":["11439091","11389485"],"confidence":"High","gaps":["Degradation pathway for unstable PEX1 not identified","No structural explanation for binding loss"]},{"year":2004,"claim":"Solved the PEX1 N-terminal domain structure, revealing a double-psi-barrel fold shared with p97/VCP and NSF and implying an adaptor-binding role analogous to other membrane AAA-ATPases.","evidence":"X-ray crystallography at 2.05 Å with structural homology analysis (mouse N-domain)","pmids":["15328346"],"confidence":"High","gaps":["Functional adaptor of the N-domain not identified","Full-length architecture unknown"]},{"year":2006,"claim":"Characterized two N-terminal/Walker-motif activities: phosphoinositide binding by the N-domain and a Walker A1 requirement for both PEX6 binding and peroxisomal targeting.","evidence":"Protein-lipid overlay with arginine mutagenesis; co-IP and localization of PEX1-G606E at permissive/non-permissive temperatures","pmids":["17018057","16723118"],"confidence":"Medium","gaps":["Physiological role of lipid binding uncharacterized","Walker A1 data from a single unreplicated study"]},{"year":2015,"claim":"Resolved the complex as an alternating PEX1/PEX6 heterohexameric double ring with an inactive D1 and active D2 ATPase ring, defining it mechanistically as a p97-like protein-extraction machine and localizing catalytic activity to Pex6 D2.","evidence":"Cryo-EM in multiple nucleotide states plus ATPase assays and Walker B mutagenesis (yeast Pex1/Pex6)","pmids":["26170309","26066397"],"confidence":"High","gaps":["Physiological substrate not yet captured","Translocation directionality and processivity unproven"]},{"year":2015,"claim":"Separated the complex's role from membrane biogenesis: Pex1 depletion blocks matrix import but spares membrane protein delivery, and pex1Δ ghosts can be matured to functional peroxisomes upon Pex1 restoration.","evidence":"Conditional Pex1 depletion with matrix vs membrane import assays; EM/tomography of ghosts with re-expression rescue (yeast)","pmids":["26644516","26644511"],"confidence":"High","gaps":["Whether ghosts are import precursors or stalled intermediates not fully resolved"]},{"year":2018,"claim":"Identified direct substrates and showed processive unfolding: the complex threads and unfolds the membrane anchor Pex15 through its pore, and monoubiquitinated PEX5 engages PEX1/PEX6 via its ubiquitin and is globally unfolded during ATP-dependent extraction.","evidence":"In vitro unfolding and pore-loop mutagenesis with cryo-EM (Pex15); cell-free system with photoaffinity cross-linking and PEGylation (Ub-PEX5)","pmids":["29321502","29884772"],"confidence":"High","gaps":["How ubiquitin recognition couples to pore engagement not fully defined","Fate of PEX5 after extraction not traced here"]},{"year":2023,"claim":"Defined the translocation mechanism at the residue level: a pore-1 loop staircase in D2 with a 'twin-seam' heterodimer that disengages to drive substrate movement, while the inactive D1 ring undergoes D2-fueled conformational cycling.","evidence":"Cryo-EM of S. cerevisiae Pex1/Pex6 with endogenous substrate trapped in the D2 pore","pmids":["37741838"],"confidence":"High","gaps":["Identity of the trapped endogenous substrate not established","Full ATPase cycle kinetics incomplete"]},{"year":2023,"claim":"Assigned membrane tethering to the Pex6 N1 domain, which binds Pex15 and contacts a Pex1 D2 loop to stabilize the heterohexamer, with ATPase-active but biologically inactive complex when N1 is deleted.","evidence":"X-ray crystallography of Pex6 N1, cryo-EM, AlphaFold2 modeling, binding assays, in vivo complementation","pmids":["38036174"],"confidence":"High","gaps":["Equivalent role of Pex1 N1 not fully resolved","Coupling of tethering to substrate extraction unclear"]},{"year":2025,"claim":"Explained the molecular pathology of G843D: it destabilizes the D2 domain and impairs PEX6 assembly while retaining residual motor activity, and impaired assembly itself triggers proteasomal degradation of PEX1, providing a rationale for stabilization-based rescue.","evidence":"In vitro ATPase assays, co-IP, proteasome inhibition, deubiquitinase-fusion stabilization, and import rescue in human cells","pmids":["40158855"],"confidence":"High","gaps":["E3 ligase mediating PEX1 degradation not identified","Therapeutic generalizability across alleles untested"]},{"year":2022,"claim":"Distinguished primary import function from secondary effects: PEX1 absence increases ghost abundance, and an N-terminally truncated isoform fails to rescue import but alters pre-peroxisomal vesicle number, hinting at a moonlighting N-terminal role.","evidence":"CRISPR PEX1-KO HEK293 with full-length and truncated isoform re-expression and import assays","pmids":["36534601"],"confidence":"Medium","gaps":["Mechanism of N-terminal vesicle-regulatory role undefined","Single lab"]},{"year":2025,"claim":"Resolved that pexophagy is downstream of import failure and mapped its