{"gene":"PEX6","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":1996,"finding":"PXAAA1 (PEX6) encodes a cytoplasmic AAA-family ATPase required for peroxisomal matrix protein import; substitution of arginine for the conserved lysine in the ATPase domain abolished biological activity, confirming ATPase activity is essential. PEX6 is also required for stability of the PTS1 receptor Pxr1p (PEX5).","method":"Functional complementation in patient fibroblasts, ATPase domain mutagenesis (K→R), immunolocalization, receptor stability assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — active-site mutagenesis abolishing activity, complementation in human patient cells, receptor stability readout; foundational paper replicated by multiple subsequent studies","pmids":["8670792"],"is_preprint":false},{"year":1998,"finding":"PEX1 and PEX6 physically interact with each other in vitro and in the yeast two-hybrid system; overexpression of PEX6 suppresses certain PEX1-deficient phenotypes in an allele-specific manner requiring partial PEX1 activity, and the common disease mutation PEX1-G843D attenuates the PEX1–PEX6 interaction.","method":"Yeast two-hybrid, in vitro binding (pull-down), allele-specific genetic suppression (overexpression rescue)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal genetic suppression plus direct in vitro binding plus two-hybrid, replicated in subsequent structural and biochemical studies","pmids":["9671729"],"is_preprint":false},{"year":2011,"finding":"AWP1/ZFAND6 interacts with PEX6 AAA ATPase (but not with PEX1–PEX6 complexes) and preferentially binds cysteine-monoubiquitinated PEX5 via its A20 zinc-finger domain; AWP1 stimulates PEX5 export in an in vitro assay and its knockdown impairs PTS1-protein import and reduces PEX5 stability, identifying AWP1 as a novel PEX6 cofactor in PEX5 recycling.","method":"In vitro Pex5 export assay, co-immunoprecipitation, RNAi knockdown, PEX5 stability assay, domain mapping","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro export assay plus co-IP plus RNAi phenotype, multiple orthogonal methods in single study","pmids":["21980954"],"is_preprint":false},{"year":2015,"finding":"Cryo-EM structures of the Pex1/Pex6 complex in different nucleotide states reveal an unprecedented heterohexameric double ring in which Pex1 and Pex6 alternate; the N-terminal D1 ring is catalytically inactive and symmetric, while the C-terminal D2 ring is active and asymmetric. N1 of Pex1 is mobile; Pex6 N1 is packed against the ring. The architecture resembles p97/NSF, suggesting an analogous role in peroxisomal protein import to p97 in ERAD.","method":"Cryo-electron microscopy, computational domain fitting (Monte Carlo placement + energy minimization), nucleotide-state comparisons","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural determination with multiple nucleotide states; foundational structure replicated and extended by subsequent cryo-EM studies","pmids":["26170309"],"is_preprint":false},{"year":2015,"finding":"PEX6 localizes to cilia of retinal photoreceptor cells and to apical extensions of secretory ameloblasts/odontoblasts in mice, as shown by immunofluorescence, linking peroxisome biogenesis to ciliary function in the retina.","method":"Immunofluorescence microscopy in mouse retinal and dental tissues","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single localization study by immunofluorescence, single lab, no direct functional manipulation of the localization","pmids":["26593283"],"is_preprint":false},{"year":2018,"finding":"The yeast Pex1/Pex6 heterohexamer is a protein translocase that unfolds and processively threads substrates through its central pore in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Pex15 (the peroxisomal membrane tether) is recruited via its C-terminal disordered region engaging Pex1/Pex6 pore loops after first binding the N-terminal domains of Pex6. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to the import machinery.","method":"In vitro unfolding/threading assay, cryo-EM structural analysis of Pex15–Pex1/Pex6 complex, pore-loop mutagenesis, ATP hydrolysis assay, co-immunoprecipitation (Pex15–Pex5 binding)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro translocase activity, cryo-EM structure of substrate-bound complex, pore-loop mutagenesis, multiple orthogonal methods","pmids":["29321502"],"is_preprint":false},{"year":2018,"finding":"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide is globally unfolded during ATP-dependent extraction by the PEX1–PEX6 complex, establishing Ub-PEX5 as a bona fide substrate of the PEX1–PEX6 translocase.","method":"Cell-free in vitro extraction system, photoaffinity cross-linking, protein PEGylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — cell-free reconstituted extraction assay combined with photoaffinity cross-linking and PEGylation to detect unfolding; multiple orthogonal methods in single study","pmids":["29884772"],"is_preprint":false},{"year":2014,"finding":"Deficiency of the exportomer components Pex1, Pex6, and Pex15 in S. cerevisiae causes enhanced pexophagy dependent on Atg11 and the pexophagy receptor Atg36; accumulation of ubiquitinated receptors at the peroxisomal membrane in pex1Δ/pex6Δ cells does not drive pexophagy in yeast (negative result for that trigger). Nearly all peroxisomal membranes associate with phagophore assembly sites in pex1Δ atg1Δ cells.","method":"Genetic screen of peroxisome import mutants, fluorescence microscopy, epistasis analysis (atg1Δ, atg11Δ, atg36Δ double mutants)","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis with multiple double mutants, fluorescence microscopy, single lab","pmids":["24657987"],"is_preprint":false},{"year":2002,"finding":"In PEX6-deficient CHO cells, peroxisomal ghosts are complex membrane structures (a central spherical body with double-membraned loops, with ER alongside); upon complementation with PEX6 cDNA, these structures become import-competent and are converted into functional peroxisomes, with catalase and acyl-CoA oxidase first accumulating in the lumen of the double-membraned loops.","method":"Genetic complementation with PEX6 cDNA, electron microscopy, immunofluorescence, biochemical fractionation","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementation plus electron microscopy plus biochemical fractionation; single lab, multiple orthogonal methods","pmids":["11854424"],"is_preprint":false},{"year":2007,"finding":"In yeast, PEX6 (multicopy) suppresses aging defects in an atp2 mutant by improving mitochondrial import kinetics of Atp2p, requiring ATP binding/hydrolysis activity of Pex6p and specific residues in Atp2p; this reveals a novel role for Pex6p in mitochondrial maintenance and age asymmetry.","method":"Multicopy suppressor screen, epistasis analysis, mitochondrial import assay, ATP binding/hydrolysis mutants","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — suppressor screen plus mitochondrial import assay plus ATPase mutant analysis; single lab","pmids":["17465979"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of yeast Pex1/Pex6 with an endogenous substrate trapped in the D2 ring central pore reveal that pairs of Pex1/Pex6 D2 subdomains engage substrate via a staircase of pore-1 loops with distinct properties; the inactive D1 ring undergoes conformational changes (widening/narrowing) fueled by D2 ATP hydrolysis; a 'twin-seam' Pex1/Pex6 D2 heterodimer