{"gene":"PEX12","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2009,"finding":"PEX12 (along with PEX2 and PEX10) functions as a RING-domain ubiquitin-protein isopeptide ligase (E3) in peroxisomal protein import. Specifically, PEX12 facilitates the PEX4 (Ubc10)-dependent monoubiquitination of the PTS1 receptor PEX5, targeting it for receptor recycling rather than proteasomal degradation.","method":"In vitro ubiquitin ligase activity assays; genetic and biochemical dissection of ubiquitin-conjugating enzyme specificity (Ubc4 vs. Pex4/Ubc10); yeast molecular and cellular biology","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic activity demonstrated, E3 ligase activity confirmed biochemically, enzyme-substrate specificity (Pex4-dependent monoubiquitination) established with multiple orthogonal approaches in a focused study","pmids":["19687296"],"is_preprint":false},{"year":1999,"finding":"The zinc RING domain of PEX12 physically interacts with PEX5 (the PTS1 receptor) and PEX10 (another integral peroxisomal membrane protein). A patient missense mutation S320F in the zinc-binding domain reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation. Loss of PEX12 does not reduce PEX5 docking to peroxisomes, placing PEX12 function downstream of receptor docking in the import pathway.","method":"Two-hybrid studies, blot overlay assays, co-immunoprecipitation, patient mutation analysis, genetic suppression by overexpression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, two-hybrid, blot overlay (three orthogonal binding methods), patient mutation corroborating functional residue, genetic epistasis via suppressor overexpression, all in one focused study","pmids":["10562279"],"is_preprint":false},{"year":1997,"finding":"Human PEX12 localizes to the peroxisome membrane and is essential for peroxisomal matrix protein import. Expression of PEX12 restores peroxisomal protein import in fibroblasts from PBD complement group 3 patients, and frameshift mutations in PEX12 are causative for this group.","method":"Subcellular fractionation/immunofluorescence for localization; functional complementation of PBD patient fibroblasts; patient mutation detection","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct complementation rescue with defined localization, replicated in patient cells and confirmed by independent group (PMID:9632816), establishing PEX12 as essential for matrix protein import","pmids":["9090384"],"is_preprint":false},{"year":1998,"finding":"PEX12 is an integral peroxisomal membrane protein with two transmembrane segments and a cytoplasm-facing C-terminal RING (zinc finger) motif. Both the N- and C-terminal cytoplasmic regions are essential for PEX12 biological function. The protein exposes both termini to the cytosol.","method":"Functional complementation of CHO mutant cells; topology mapping by expression of epitope-tagged and truncation mutants; GFP-based peroxisome restoration assay; patient mutation analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — topology established by systematic epitope-tag/truncation mapping with functional readout, complemented by patient mutations confirming essential domains, replicated across labs","pmids":["9632816"],"is_preprint":false},{"year":2002,"finding":"The PEX12 zinc RING domain does not detectably affect the kinetics of PEX5 binding to PTS1 peptide, demonstrating that PEX12 interaction with PEX5 does not regulate the initial cargo recognition step.","method":"Fluorescence anisotropy-based in vitro binding assay with purified recombinant PEX5 and PEX12 zinc RING domain added as competitor/modulator","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstituted binding assay with purified components and rigorous controls, but single lab and single method; reports a negative result (no effect on PEX5-PTS1 binding kinetics)","pmids":["12456682"],"is_preprint":false},{"year":2025,"finding":"PEX12 is part of the peroxisomal PEX2/PEX10/PEX12 E3-ubiquitin ligase complex. Structural and functional data show that the Pex5/Pex8 complex assembles with this E3 complex to initiate receptor recycling after cargo release, placing PEX12 in the translocation-to-recycling transition step.","method":"Cryo-EM/X-ray structure of Pex8-Pex5 complex; functional translocation assays in yeast; mutagenesis disrupting Pex8-Pex5 interaction; biochemical assembly assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural and functional evidence from a single preprint study; the specific PEX12 mechanistic placement is contextual within a broader Pex8-Pex5 study and not independently replicated yet","pmids":["bio_10.1101_2025.08.30.673231"],"is_preprint":true}],"current_model":"PEX12 is an integral peroxisomal membrane protein with two transmembrane segments and cytoplasm-facing N- and C-terminal regions; its C-terminal RING (zinc finger) domain acts as an E3 ubiquitin ligase that, in partnership with the E2 enzyme PEX4/Ubc10, monoubiquitinates the PTS1 import receptor PEX5 to drive receptor recycling after cargo release—a step that occurs downstream of PEX5 docking and is coordinated through direct RING-domain interactions with both PEX5 and PEX10 within the PEX2/PEX10/PEX12 E3 complex."