molecular triggers: PEX1 loss drives ROS, activating a TBK1-MARCHF7-PXMP4-NBR1 axis, while blocking pexophagy does not restore import or β-oxidation.","evidence":"Yeast epistasis with β-oxidation/import assays; HeLa knockdown with co-IP, ubiquitination, phosphorylation and flux assays","pmids":["32013259","41267209"],"confidence":"Medium","gaps":["Conservation of the TBK1-MARCHF7-PXMP4-NBR1 axis across cell types untested","How ROS arises from import failure not detailed"]},{"year":null,"claim":"How PEX1 dysfunction produces tissue-specific phenotypes such as inner hair cell synaptic defects, and the in vivo significance of N-domain lipid binding, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Mechanism linking peroxisome loss to ribbon synapse/exocytosis defects unknown","Physiological role of phosphoinositide binding by the N-domain uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,9,21]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[13,14,15]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6]}],"localization":[{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[11,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,10,14]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[12,18,22]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[11,20]}],"complexes":["PEX1–PEX6 AAA-ATPase heterohexamer"],"partners":["PEX6","PEX5","PEX15","PEX26","HNRNPA1"],"other_free_text":[]}},"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 G843D mutation in PEX1 attenuates the PEX1–PEX6 interaction in both yeast two-hybrid and in vitro binding assays, and overexpression of PEX6 can suppress PEX1 G843D phenotypes in an allele-specific manner.\",\n      \"method\": \"Yeast two-hybrid assay; in vitro binding (pulldown); 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 / Strong — reciprocal genetic suppression plus two orthogonal physical interaction methods (Y2H + in vitro binding), replicated in subsequent work\",\n      \"pmids\": [\"9671729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The PEX1 G843D missense mutation causes temperature-sensitive peroxisome assembly: peroxisomes form at 30°C but not 37°C in patient fibroblasts and CHO cell mutants transfected with PEX1-G843D, demonstrating the mutation produces a misfolded but partially functional protein.\",\n      \"method\": \"Immunofluorescence microscopy and biochemical peroxisome assays in patient fibroblasts and transfected CHO mutants at 30°C vs. 37°C\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — demonstrated in both patient fibroblasts and CHO cell model with morphological and biochemical readouts, replicated across multiple studies\",\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 present at normal levels at 30°C; ZS-associated PEX1 mutants (L664P, deletion 634–690) are stably expressed at both temperatures but fail to bind PEX6, whereas G843D retains ~50% of wild-type PEX6 binding. PEX1–PEX6 interaction is thus the key determinant of phenotypic severity.\",\n      \"method\": \"Western blot of patient fibroblasts at permissive/non-permissive temperatures; co-immunoprecipitation of PEX1 mutants with PEX6\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mutant alleles tested by co-IP + protein stability assay, consistent with genetic evidence from PMID:9671729\",\n      \"pmids\": [\"11439091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Residual PEX1 protein levels correlate with clinical phenotype severity: complete absence of PEX1 protein is associated with severe Zellweger syndrome, whereas NALD/IRD patients retain residual protein (especially those carrying the G843D allele). Growing G843D fibroblasts at 30°C increases PEX1 protein 2–3-fold with recovery of peroxisomal function, indicating G843D generates a misfolded, thermolabile protein.\",\n      \"method\": \"Western blot quantification of PEX1 protein in patient fibroblasts at 37°C and 30°C; biochemical peroxisome function assays\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic analysis across multiple patient alleles with orthogonal biochemical and protein level measurements\",\n      \"pmids\": [\"11389485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The crystal structure of the mouse PEX1 N-terminal domain was determined at 2.05-Å resolution, revealing a double-psi-barrel fold structurally similar to the N-terminal domains of p97/VCP and NSF (RMSD ~2.1–2.7 Å), suggesting a common adaptor-binding function in membrane-associated AAA-ATPases.\",\n      \"method\": \"X-ray crystallography at 2.05-Å resolution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination with structural homology analysis; single study but direct structural data\",\n      \"pmids\": [\"15328346\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal domain of PEX1 binds phosphoinositides, preferentially phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate; a conserved arginine residue (surrounded by hydrophobic residues) is essential for this lipid-binding activity, as shown by mutational analysis.