disengages from the staircase to propagate mechanical force in a manner unique to heteromeric AAA-ATPases.","method":"Cryo-EM with endogenous substrate trap, structural analysis of nucleotide states","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution cryo-EM with substrate-bound complex, single lab but rigorous structural methodology with mechanistic interpretation","pmids":["37741838"],"is_preprint":false},{"year":2023,"finding":"The N1 domain of Pex6 is required for Pex1/Pex6 function in vivo at the peroxisome, even though ΔN1-Pex6 retains ATPase activity in vitro. Crystal structure of the Pex6 N1 domain reveals the same fold as N-terminal domains of PEX1, CDC48, and NSF. Biochemical assays show Pex6 N1 mediates binding to the peroxisomal membrane tether Pex15 and also contacts an extended loop of the Pex1 D2 ATPase domain that stabilizes the heterohexamer.","method":"X-ray crystallography of isolated Pex6 N1 domain, cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 modeling, in vitro ATPase assay, biochemical binding assays, in vivo functional complementation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — X-ray crystal structure plus cryo-EM plus biochemical binding assays plus in vivo complementation; multiple orthogonal methods","pmids":["38036174"],"is_preprint":false},{"year":2003,"finding":"In Hansenula polymorpha, a point mutation G737E in the first AAA module of Pex6p confers cold-sensitive peroxisome biogenesis; a second intragenic mutation R1000G in the second AAA domain acts as an intragenic suppressor restoring growth at permissive temperature. These residues in both AAA domains are important for Pex6p function.","method":"UV mutagenesis, intragenic suppressor analysis, electron microscopy of peroxisome formation","journal":"FEMS yeast research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Weak — intragenic suppressor analysis with EM validation, single lab, yeast ortholog","pmids":["14613878"],"is_preprint":false},{"year":2000,"finding":"A temperature-sensitive missense mutation L57P in PEX6 (but not the counterpart L111P in PEX1, nor G708D in PEX6) causes temperature-sensitive peroxisome biogenesis in patient fibroblasts and CHO transfectants, demonstrating that L57 in the N-terminal region of PEX6 is critical for its activity and that temperature-sensitive residues are not conserved between PEX6 and PEX1.","method":"Temperature-shift experiments in patient fibroblasts and CHO transfectants, morphological and biochemical peroxisome assays","journal":"Pediatric research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — temperature-sensitive mutagenesis in multiple cell systems with morphological and biochemical readouts, single lab","pmids":["11004248"],"is_preprint":false},{"year":2026,"finding":"Pex6 (together with Pex26) regulates reversible, ATP-dependent topological remodeling of the peroxisomal membrane protein Pex14: under normal conditions Pex14's N-terminal domain faces the lumen, but loss of Pex6 or Pex26, or pharmacological inhibition of AAA+ ATPases, causes the Pex14 N-terminus to become cytoplasm-facing. Inhibition of ubiquitin activation (blocking Pex5 ubiquitination and extraction) prevented this reorientation, placing Pex5 ubiquitination and Pex6-mediated extraction upstream of Pex14 topological remodeling.","method":"Immunofluorescence microscopy, protease protection assay, pharmacological inhibitors (AAA+ ATPase inhibitor, MLN-7243 ubiquitin E1 inhibitor), Pex6/Pex26 deficiency cell lines","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protease protection plus immunofluorescence plus pharmacological perturbations; multiple orthogonal methods, single lab","pmids":["41581879"],"is_preprint":false}],"current_model":"PEX6 is a cytoplasmic AAA+ ATPase that forms a heterohexameric complex with PEX1 (alternating subunits in a double ring), anchored to the peroxisomal membrane via Pex15/Pex26; the catalytically active D2 ring uses ATP hydrolysis to processively thread and unfold substrates—principally monoubiquitinated PEX5—through its central pore, thereby extracting the receptor from the peroxisomal docking/translocation module to reset the matrix protein import cycle, while the Pex6 N1 domain mediates membrane recruitment via Pex15 and hexamer stabilization via Pex1, and Pex6 activity additionally regulates reversible topological remodeling of Pex14 during each import cycle."},"narrative":{"mechanistic_narrative":"PEX6 is a cytoplasmic AAA+ ATPase essential for peroxisomal matrix protein import, where its catalytic activity drives recycling of the cargo receptor PEX5 [PMID:8670792]. It functions not alone but as a heterohexameric double-ring complex with PEX1, in which the two proteins alternate; the N-terminal D1 ring is catalytically inactive while the C-terminal D2 ring is the active motor, an architecture resembling p97/NSF [PMID:9671729, PMID:26170309]. The active D2 ring uses ATP hydrolysis to processively thread substrate through its central pore via a staircase of pore loops, with a 'twin-seam' D2 heterodimer disengaging to propagate force in a manner specific to heteromeric AAA-ATPases [PMID:29321502, PMID:37741838]. Its principal substrate is monoubiquitinated PEX5, which engages PEX1 and PEX6 through its ubiquitin moiety and is globally unfolded during ATP-dependent extraction from the docking/translocation module, thereby resetting the import cycle [PMID:21980954, PMID:29884772]. The complex is recruited to the peroxisomal membrane through the tether Pex15/PEX26, which binds the PEX6 N-terminal (N1) domain before engaging the pore loops and also bridges to PEX5; the PEX6 N1 domain additionally stabilizes the heterohexamer by contacting PEX1 and is required for function in vivo independent of intrinsic ATPase activity [PMID:29321502, PMID:38036174]. Beyond receptor extraction, PEX6 activity governs reversible topological remodeling of Pex14 during the import cycle, with PEX5 ubiquitination and PEX6-mediated extraction acting upstream of this reorientation [PMID:41581879]. PEX6 mutations that attenuate the PEX1–PEX6 interaction or impair ATPase function underlie defective peroxisome biogenesis [PMID:9671729, PMID:11004248].","teleology":[{"year":1996,"claim":"Established that PEX6 is an essential AAA-family ATPase for peroxisomal matrix protein import and that its catalytic activity is required, answering whether the gene's ATPase function is biologically necessary.","evidence":"Functional complementation in patient fibroblasts with active-site (K→R) mutagenesis, immunolocalization and PEX5 receptor stability assay","pmids":["8670792"],"confidence":"High","gaps":["Did not define the complex partners or substrate","Mechanism of how ATPase activity supports import unresolved"]},{"year":1998,"claim":"Identified PEX1 as the direct physical and functional partner of PEX6, framing the two as a cooperating unit and linking a disease mutation to a weakened interaction.","evidence":"Yeast two-hybrid, in vitro pull-down, and allele-specific overexpression suppression in human cells","pmids":["9671729"],"confidence":"High","gaps":["Stoichiometry and architecture of the PEX1–PEX6 complex unknown","Functional output of the interaction not defined"]},{"year":2000,"claim":"Showed that the N-terminal region of PEX6 is functionally critical and that key residues differ from PEX1, establishing non-equivalence of the two paralogs.","evidence":"Temperature-sensitive