\",\n      \"method\": \"Lipid-binding assays (protein–lipid overlay); site-directed mutagenesis of the conserved arginine\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro lipid binding with mutagenesis validation, but physiological significance of this lipid interaction remains uncharacterized\",\n      \"pmids\": [\"17018057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"A mutation in the Walker A1 motif of PEX1 (G606E) abolishes PEX1–PEX6 interaction at 37°C but not 30°C, and causes cytosolic mislocalization of PEX1 at the non-permissive temperature, demonstrating that the Walker A1 motif is essential for both PEX1–PEX6 interaction and peroxisomal targeting of PEX1.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence localization of PEX1 in CHO cell mutants transfected with PEX1-G606E at 30°C vs. 37°C\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP and localization data in a single study without independent replication\",\n      \"pmids\": [\"16723118\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cryo-EM structures of the Pex1/Pex6 complex reveal an unprecedented heterohexameric double ring in which Pex1 and Pex6 alternate. Each subunit has two N-terminal domains (N1, N2); N1 of Pex1 is mobile. The N-terminal D1 ATPase ring is inactive and symmetric; the C-terminal D2 ring is active and asymmetric with subunits likely in different nucleotide states. This organization is analogous to p97, suggesting a role in protein extraction from the peroxisomal membrane.\",\n      \"method\": \"Cryo-electron microscopy with computational model building (Monte Carlo placement + energy minimization); nucleotide state variation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure determined in multiple nucleotide states, independently confirmed by parallel cryo-EM study (PMID:26066397)\",\n      \"pmids\": [\"26170309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Biochemical analysis of yeast Pex1/Pex6 heterohexamer shows that the C-terminal D2 domains of Pex6 constitute the main ATPase activity; ATP hydrolysis drives a pumping motion of the complex consistent with substrate translocation through the central channel. Walker B mutation in one D2 domain promotes ATP hydrolysis in the neighboring domain, revealing inter-domain communication.\",\n      \"method\": \"ATPase activity assays; cryo-EM structural analysis; Walker B mutagenesis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ATPase reconstitution with mutagenesis plus cryo-EM, independently confirmed by PMID:26170309\",\n      \"pmids\": [\"26066397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Induced depletion of Pex1 in yeast blocks import of matrix proteins but does not affect peroxisomal membrane protein delivery, confirming that the primary role of the Pex1/Pex6 complex is in matrix protein import rather than peroxisomal membrane biogenesis or vesicle fusion.\",\n      \"method\": \"Conditional Pex1 depletion; immunofluorescence and biochemical assays for matrix vs. membrane protein import\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean inducible depletion with defined phenotypic readouts separating matrix vs. membrane import, single lab\",\n      \"pmids\": [\"26644516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Yeast pex1Δ cells contain peroxisomal membrane remnants (ghosts) lacking matrix proteins but retaining major membrane proteins (Pex2, Pex10, Pex11, Pex13, Pex14). Upon re-introduction of Pex1, these ghosts rapidly incorporated peroxisomal matrix proteins and developed into functional peroxisomes.\",\n      \"method\": \"Electron microscopy (ultrathin sections, electron tomography); immunocytochemistry; Pex1 re-expression rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ultrastructural characterization with functional rescue, multiple orthogonal microscopy methods\",\n      \"pmids\": [\"26644511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deficiency of Pex1 (along with Pex6 and Pex15) leads to enhanced pexophagy in S. cerevisiae, with almost all peroxisomal membranes associating with phagophore assembly sites in pex1Δ atg1Δ cells. This pexophagy depends on Atg11 and the pexophagy receptor Atg36. Preventing accumulation of ubiquitinated receptors at the peroxisomal membrane does not abolish pexophagy in yeast.\",\n      \"method\": \"Genetic analysis (double mutants); fluorescence microscopy; epistasis with atg mutants\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined cellular phenotype, multiple mutant combinations tested\",\n      \"pmids\": [\"24657987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Pex1/Pex6 is a protein translocase that unfolds Pex15 (its membrane anchor) in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Pex15 binds the N-terminal domains of Pex6, then its C-terminal disordered region engages with the pore loops of the motor, which processively threads Pex15 through the central pore. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to the import machinery.\",\n      \"method\": \"In vitro unfolding assays; cryo-EM structural analysis of Pex15 alone and in complex with Pex1/Pex6; pore-loop mutagenesis; pulldown assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro unfolding, structural data, and mutagenesis in one study with multiple orthogonal methods\",\n      \"pmids\": [\"29321502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Peroxisomal monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide chain is globally unfolded during ATP-dependent extraction from the peroxisomal membrane, identifying DTM-embedded Ub-PEX5 as a direct substrate of the PEX1–PEX6 complex.\",\n      \"method\": \"Cell-free in vitro system; photoaffinity cross-linking; protein PEGylation assays (to detect unfolding)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with multiple orthogonal biochemical methods (photoaffinity cross-linking + PEGylation) in a single rigorous study\",\n      \"pmids\": [\"29884772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CryoEM structures of S. cerevisiae Pex1/Pex6 with an endogenous substrate trapped in the D2 pore reveal that Pex1/Pex6(D2) subdomains engage substrate via a staircase of pore-1 loops with distinct properties. The D1 ring is catalytically inactive but undergoes conformational changes (alternating pore widening/narrowing) fueled by D2 ATP hydrolysis. A 'twin-seam' Pex1/Pex6(D2) heterodimer disengages from the staircase to drive substrate translocation through a unique inter-subunit communication mechanism.\",\n      \"method\": \"Cryo-EM with endogenous substrate trapped in the pore\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution cryo-EM with substrate-engaged complex, revealing detailed translocation mechanism\",\n      \"pmids\": [\"37741838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The N1 domain of Pex6 (not Pex1) directly mediates binding to the peroxisomal membrane tether Pex15 and also contacts an extended loop from the D2 ATPase domain of Pex1, influencing Pex1/Pex6 heterohexamer stability. Pex1/ΔN1-Pex6 retains ATPase activity in vitro but fails to support peroxisome function in vivo.\",\n      \"method\": \"X-ray crystallography of Pex6 N1 domain; cryo-EM of Pex1/Pex6; AlphaFold2 modeling; biochemical binding assays; in vivo complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus cryo-EM plus biochemical assays plus in vivo complementation, multiple orthogonal methods\",\n      \"pmids\": [\"38036174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Arabidopsis, PEX1 stabilizes PEX6 protein levels and vice versa. A pex1 missense allele (pex1-2) reduces both PEX1 and PEX6 protein levels and causes peroxisome dysfunction that is temperature-sensitive and partially rescued by PEX6 overexpression. A second allele (pex1-3) is semidominant, consistent with PEX1 forming a heterooligomer with PEX6 that is poisoned by mutant subunits. Blocking autophagy partially rescues pex1-3 defects and restores normal peroxisome size.\",\n      \"method\": \"Genetic analysis of Arabidopsis pex1 alleles; Western blot of PEX1 and PEX6 levels; PEX6 overexpression rescue; autophagy mutant epistasis\",\n      \"journal\": \"Plant physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and biochemical approaches in plant ortholog, consistent with mammalian/yeast findings\",\n      \"pmids\": [\"28600347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Blocking pexophagy specifically in pex1Δ yeast (via deletion of pexophagy genes) does not restore peroxisomal matrix protein import or β-oxidation function, demonstrating that PEX1 is directly and essentially involved in peroxisomal matrix protein import and that pex1-induced pexophagy is a consequence rather than the cause of the import defect.\",\n      \"method\": \"Genetic analysis in S. cerevisiae (pex1Δ combined with pexophagy-null mutants); β-oxidation functional assays; matrix protein import assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis analysis with defined functional readouts, single lab\",\n      \"pmids\": [\"32013259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HNRNPA1 controls PEX1 expression at the post-transcriptional level; depletion of HNRNPA1 reduces PEX1 protein levels, increases peroxisomal ROS, and induces pexophagy (blocked in ATG5-KO cells). Inhibiting ROS with NAC suppresses pexophagy in HNRNPA1-deficient cells.