missense mutation (L57P) analysis in patient fibroblasts and CHO transfectants","pmids":["11004248"],"confidence":"Medium","gaps":["Molecular role of the N-terminal region not defined","No structural basis at the time"]},{"year":2002,"claim":"Demonstrated that PEX6 deficiency arrests peroxisomes as import-incompetent membrane ghosts that become functional upon complementation, clarifying PEX6's role at the matrix import step.","evidence":"Genetic complementation with PEX6 cDNA, electron microscopy and biochemical fractionation in CHO cells","pmids":["11854424"],"confidence":"Medium","gaps":["Did not address the molecular import event PEX6 enables","Origin of double-membraned loop structures unexplained"]},{"year":2003,"claim":"Genetic dissection of both AAA modules showed each contributes to function and can be modulated by intragenic suppression, supporting cooperativity between the two nucleotide-binding domains.","evidence":"UV mutagenesis and intragenic suppressor analysis with EM in Hansenula polymorpha","pmids":["14613878"],"confidence":"Medium","gaps":["Catalytic versus structural roles of the two AAA rings not separated","Yeast ortholog; human relevance inferred"]},{"year":2007,"claim":"Revealed an unexpected moonlighting role for Pex6 in mitochondrial protein import kinetics and age asymmetry, broadening its functional scope beyond peroxisomes.","evidence":"Multicopy suppressor screen, mitochondrial import assay and ATP binding/hydrolysis mutants in yeast","pmids":["17465979"],"confidence":"Medium","gaps":["Mechanism connecting Pex6 to mitochondrial import unclear","Not shown to be a direct mitochondrial activity"]},{"year":2011,"claim":"Identified AWP1/ZFAND6 as a PEX6-specific cofactor that recognizes monoubiquitinated PEX5, linking the ubiquitin signal on the receptor to PEX6-driven export.","evidence":"In vitro PEX5 export assay, co-IP, RNAi knockdown and domain mapping","pmids":["21980954"],"confidence":"High","gaps":["How AWP1 hands off Ub-PEX5 to the motor unresolved","Structural basis of AWP1–PEX6 interaction unknown"]},{"year":2015,"claim":"Solved the heterohexameric double-ring architecture, defining D1 as inactive/symmetric and D2 as active/asymmetric and revealing a p97/NSF-like organization that implied a translocase mechanism.","evidence":"Cryo-EM of Pex1/Pex6 in multiple nucleotide states with computational domain fitting","pmids":["26170309"],"confidence":"High","gaps":["No substrate visualized at the time","Translocase activity was inferred, not demonstrated"]},{"year":2015,"claim":"Localized PEX6 to photoreceptor cilia and apical extensions of ameloblasts/odontoblasts, connecting peroxisome biogenesis to ciliary tissue contexts.","evidence":"Immunofluorescence microscopy in mouse retinal and dental tissues","pmids":["26593283"],"confidence":"Medium","gaps":["No functional manipulation of the ciliary localization","Single-lab descriptive finding"]},{"year":2018,"claim":"Reconstituted Pex1/Pex6 as a processive, pore-loop-dependent translocase and defined how the Pex15 tether is engaged and links the motor to PEX5, converting the structural model into a demonstrated mechanism.","evidence":"In vitro unfolding/threading assay, cryo-EM of the Pex15–Pex1/Pex6 complex, pore-loop mutagenesis and co-IP","pmids":["29321502"],"confidence":"High","gaps":["Native substrate engagement in cells not directly captured","Coupling to membrane retro-translocation step incomplete"]},{"year":2018,"claim":"Established monoubiquitinated PEX5 as a bona fide substrate that is globally unfolded during ATP-dependent extraction, directly demonstrating receptor recycling by the motor.","evidence":"Cell-free extraction system, photoaffinity cross-linking and PEGylation unfolding assay","pmids":["29884772"],"confidence":"High","gaps":["Fate of PEX5 after extraction not tracked","Role of deubiquitination in release unresolved"]},{"year":2023,"claim":"Captured an endogenous substrate in the D2 pore and defined a 'twin-seam' heterodimer mechanism, explaining how a heteromeric AAA-ATPase propagates mechanical force during threading.","evidence":"Cryo-EM with endogenous substrate trap and nucleotide-state analysis in yeast","pmids":["37741838"],"confidence":"High","gaps":["Identity/specificity of all endogenous substrates not enumerated","How D1 conformational cycling contributes functionally unclear"]},{"year":2023,"claim":"Showed the Pex6 N1 domain is essential in vivo independent of ATPase activity, mediating both Pex15 membrane binding and heterohexamer stabilization, resolving the long-standing functional importance of the N-terminus.","evidence":"X-ray crystallography of N1, cryo-EM, AlphaFold2 modeling, in vitro ATPase and binding assays, in vivo complementation","pmids":["38036174"],"confidence":"High","gaps":["Dynamics of N1–Pex15 engagement during the cycle not resolved","How N1 binding is regulated unknown"]},{"year":2026,"claim":"Linked PEX6 extraction activity to reversible topological remodeling of Pex14, placing PEX5 ubiquitination and PEX6 extraction upstream of membrane protein reorientation during import cycles.","evidence":"Protease protection, immunofluorescence and pharmacological inhibition (AAA+ ATPase and ubiquitin E1 inhibitors) in Pex6/Pex26-deficient cells","pmids":["41581879"],"confidence":"Medium","gaps":["Direct mechanism coupling extraction to Pex14 topology unresolved","Functional consequence of Pex14 reorientation unclear"]},{"year":null,"claim":"How the PEX6/PEX1 extraction cycle, receptor deubiquitination, and Pex14 topological remodeling are temporally coordinated into one import cycle, and the full substrate repertoire beyond PEX5, remain to be defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Complete substrate set unknown","Temporal coordination of extraction and membrane remodeling undefined","Regulation of cofactor (AWP1) handoff unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,3,5,6,10]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,6,10]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[8,11]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[8]}],"complexes":["PEX1-PEX6 heterohexamer"],"partners":["PEX1","PEX5","PEX26","ZFAND6","PEX14"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13608","full_name":"Peroxisomal ATPase PEX6","aliases":["Peroxin-6","Peroxisomal biogenesis factor 6","Peroxisomal-type ATPase 1","Peroxisome assembly factor 2","PAF-2"],"length_aa":980,"mass_kda":104.1,"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: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; Cell projection, cilium, photoreceptor outer segment","url":"https://www.uniprot.org/uniprotkb/Q13608/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX6","classification":"Not Classified","n_dependent_lines":187,"n_total_lines":1208,"dependency_fraction":0.15480132450331127},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PEX6","total_profiled":1310},"omim":[{"mim_id":"619518","title":"MUSCULAR DYSTROPHY, CONGENITAL HEARING LOSS, AND OVARIAN INSUFFICIENCY SYNDROME; MDHLO","url":"https://www.omim.org/entry/619518"},{"mim_id":"616617","title":"HEIMLER SYNDROME 2; HMLR2","url":"https://www.omim.org/entry/616617"},{"mim_id":"614885","title":"PEROXISOME BIOGENESIS DISORDER 11B; PBD11B","url":"https://www.omim.org/entry/614885"},{"mim_id":"614870","title":"PEROXISOME BIOGENESIS DISORDER 6A (ZELLWEGER); PBD6A","url":"https://www.omim.org/entry/614870"},{"mim_id":"614863","title":"PEROXISOME BIOGENESIS DISORDER 4B; PBD4B","url":"https://www.omim.org/entry/614863"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"pancreas","ntpm":71.9}],"url":"https://www.proteinatlas.org/search/PEX6"},"hgnc":{"alias_symbol":["PXAAA1","PAF-2"],"prev_symbol":[]},"alphafold":{"accession":"Q13608","domains":[{"cath_id":"-","chopping":"1-62_69-106","consensus_level":"medium","plddt":33.2914,"start":1,"end":106},{"cath_id":"2.40.40.20","chopping":"191-294","consensus_level":"medium","plddt":63.0966,"start":191,"end":294},{"cath_id":"3.10.330.10","chopping":"319-414","consensus_level":"medium","plddt":75.9725,"start":319,"end":414},{"cath_id":"3.40.50.300","chopping":"437-591","consensus_level":"high","plddt":79.9297,"start":437,"end":591},{"cath_id":"1.10.8.60","chopping":"597-654_671-690","consensus_level":"medium","plddt":84.5042,"start":597,"end":690},{"cath_id":"3.40.50.300","chopping":"696-868","consensus_level":"high","plddt":82.4492,"start":696,"end":868},{"cath_id":"1.10.8.60","chopping":"875-961","consensus_level":"high","plddt":87.7785,"start":875,"end":961}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13608","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13608-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13608-F1-predicted_aligned_error_v6.png","plddt_mean":69.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PEX6","jax_strain_url":"https://www.jax.org/strain/search?query=PEX6"},"sequence":{"accession":"Q13608","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13608.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13608/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13608"}},"corpus_meta":[{"pmid":"8670792","id":"PMC_8670792","title":"The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor.","date":"1996","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/8670792","citation_count":165,"is_preprint":false},{"pmid":"9671729","id":"PMC_9671729","title":"Disruption of a PEX1-PEX6 interaction is the most common cause of the neurologic disorders Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9671729","citation_count":105,"is_preprint":false},{"pmid":"8940266","id":"PMC_8940266","title":"Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans.","date":"1996","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/8940266","citation_count":96,"is_preprint":false},{"pmid":"26170309","id":"PMC_26170309","title":"Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26170309","citation_count":71,"is_preprint":false},{"pmid":"11873320","id":"PMC_11873320","title":"A PEX6-defective peroxisomal biogenesis disorder with severe phenotype in an infant, versus mild phenotype resembling Usher syndrome in the affected parents.","date":"2002","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11873320","citation_count":60,"is_preprint":false},{"pmid":"29321502","id":"PMC_29321502","title":"The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29321502","citation_count":57,"is_preprint":false},{"pmid":"24657987","id":"PMC_24657987","title":"Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae.","date":"2014","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/24657987","citation_count":57,"is_preprint":false},{"pmid":"21980954","id":"PMC_21980954","title":"AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase.","date":"2011","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/21980954","citation_count":46,"is_preprint":false},{"pmid":"29220678","id":"PMC_29220678","title":"Allelic Expression Imbalance Promoting a Mutant PEX6 Allele Causes Zellweger Spectrum Disorder.","date":"2017","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29220678","citation_count":41,"is_preprint":false},{"pmid":"29884772","id":"PMC_29884772","title":"Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29884772","citation_count":40,"is_preprint":false},{"pmid":"17041890","id":"PMC_17041890","title":"Identification of novel mutations in PEX2, PEX6, PEX10, PEX12, and PEX13 in Zellweger spectrum patients.","date":"2006","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/17041890","citation_count":39,"is_preprint":false},{"pmid":"16388862","id":"PMC_16388862","title":"Identification and characterization of three peroxins--PEX6, PEX10 and PEX12--involved in glycosome biogenesis in Trypanosoma brucei.","date":"2005","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/16388862","citation_count":37,"is_preprint":false},{"pmid":"26593283","id":"PMC_26593283","title":"PEX6 is Expressed in Photoreceptor Cilia and Mutated in Deafblindness with Enamel Dysplasia and Microcephaly.","date":"2015","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/26593283","citation_count":35,"is_preprint":false},{"pmid":"19877282","id":"PMC_19877282","title":"Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients.","date":"2010","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/19877282","citation_count":32,"is_preprint":false},{"pmid":"22894767","id":"PMC_22894767","title":"A founder mutation in the PEX6 gene is responsible for increased incidence of Zellweger syndrome in a French Canadian population.","date":"2012","source":"BMC medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22894767","citation_count":32,"is_preprint":false},{"pmid":"10408779","id":"PMC_10408779","title":"Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor-2) gene in patients with peroxisome biogenesis disorders.","date":"1999","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/10408779","citation_count":25,"is_preprint":false},{"pmid":"17465979","id":"PMC_17465979","title":"A novel role of peroxin PEX6: suppression of aging defects in mitochondria.","date":"2007","source":"Aging cell","url":"https://pubmed.ncbi.nlm.nih.gov/17465979","citation_count":24,"is_preprint":false},{"pmid":"11854424","id":"PMC_11854424","title":"Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation.","date":"2002","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/11854424","citation_count":24,"is_preprint":false},{"pmid":"28430524","id":"PMC_28430524","title":"miRNA-Related Polymorphisms in miR-423 (rs6505162) and PEX6 (rs1129186) and Risk of Esophageal Squamous Cell Carcinoma in an Iranian Cohort.","date":"2017","source":"Genetic testing and molecular biomarkers","url":"https://pubmed.ncbi.nlm.nih.gov/28430524","citation_count":23,"is_preprint":false},{"pmid":"31374812","id":"PMC_31374812","title":"Structural Mapping of Missense Mutations in the Pex1/Pex6 Complex.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31374812","citation_count":20,"is_preprint":false},{"pmid":"11004248","id":"PMC_11004248","title":"Temperature-sensitive mutation of PEX6 in peroxisome biogenesis disorders in complementation group C (CG-C): comparative study of PEX6 and PEX1.","date":"2000","source":"Pediatric research","url":"https://pubmed.ncbi.nlm.nih.gov/11004248","citation_count":19,"is_preprint":false},{"pmid":"28600347","id":"PMC_28600347","title":"The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology.","date":"2017","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/28600347","citation_count":18,"is_preprint":false},{"pmid":"37741838","id":"PMC_37741838","title":"Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37741838","citation_count":17,"is_preprint":false},{"pmid":"29676688","id":"PMC_29676688","title":"Ophthalmic manifestations of Heimler syndrome due to PEX6 mutations.","date":"2018","source":"Ophthalmic genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29676688","citation_count":17,"is_preprint":false},{"pmid":"25079577","id":"PMC_25079577","title":"Late-onset Zellweger spectrum disorder caused by PEX6 mutations mimicking X-linked adrenoleukodystrophy.","date":"2014","source":"Pediatric neurology","url":"https://pubmed.ncbi.nlm.nih.gov/25079577","citation_count":17,"is_preprint":false},{"pmid":"29047053","id":"PMC_29047053","title":"Mild Zellweger syndrome due to a novel PEX6 mutation: correlation between clinical phenotype and in silico prediction of variant pathogenicity.","date":"2017","source":"Journal of applied genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29047053","citation_count":13,"is_preprint":false},{"pmid":"31652724","id":"PMC_31652724","title":"A Mechanistic Perspective on PEX1 and PEX6, Two AAA+ Proteins of the Peroxisomal Protein Import Machinery.