\",\n      \"method\": \"siRNA knockdown of HNRNPA1; Western blot of PEX1; ROS measurement; pexophagy assays; ATG5-KO epistasis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — knockdown with functional readouts and genetic epistasis, but mechanism of HNRNPA1 regulation of PEX1 mRNA not fully characterized\",\n      \"pmids\": [\"33545634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In PEX1-KO HEK293 cells, complete absence of PEX1 significantly increases the number of peroxisomal ghosts (import-incompetent membrane vesicles). Re-expression of full-length PEX1 restores peroxisome import competence and abundance. An alternatively spliced PEX1 isoform lacking 321 N-terminal amino acids fails to rescue peroxisomal import defects but reduces the number of peroxisomal vesicles, suggesting a 'moonlighting' role for the N-terminal region of PEX1 in regulating pre-peroxisomal vesicles.\",\n      \"method\": \"CRISPR/Cas9 PEX1-KO in HEK293 cells; re-expression of full-length and truncated PEX1 isoforms; immunofluorescence quantification of peroxisomal ghosts; matrix protein import assays\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean CRISPR KO with defined rescue experiments using domain-truncated isoforms, single lab\",\n      \"pmids\": [\"36534601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PEX1-G843D (and its yeast homolog ScPex1-G700D) destabilizes Pex1's active D2 ATPase domain and impairs assembly with Pex6, but retains residual AAA-ATPase motor activity in vitro. In human cells, PEX1-G843D is rapidly degraded by the proteasome; impaired Pex1/Pex6 assembly is itself sufficient to trigger proteasomal degradation of PEX1-WT. Fusing a deubiquitinase to PEX1-G843D substantially stabilizes the protein. Induced overexpression of PEX1-G843D restores peroxisome import.\",\n      \"method\": \"In vitro ATPase assays; co-IP for PEX1–PEX6 assembly; proteasome inhibitor treatment; deubiquitinase fusion stabilization; peroxisome import assays in human cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution plus cell-based degradation assays with multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"40158855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PEX1 depletion in HeLa cells activates a TBK1-MARCHF7-PXMP4-NBR1 pexophagy axis: PEX1 deficiency induces ROS accumulation that activates TBK1 (phospho-S172), which phosphorylates MARCHF7 (an E3 ligase); MARCHF7 ubiquitinates PXMP4 at K20, and ubiquitinated PXMP4 recruits NBR1 as a pexophagy receptor.\",\n      \"method\": \"siRNA knockdown; functional screening; co-IP; ubiquitination assays; phosphorylation analysis; pexophagy flux assays; PXMP4 K20R mutant rescue\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical and genetic approaches in a single study defining a pathway downstream of PEX1 depletion\",\n      \"pmids\": [\"41267209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Conditional deletion of Pex1 specifically in inner hair cells (IHCs) of the mouse inner ear causes progressive hearing loss, decreased ABR wave I amplitude (indicative of synaptic defects), decreased ribbon synapse volume, altered IHC exocytosis, and reduced peroxisome number in IHCs, establishing a direct role for PEX1 in IHC synapse development and auditory function.\",\n      \"method\": \"Conditional knockout mice (Gfi1-Cre or VGlut3-Cre × floxed Pex1); ABR electrophysiology; IHC synapse immunofluorescence; exocytosis assays; peroxisome counting\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific KO with defined electrophysiological and synaptic phenotypes, single lab\",\n      \"pmids\": [\"36552747\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX1 is an AAA-ATPase that forms a heterohexameric complex with PEX6 (alternating subunits in a double ring), anchored to the peroxisomal membrane via PEX15/PEX26; this complex uses ATP hydrolysis in its active D2 ring to processively thread and unfold substrates through its central pore—most critically monoubiquitinated PEX5—thereby extracting and recycling the peroxisomal matrix protein import receptor from the membrane; PEX1–PEX6 assembly is mutually stabilizing (reduced affinity triggers proteasomal degradation of PEX1), and loss of PEX1 function causes peroxisomal matrix protein import failure, accumulation of import-incompetent peroxisomal ghosts, and secondary pexophagy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PEX1 is an AAA-family ATPase essential for peroxisomal matrix protein import; its loss produces import-incompetent peroxisomal membrane remnants (\\\"ghosts\\\") that retain membrane proteins but lack matrix content and can be rescued by PEX1 re-expression [#0, #11, #20]. PEX1 functions as an obligate partner of PEX6, with which it forms a heterohexameric double-ring complex in which the two subunits alternate; the N-terminal D1 ring is catalytically inactive and the C-terminal D2 ring carries out the ATP hydrolysis that powers the motor [#8, #9]. This complex acts as a protein translocase that processively threads and unfolds substrates through its central pore via a pore-loop staircase, driven by D2 ATP hydrolysis and inter-subunit communication [#13, #15]. Its critical substrate is monoubiquitinated PEX5, the recycled PTS1 import receptor, which engages the complex through its ubiquitin moiety and is globally unfolded during ATP-dependent extraction from the peroxisomal membrane [#14]; the complex similarly unfolds and is anchored to the membrane via the tether Pex15/PEX26, bound by the Pex6 N1 domain [#13, #16]. PEX1 and PEX6 are mutually stabilizing, and disrupted assembly triggers proteasomal degradation of PEX1, a mechanism that underlies the common disease allele G843D, which destabilizes the D2 domain and impairs assembly while retaining residual motor activity [#3, #21]. Mutations spanning loss of PEX1 protein to attenuated PEX1\\u2013PEX6 interaction cause the peroxisome biogenesis disorder spectrum from severe Zellweger syndrome to milder NALD/IRD, with residual protein levels and PEX6-binding capacity determining phenotypic severity [#1, #3, #4]. Loss of PEX1 secondarily activates ROS-driven pexophagy, which is a consequence rather than the cause of the import defect [#18, #22].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established PEX1 as an AAA-ATPase whose loss specifically abolishes matrix protein import while leaving peroxisomal membranes intact, distinguishing import failure from organelle absence.\",\n      \"evidence\": \"Functional complementation in CG1 patient fibroblasts with PEX5 Western blot\",\n      \"pmids\": [\"9398847\", \"9398848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PEX1 acts on PEX5 not yet defined\", \"No partner protein identified at this stage\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified PEX6 as the direct physical partner of PEX1 and showed the interaction is required for biogenesis, framing PEX1 function as dependent on heterocomplex formation.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pulldown, and allele-specific genetic suppression by PEX6 overexpression\",\n      \"pmids\": [\"9671729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the complex unknown\", \"How the interaction enables import not addressed\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated that the common G843D allele is temperature-sensitive, defining it as a misfolded but partially functional protein rather than a null.\",\n      \"evidence\": \"Immunofluorescence and biochemical peroxisome assays at 30\\u00b0C vs 37\\u00b0C in patient fibroblasts and CHO mutants\",\n      \"pmids\": [\"9817926\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of thermolability unresolved\", \"Fate of misfolded protein not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Linked PEX1\\u2013PEX6 binding capacity and residual protein level to clinical severity, establishing the genotype-phenotype logic of the disease spectrum.\",\n      \"evidence\": \"Co-IP of PEX1 mutants with PEX6 and protein stability quantification across multiple alleles in patient fibroblasts\",\n      \"pmids\": [\"11439091\", \"11389485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degradation pathway for unstable PEX1 not identified\", \"No structural explanation for binding loss\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Solved the PEX1 N-terminal domain structure, revealing a double-psi-barrel fold shared with p97/VCP and NSF and implying an adaptor-binding role analogous to other membrane AAA-ATPases.\",\n      \"evidence\": \"X-ray crystallography at 2.05 \\u00c5 with structural homology analysis (mouse N-domain)\",\n      \"pmids\": [\"15328346\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional adaptor of the N-domain not identified\", \"Full-length architecture unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Characterized two N-terminal/Walker-motif activities: phosphoinositide binding by the N-domain and a Walker A1 requirement for both PEX6 binding and peroxisomal targeting.\",\n      \"evidence\": \"Protein-lipid overlay with arginine mutagenesis; co-IP and localization of PEX1-G606E at permissive/non-permissive temperatures\",\n      \"pmids\": [\"17018057\", \"16723118\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological role of lipid binding uncharacterized\", \"Walker A1 data from a single unreplicated study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the complex as an alternating PEX1/PEX6 heterohexameric double ring with an inactive D1 and active D2 ATPase ring, defining it mechanistically as a p97-like protein-extraction machine and localizing catalytic activity to Pex6 D2.\",\n      \"evidence\": \"Cryo-EM in multiple nucleotide states plus ATPase assays and Walker B mutagenesis (yeast Pex1/Pex6)\",\n      \"pmids\": [\"26170309\", \"26066397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrate not yet captured\", \"Translocation directionality and processivity unproven\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Separated the complex's role from membrane biogenesis: Pex1 depletion blocks matrix import but spares membrane protein delivery, and pex1\\u0394 ghosts can be matured to functional peroxisomes upon Pex1 restoration.