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31652724","citation_count":12,"is_preprint":false},{"pmid":"36249295","id":"PMC_36249295","title":"PEX6 Mutations in Peroxisomal Biogenesis Disorders: An Usher Syndrome Mimic.","date":"2021","source":"Ophthalmology science","url":"https://pubmed.ncbi.nlm.nih.gov/36249295","citation_count":9,"is_preprint":false},{"pmid":"32214787","id":"PMC_32214787","title":"Exome sequencing identifies PEX6 mutations in three cases diagnosed with Retinitis Pigmentosa and hearing impairment.","date":"2020","source":"Molecular vision","url":"https://pubmed.ncbi.nlm.nih.gov/32214787","citation_count":8,"is_preprint":false},{"pmid":"31555682","id":"PMC_31555682","title":"Two novel mutations of PEX6 in one Chinese Zellweger spectrum disorder and their clinical characteristics.","date":"2019","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31555682","citation_count":7,"is_preprint":false},{"pmid":"34631344","id":"PMC_34631344","title":"RNAi-mediated silencing of PEX6 and GAS1 genes of Fusarium oxysporum f. sp. lycopersici confers resistance against Fusarium wilt in tomato.","date":"2021","source":"3 Biotech","url":"https://pubmed.ncbi.nlm.nih.gov/34631344","citation_count":6,"is_preprint":false},{"pmid":"38036174","id":"PMC_38036174","title":"The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38036174","citation_count":5,"is_preprint":false},{"pmid":"11389979","id":"PMC_11389979","title":"Identification and characterisation of PEX6 orthologues from plants.","date":"2001","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11389979","citation_count":5,"is_preprint":false},{"pmid":"36980088","id":"PMC_36980088","title":"PEX6 Mutation in a Child with Infantile Refsum Disease-A Case Report and Literature Review.","date":"2023","source":"Children (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/36980088","citation_count":3,"is_preprint":false},{"pmid":"28033303","id":"PMC_28033303","title":"Strong cis-acting expression quantitative trait loci for the genes encoding SNHG5 and PEX6.","date":"2016","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28033303","citation_count":3,"is_preprint":false},{"pmid":"37842507","id":"PMC_37842507","title":"Zellweger's Syndrome With PEX6 Gene Mutation in Mixteco Neonates Due to Possible Founder Effect.","date":"2023","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/37842507","citation_count":1,"is_preprint":false},{"pmid":"39013483","id":"PMC_39013483","title":"A novel splice variant in intron 10 of PEX6 is associated with Zellweger Syndrome in a Chinese neonate.","date":"2024","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/39013483","citation_count":1,"is_preprint":false},{"pmid":"14613878","id":"PMC_14613878","title":"Identification of intragenic mutations in the Hansenula polymorpha PEX6 gene that affect peroxisome biogenesis and methylotrophic growth.","date":"2003","source":"FEMS yeast research","url":"https://pubmed.ncbi.nlm.nih.gov/14613878","citation_count":1,"is_preprint":false},{"pmid":"38154976","id":"PMC_38154976","title":"[Zellweger syndrome caused by PEX6 gene variation in 2 cases and literature review].","date":"2024","source":"Zhonghua er ke za zhi = Chinese journal of pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/38154976","citation_count":0,"is_preprint":false},{"pmid":"41581879","id":"PMC_41581879","title":"Pex6 and ubiquitination regulate topological remodeling of the peroxisomal membrane protein Pex14.","date":"2026","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41581879","citation_count":0,"is_preprint":false},{"pmid":"37745580","id":"PMC_37745580","title":"The Pex6 N1 domain is required for Pex15 binding and proper assembly with Pex1.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37745580","citation_count":0,"is_preprint":false},{"pmid":"36952198","id":"PMC_36952198","title":"Purification of a Recombinant Human PEX1/PEX6 AAA+ ATPase Complex from HEK293TT Cells.","date":"2023","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/36952198","citation_count":0,"is_preprint":false},{"pmid":"41787707","id":"PMC_41787707","title":"Unraveling PEX6: insights into very-long-chain fatty acid levels and peroxisome biogenesis disorders in pediatric populations.","date":"2026","source":"Annals of pediatric endocrinology & metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/41787707","citation_count":0,"is_preprint":false},{"pmid":"42074478","id":"PMC_42074478","title":"Heimler Syndrome Caused by Novel PEX6 Variants: Clinical and Genetic Characterization in a Saudi Cohort.","date":"2026","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/42074478","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.03.680009","title":"Identification of small molecule inhibitors of  <i>Trypanosoma</i>  PEX15– PEX6 interaction","date":"2025-10-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.03.680009","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.10.14.682039","title":"<i>ATAD1</i>  Overexpression Enhances Mitochondrial and Peroxisomal Function in Zellweger Syndrome Disorder Models","date":"2025-10-15","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.14.682039","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.10.627778","title":"PEX1  <sup>G843D</sup>  remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome","date":"2024-12-13","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.10.627778","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.01.25332822","title":"Genetically determined platelet traits impact stroke risk through multiple mechanisms and cell types","date":"2025-08-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.01.25332822","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.11.06.622258","title":"Complementation of a human disease phenotype in vitro by intercellular mRNA transfer","date":"2024-11-06","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.06.622258","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.20.629653","title":"Single Cell Transcriptomic Modelling of the Fallopian Tube Epithelium Identifies Cellular Specialisation, Novel Differentiation Trajectories, and Gene Network Associations with Ectopic Pregnancy","date":"2024-12-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.20.629653","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26767,"output_tokens":4250,"usd":0.072026,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12029,"output_tokens":4282,"usd":0.083598,"stage2_stop_reason":"end_turn"},"total_usd":0.155624,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"PXAAA1 (PEX6) encodes a cytoplasmic AAA-family ATPase required for peroxisomal matrix protein import; substitution of arginine for the conserved lysine in the ATPase domain abolished biological activity, confirming ATPase activity is essential. PEX6 is also required for stability of the PTS1 receptor Pxr1p (PEX5).