\",\n      \"evidence\": \"Conditional Pex1 depletion with matrix vs membrane import assays; EM/tomography of ghosts with re-expression rescue (yeast)\",\n      \"pmids\": [\"26644516\", \"26644511\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ghosts are import precursors or stalled intermediates not fully resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified direct substrates and showed processive unfolding: the complex threads and unfolds the membrane anchor Pex15 through its pore, and monoubiquitinated PEX5 engages PEX1/PEX6 via its ubiquitin and is globally unfolded during ATP-dependent extraction.\",\n      \"evidence\": \"In vitro unfolding and pore-loop mutagenesis with cryo-EM (Pex15); cell-free system with photoaffinity cross-linking and PEGylation (Ub-PEX5)\",\n      \"pmids\": [\"29321502\", \"29884772\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ubiquitin recognition couples to pore engagement not fully defined\", \"Fate of PEX5 after extraction not traced here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the translocation mechanism at the residue level: a pore-1 loop staircase in D2 with a 'twin-seam' heterodimer that disengages to drive substrate movement, while the inactive D1 ring undergoes D2-fueled conformational cycling.\",\n      \"evidence\": \"Cryo-EM of S. cerevisiae Pex1/Pex6 with endogenous substrate trapped in the D2 pore\",\n      \"pmids\": [\"37741838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the trapped endogenous substrate not established\", \"Full ATPase cycle kinetics incomplete\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Assigned membrane tethering to the Pex6 N1 domain, which binds Pex15 and contacts a Pex1 D2 loop to stabilize the heterohexamer, with ATPase-active but biologically inactive complex when N1 is deleted.\",\n      \"evidence\": \"X-ray crystallography of Pex6 N1, cryo-EM, AlphaFold2 modeling, binding assays, in vivo complementation\",\n      \"pmids\": [\"38036174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Equivalent role of Pex1 N1 not fully resolved\", \"Coupling of tethering to substrate extraction unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Explained the molecular pathology of G843D: it destabilizes the D2 domain and impairs PEX6 assembly while retaining residual motor activity, and impaired assembly itself triggers proteasomal degradation of PEX1, providing a rationale for stabilization-based rescue.\",\n      \"evidence\": \"In vitro ATPase assays, co-IP, proteasome inhibition, deubiquitinase-fusion stabilization, and import rescue in human cells\",\n      \"pmids\": [\"40158855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating PEX1 degradation not identified\", \"Therapeutic generalizability across alleles untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Distinguished primary import function from secondary effects: PEX1 absence increases ghost abundance, and an N-terminally truncated isoform fails to rescue import but alters pre-peroxisomal vesicle number, hinting at a moonlighting N-terminal role.\",\n      \"evidence\": \"CRISPR PEX1-KO HEK293 with full-length and truncated isoform re-expression and import assays\",\n      \"pmids\": [\"36534601\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of N-terminal vesicle-regulatory role undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved that pexophagy is downstream of import failure and mapped its molecular triggers: PEX1 loss drives ROS, activating a TBK1-MARCHF7-PXMP4-NBR1 axis, while blocking pexophagy does not restore import or \\u03b2-oxidation.\",\n      \"evidence\": \"Yeast epistasis with \\u03b2-oxidation/import assays; HeLa knockdown with co-IP, ubiquitination, phosphorylation and flux assays\",\n      \"pmids\": [\"32013259\", \"41267209\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conservation of the TBK1-MARCHF7-PXMP4-NBR1 axis across cell types untested\", \"How ROS arises from import failure not detailed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PEX1 dysfunction produces tissue-specific phenotypes such as inner hair cell synaptic defects, and the in vivo significance of N-domain lipid binding, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking peroxisome loss to ribbon synapse/exocytosis defects unknown\", \"Physiological role of phosphoinositide binding by the N-domain uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 9, 21]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [13, 14, 15]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005778\", \"supporting_discovery_ids\": [7, 16]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [11, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 10, 14]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [12, 18, 22]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [11, 20]}\n    ],\n    \"complexes\": [\"PEX1\\u2013PEX6 AAA-ATPase heterohexamer\"],\n    \"partners\": [\"PEX6\", \"PEX5\", \"PEX15\", \"PEX26\", \"HNRNPA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}