\",\n      \"method\": \"Functional complementation in patient fibroblasts, ATPase domain mutagenesis (K→R), immunolocalization, receptor stability assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — active-site mutagenesis abolishing activity, complementation in human patient cells, receptor stability readout; foundational paper replicated by multiple subsequent studies\",\n      \"pmids\": [\"8670792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PEX1 and PEX6 physically interact with each other in vitro and in the yeast two-hybrid system; overexpression of PEX6 suppresses certain PEX1-deficient phenotypes in an allele-specific manner requiring partial PEX1 activity, and the common disease mutation PEX1-G843D attenuates the PEX1–PEX6 interaction.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding (pull-down), allele-specific genetic suppression (overexpression rescue)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal genetic suppression plus direct in vitro binding plus two-hybrid, replicated in subsequent structural and biochemical studies\",\n      \"pmids\": [\"9671729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AWP1/ZFAND6 interacts with PEX6 AAA ATPase (but not with PEX1–PEX6 complexes) and preferentially binds cysteine-monoubiquitinated PEX5 via its A20 zinc-finger domain; AWP1 stimulates PEX5 export in an in vitro assay and its knockdown impairs PTS1-protein import and reduces PEX5 stability, identifying AWP1 as a novel PEX6 cofactor in PEX5 recycling.\",\n      \"method\": \"In vitro Pex5 export assay, co-immunoprecipitation, RNAi knockdown, PEX5 stability assay, domain mapping\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro export assay plus co-IP plus RNAi phenotype, multiple orthogonal methods in single study\",\n      \"pmids\": [\"21980954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cryo-EM structures of the Pex1/Pex6 complex in different nucleotide states reveal an unprecedented heterohexameric double ring in which Pex1 and Pex6 alternate; the N-terminal D1 ring is catalytically inactive and symmetric, while the C-terminal D2 ring is active and asymmetric. N1 of Pex1 is mobile; Pex6 N1 is packed against the ring. The architecture resembles p97/NSF, suggesting an analogous role in peroxisomal protein import to p97 in ERAD.\",\n      \"method\": \"Cryo-electron microscopy, computational domain fitting (Monte Carlo placement + energy minimization), nucleotide-state comparisons\",\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 structural determination with multiple nucleotide states; foundational structure replicated and extended by subsequent cryo-EM studies\",\n      \"pmids\": [\"26170309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PEX6 localizes to cilia of retinal photoreceptor cells and to apical extensions of secretory ameloblasts/odontoblasts in mice, as shown by immunofluorescence, linking peroxisome biogenesis to ciliary function in the retina.\",\n      \"method\": \"Immunofluorescence microscopy in mouse retinal and dental tissues\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single localization study by immunofluorescence, single lab, no direct functional manipulation of the localization\",\n      \"pmids\": [\"26593283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The yeast Pex1/Pex6 heterohexamer is a protein translocase that unfolds and processively threads substrates through its central pore in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Pex15 (the peroxisomal membrane tether) is recruited via its C-terminal disordered region engaging Pex1/Pex6 pore loops after first binding the N-terminal domains of Pex6. Pex15 also directly binds the cargo receptor Pex5, linking Pex1/Pex6 to the import machinery.\",\n      \"method\": \"In vitro unfolding/threading assay, cryo-EM structural analysis of Pex15–Pex1/Pex6 complex, pore-loop mutagenesis, ATP hydrolysis assay, co-immunoprecipitation (Pex15–Pex5 binding)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro translocase activity, cryo-EM structure of substrate-bound complex, pore-loop mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"29321502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"DTM-embedded monoubiquitinated PEX5 (Ub-PEX5) directly interacts with both PEX1 and PEX6 through its ubiquitin moiety, and the PEX5 polypeptide is globally unfolded during ATP-dependent extraction by the PEX1–PEX6 complex, establishing Ub-PEX5 as a bona fide substrate of the PEX1–PEX6 translocase.\",\n      \"method\": \"Cell-free in vitro extraction system, photoaffinity cross-linking, protein PEGylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cell-free reconstituted extraction assay combined with photoaffinity cross-linking and PEGylation to detect unfolding; multiple orthogonal methods in single study\",\n      \"pmids\": [\"29884772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Deficiency of the exportomer components Pex1, Pex6, and Pex15 in S. cerevisiae causes enhanced pexophagy dependent on Atg11 and the pexophagy receptor Atg36; accumulation of ubiquitinated receptors at the peroxisomal membrane in pex1Δ/pex6Δ cells does not drive pexophagy in yeast (negative result for that trigger). Nearly all peroxisomal membranes associate with phagophore assembly sites in pex1Δ atg1Δ cells.\",\n      \"method\": \"Genetic screen of peroxisome import mutants, fluorescence microscopy, epistasis analysis (atg1Δ, atg11Δ, atg36Δ double mutants)\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis with multiple double mutants, fluorescence microscopy, single lab\",\n      \"pmids\": [\"24657987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In PEX6-deficient CHO cells, peroxisomal ghosts are complex membrane structures (a central spherical body with double-membraned loops, with ER alongside); upon complementation with PEX6 cDNA, these structures become import-competent and are converted into functional peroxisomes, with catalase and acyl-CoA oxidase first accumulating in the lumen of the double-membraned loops.\",\n      \"method\": \"Genetic complementation with PEX6 cDNA, electron microscopy, immunofluorescence, biochemical fractionation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementation plus electron microscopy plus biochemical fractionation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"11854424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In yeast, PEX6 (multicopy) suppresses aging defects in an atp2 mutant by improving mitochondrial import kinetics of Atp2p, requiring ATP binding/hydrolysis activity of Pex6p and specific residues in Atp2p; this reveals a novel role for Pex6p in mitochondrial maintenance and age asymmetry.\",\n      \"method\": \"Multicopy suppressor screen, epistasis analysis, mitochondrial import assay, ATP binding/hydrolysis mutants\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — suppressor screen plus mitochondrial import assay plus ATPase mutant analysis; single lab\",\n      \"pmids\": [\"17465979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of yeast Pex1/Pex6 with an endogenous substrate trapped in the D2 ring central pore reveal that pairs of Pex1/Pex6 D2 subdomains engage substrate via a staircase of pore-1 loops with distinct properties; the inactive D1 ring undergoes conformational changes (widening/narrowing) fueled by D2 ATP hydrolysis; a 'twin-seam' Pex1/Pex6 D2 heterodimer disengages from the staircase to propagate mechanical force in a manner unique to heteromeric AAA-ATPases.\",\n      \"method\": \"Cryo-EM with endogenous substrate trap, structural analysis of nucleotide states\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution cryo-EM with substrate-bound complex, single lab but rigorous structural methodology with mechanistic interpretation\",\n      \"pmids\": [\"37741838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The N1 domain of Pex6 is required for Pex1/Pex6 function in vivo at the peroxisome, even though ΔN1-Pex6 retains ATPase activity in vitro. Crystal structure of the Pex6 N1 domain reveals the same fold as N-terminal domains of PEX1, CDC48, and NSF. Biochemical assays show Pex6 N1 mediates binding to the peroxisomal membrane tether Pex15 and also contacts an extended loop of the Pex1 D2 ATPase domain that stabilizes the heterohexamer.\",\n      \"method\": \"X-ray crystallography of isolated Pex6 N1 domain, cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 modeling, in vitro ATPase assay, biochemical binding assays, in vivo functional complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — X-ray crystal structure plus cryo-EM plus biochemical binding assays plus in vivo complementation; multiple orthogonal methods\",\n      \"pmids\": [\"38036174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In Hansenula polymorpha, a point mutation G737E in the first AAA module of Pex6p confers cold-sensitive peroxisome biogenesis; a second intragenic mutation R1000G in the second AAA domain acts as an intragenic suppressor restoring growth at permissive temperature. These residues in both AAA domains are important for Pex6p function.\",\n      \"method\": \"UV mutagenesis, intragenic suppressor analysis, electron microscopy of peroxisome formation\",\n      \"journal\": \"FEMS yeast research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Weak — intragenic suppressor analysis with EM validation, single lab, yeast ortholog\",\n      \"pmids\": [\"14613878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"A temperature-sensitive missense mutation L57P in PEX6 (but not the counterpart L111P in PEX1, nor G708D in PEX6) causes temperature-sensitive peroxisome biogenesis in patient fibroblasts and CHO transfectants, demonstrating that L57 in the N-terminal region of PEX6 is critical for its activity and that temperature-sensitive residues are not conserved between PEX6 and PEX1.\",\n      \"method\": \"Temperature-shift experiments in patient fibroblasts and CHO transfectants, morphological and biochemical peroxisome assays\",\n      \"journal\": \"Pediatric research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — temperature-sensitive mutagenesis in multiple cell systems with morphological and biochemical readouts, single lab\",\n      \"pmids\": [\"11004248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Pex6 (together with Pex26) regulates reversible, ATP-dependent topological remodeling of the peroxisomal membrane protein Pex14: under normal conditions Pex14's N-terminal domain faces the lumen, but loss of Pex6 or Pex26, or pharmacological inhibition of AAA+ ATPases, causes the Pex14 N-terminus to become cytoplasm-facing. Inhibition of ubiquitin activation (blocking Pex5 ubiquitination and extraction) prevented this reorientation, placing Pex5 ubiquitination and Pex6-mediated extraction upstream of Pex14 topological remodeling.\",\n      \"method\": \"Immunofluorescence microscopy, protease protection assay, pharmacological inhibitors (AAA+ ATPase inhibitor, MLN-7243 ubiquitin E1 inhibitor), Pex6/Pex26 deficiency cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protease protection plus immunofluorescence plus pharmacological perturbations; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"41581879\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX6 is a cytoplasmic AAA+ ATPase that forms a heterohexameric complex with PEX1 (alternating subunits in a double ring), anchored to the peroxisomal membrane via Pex15/Pex26; the catalytically active D2 ring uses ATP hydrolysis to processively thread and unfold substrates—principally monoubiquitinated PEX5—through its central pore, thereby extracting the receptor from the peroxisomal docking/translocation module to reset the matrix protein import cycle, while the Pex6 N1 domain mediates membrane recruitment via Pex15 and hexamer stabilization via Pex1, and Pex6 activity additionally regulates reversible topological remodeling of Pex14 during each import cycle.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PEX6 is a cytoplasmic AAA+ ATPase essential for peroxisomal matrix protein import, where its catalytic activity drives recycling of the cargo receptor PEX5 [#0]. It functions not alone but as a heterohexameric double-ring complex with PEX1, in which the two proteins alternate; the N-terminal D1 ring is catalytically inactive while the C-terminal D2 ring is the active motor, an architecture resembling p97/NSF [#1, #3]. The active D2 ring uses ATP hydrolysis to processively thread substrate through its central pore via a staircase of pore loops, with a 'twin-seam' D2 heterodimer disengaging to propagate force in a manner specific to heteromeric AAA-ATPases [#5, #10]. Its principal substrate is monoubiquitinated PEX5, which engages PEX1 and PEX6 through its ubiquitin moiety and is globally unfolded during ATP-dependent extraction from the docking/translocation module, thereby resetting the import cycle [#2, #6]. The complex is recruited to the peroxisomal membrane through the tether Pex15/PEX26, which binds the PEX6 N-terminal (N1) domain before engaging the pore loops and also bridges to PEX5; the PEX6 N1 domain additionally stabilizes the heterohexamer by contacting PEX1 and is required for function in vivo independent of intrinsic ATPase activity [#5, #11]. Beyond receptor extraction, PEX6 activity governs reversible topological remodeling of Pex14 during the import cycle, with PEX5 ubiquitination and PEX6-mediated extraction acting upstream of this reorientation [#14]. PEX6 mutations that attenuate the PEX1\\u2013PEX6 interaction or impair ATPase function underlie defective peroxisome biogenesis [#1, #13].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that PEX6 is an essential AAA-family ATPase for peroxisomal matrix protein import and that its catalytic activity is required, answering whether the gene's ATPase function is biologically necessary.\",\n      \"evidence\": \"Functional complementation in patient fibroblasts with active-site (K\\u2192R) mutagenesis, immunolocalization and PEX5 receptor stability assay\",\n      \"pmids\": [\"8670792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the complex partners or substrate\", \"Mechanism of how ATPase activity supports import unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified PEX1 as the direct physical and functional partner of PEX6, framing the two as a cooperating unit and linking a disease mutation to a weakened interaction.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pull-down, and allele-specific overexpression suppression in human cells\",\n      \"pmids\": [\"9671729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the PEX1\\u2013PEX6 complex unknown\", \"Functional output of the interaction not defined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed that the N-terminal region of PEX6 is functionally critical and that key residues differ from PEX1, establishing non-equivalence of the two paralogs.\",\n      \"evidence\": \"Temperature-sensitive missense mutation (L57P) analysis in patient fibroblasts and CHO transfectants\",\n      \"pmids\": [\"11004248\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular role of the N-terminal region not defined\", \"No structural basis at the time\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrated that PEX6 deficiency arrests peroxisomes as import-incompetent membrane ghosts that become functional upon complementation, clarifying PEX6's role at the matrix import step.\",\n      \"evidence\": \"Genetic complementation with PEX6 cDNA, electron microscopy and biochemical fractionation in CHO cells\",\n      \"pmids\": [\"11854424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address the molecular import event PEX6 enables\", \"Origin of double-membraned loop structures unexplained\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic dissection of both AAA modules showed each contributes to function and can be modulated by intragenic suppression, supporting cooperativity between the two nucleotide-binding domains.\",\n      \"evidence\": \"UV mutagenesis and intragenic suppressor analysis with EM in Hansenula polymorpha\",\n      \"pmids\": [\"14613878\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Catalytic versus structural roles of the two AAA rings not separated\", \"Yeast ortholog; human relevance inferred\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed an unexpected moonlighting role for Pex6 in mitochondrial protein import kinetics and age asymmetry, broadening its functional scope beyond peroxisomes.\",\n      \"evidence\": \"Multicopy suppressor screen, mitochondrial import assay and ATP binding/hydrolysis mutants in yeast\",\n      \"pmids\": [\"17465979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting Pex6 to mitochondrial import unclear\", \"Not shown to be a direct mitochondrial activity\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified AWP1/ZFAND6 as a PEX6-specific cofactor that recognizes monoubiquitinated PEX5, linking the ubiquitin signal on the receptor to PEX6-driven export.\",\n      \"evidence\": \"In vitro PEX5 export assay, co-IP, RNAi knockdown and domain mapping\",\n      \"pmids\": [\"21980954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AWP1 hands off Ub-PEX5 to the motor unresolved\", \"Structural basis of AWP1\\u2013PEX6 interaction unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Solved the heterohexameric double-ring architecture, defining D1 as inactive/symmetric and D2 as active/asymmetric and revealing a p97/NSF-like organization that implied a translocase mechanism.\",\n      \"evidence\": \"Cryo-EM of Pex1/Pex6 in multiple nucleotide states with computational domain fitting\",\n      \"pmids\": [\"26170309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrate visualized at the time\", \"Translocase activity was inferred, not demonstrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Localized PEX6 to photoreceptor cilia and apical extensions of ameloblasts/odontoblasts, connecting peroxisome biogenesis to ciliary tissue contexts.\",\n      \"evidence\": \"Immunofluorescence microscopy in mouse retinal and dental tissues\",\n      \"pmids\": [\"26593283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional manipulation of the ciliary localization\", \"Single-lab descriptive finding\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Reconstituted Pex1/Pex6 as a processive, pore-loop-dependent translocase and defined how the Pex15 tether is engaged and links the motor to PEX5, converting the structural model into a demonstrated mechanism.\",\n      \"evidence\": \"In vitro unfolding/threading assay, cryo-EM of the Pex15\\u2013Pex1/Pex6 complex, pore-loop mutagenesis and co-IP\",\n      \"pmids\": [\"29321502\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Native substrate engagement in cells not directly captured\", \"Coupling to membrane retro-translocation step incomplete\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established monoubiquitinated PEX5 as a bona fide substrate that is globally unfolded during ATP-dependent extraction, directly demonstrating receptor recycling by the motor.\",\n      \"evidence\": \"Cell-free extraction system, photoaffinity cross-linking and PEGylation unfolding assay\",\n      \"pmids\": [\"29884772\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Fate of PEX5 after extraction not tracked\", \"Role of deubiquitination in release unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Captured an endogenous substrate in the D2 pore and defined a 'twin-seam' heterodimer mechanism, explaining how a heteromeric AAA-ATPase propagates mechanical force during threading.\",\n      \"evidence\": \"Cryo-EM with endogenous substrate trap and nucleotide-state analysis in yeast\",\n      \"pmids\": [\"37741838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity/specificity of all endogenous substrates not enumerated\", \"How D1 conformational cycling contributes functionally unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed the Pex6 N1 domain is essential in vivo independent of ATPase activity, mediating both Pex15 membrane binding and heterohexamer stabilization, resolving the long-standing functional importance of the N-terminus.\",\n      \"evidence\": \"X-ray crystallography of N1, cryo-EM, AlphaFold2 modeling, in vitro ATPase and binding assays, in vivo complementation\",\n      \"pmids\": [\"38036174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of N1\\u2013Pex15 engagement during the cycle not resolved\", \"How N1 binding is regulated unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linked PEX6 extraction activity to reversible topological remodeling of Pex14, placing PEX5 ubiquitination and PEX6 extraction upstream of membrane protein reorientation during import cycles.\",\n      \"evidence\": \"Protease protection, immunofluorescence and pharmacological inhibition (AAA+ ATPase and ubiquitin E1 inhibitors) in Pex6/Pex26-deficient cells\",\n      \"pmids\": [\"41581879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism coupling extraction to Pex14 topology unresolved\", \"Functional consequence of Pex14 reorientation unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the PEX6/PEX1 extraction cycle, receptor deubiquitination, and Pex14 topological remodeling are temporally coordinated into one import cycle, and the full substrate repertoire beyond PEX5, remain to be defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Complete substrate set unknown\", \"Temporal coordination of extraction and membrane remodeling undefined\", \"Regulation of cofactor (AWP1) handoff unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 3, 5, 6, 10]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 6, 10]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"PEX1-PEX6 heterohexamer\"],\n    \"partners\": [\"PEX1\", \"PEX5\", \"PEX26\", \"ZFAND6\", \"PEX14\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}