},"narrative":{"mechanistic_narrative":"PEX12 is an integral peroxisomal membrane protein essential for peroxisomal matrix protein import, and its loss is causative for the complementation group 3 form of peroxisome biogenesis disorder, with import restored by complementation in patient fibroblasts [PMID:9090384]. The protein spans the membrane twice and exposes both its N- and C-terminal cytoplasmic regions—each required for function—with the C-terminus carrying a RING (zinc finger) motif [PMID:9632816]. Through this RING domain PEX12 directly binds the PTS1 import receptor PEX5 and the partner membrane protein PEX10, and a patient S320F mutation in the zinc-binding domain weakens both interactions; because loss of PEX12 does not impair PEX5 docking, PEX12 acts downstream of receptor docking [PMID:10562279]. Mechanistically, PEX12 functions as a RING-domain E3 ubiquitin ligase that, together with PEX2 and PEX10, drives PEX4 (Ubc10)-dependent monoubiquitination of PEX5 to target the receptor for recycling rather than proteasomal degradation [PMID:19687296]. This activity is confined to the recycling step rather than cargo recognition, as the PEX12 RING domain does not alter PEX5 binding to PTS1 peptide [PMID:12456682].","teleology":[{"year":1997,"claim":"Established that PEX12 is a peroxisomal membrane protein required for matrix protein import and the gene defective in a defined human disease group, answering whether PEX12 is essential for import at all.","evidence":"Functional complementation of PBD complementation group 3 patient fibroblasts with localization by fractionation/immunofluorescence and patient mutation detection","pmids":["9090384"],"confidence":"High","gaps":["Did not define membrane topology","Did not assign a biochemical activity to the protein"]},{"year":1998,"claim":"Defined PEX12 topology, showing two transmembrane segments with both termini cytosolic and a C-terminal RING motif, establishing where its functional domains face and act.","evidence":"Topology mapping via epitope-tagged and truncation mutants with GFP-based peroxisome restoration in CHO mutant cells, plus patient mutation analysis","pmids":["9632816"],"confidence":"High","gaps":["Did not establish the molecular activity of the RING domain","Binding partners not identified"]},{"year":1999,"claim":"Identified PEX5 and PEX10 as direct RING-domain binding partners and placed PEX12 downstream of receptor docking, clarifying its position in the import pathway.","evidence":"Two-hybrid, blot overlay, co-immunoprecipitation, patient S320F mutation analysis, and genetic suppression by PEX5/PEX10 overexpression","pmids":["10562279"],"confidence":"High","gaps":["Did not demonstrate enzymatic activity","Functional consequence of the interactions not resolved"]},{"year":2002,"claim":"Showed the PEX12 RING domain does not affect PEX5–PTS1 binding kinetics, excluding a role in initial cargo recognition and localizing PEX12 function to a later step.","evidence":"Fluorescence anisotropy in vitro binding assay with purified recombinant PEX5 and PEX12 RING domain","pmids":["12456682"],"confidence":"Medium","gaps":["Single lab and single method","Negative result; positive function not assigned here"]},{"year":2009,"claim":"Established PEX12 as a RING E3 ubiquitin ligase driving PEX4/Ubc10-dependent monoubiquitination of PEX5 for receptor recycling, defining its catalytic role in import.","evidence":"In vitro ubiquitin ligase assays and genetic/biochemical dissection of E2 specificity in yeast","pmids":["19687296"],"confidence":"High","gaps":["Precise PEX5 ubiquitination site/structural basis not resolved","Stoichiometry within the E3 complex not defined"]},{"year":2025,"claim":"Placed PEX12 within a PEX2/PEX10/PEX12 E3 complex that engages the Pex5/Pex8 module at the translocation-to-recycling transition, structurally framing the assembly that initiates recycling.","evidence":"Cryo-EM/X-ray structure of Pex8–Pex5, yeast translocation assays, and mutagenesis disrupting Pex8–Pex5 (preprint)","pmids":["bio_10.1101_2025.08.30.673231"],"confidence":"Medium","gaps":["PEX12-specific placement is contextual within a broader study","Not independently replicated"]},{"year":null,"claim":"How the PEX2/PEX10/PEX12 E3 complex is structurally organized in the membrane and how it coordinates E2 charging, substrate positioning, and the retrotranslocation machinery remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of the intact human E3 complex in the timeline","Mechanism linking monoubiquitination to receptor extraction unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[2,3]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0]}],"complexes":["PEX2/PEX10/PEX12 E3 ubiquitin ligase complex"],"partners":["PEX5","PEX10","PEX2","PEX4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00623","full_name":"Peroxisome assembly protein 12","aliases":["Peroxin-12","Peroxisome assembly factor 3","PAF-3"],"length_aa":359,"mass_kda":40.8,"function":"Component of a retrotranslocation channel required for peroxisome organization by mediating export of the PEX5 receptor from peroxisomes to the cytosol, thereby promoting PEX5 recycling (PubMed:24662292, PubMed:9354782, PubMed:9632816). The retrotranslocation channel is composed of PEX2, PEX10 and PEX12; each subunit contributing transmembrane segments that coassemble into an open channel that specifically allows the passage of PEX5 through the peroxisomal membrane (By similarity). PEX12 also regulates PEX5 recycling by activating the E3 ubiquitin-protein ligase activity of PEX10 (PubMed:24662292). When PEX5 recycling is compromised, PEX12 stimulates PEX10-mediated polyubiquitination of PEX5, leading to its subsequent degradation (By similarity)","subcellular_location":"Peroxisome membrane","url":"https://www.uniprot.org/uniprotkb/O00623/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX12","classification":"Not Classified","n_dependent_lines":14,"n_total_lines":1208,"dependency_fraction":0.011589403973509934},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PEX12","total_profiled":1310},"omim":[{"mim_id":"614958","title":"SCHLAFEN FAMILY, MEMBER 14; SLFN14","url":"https://www.omim.org/entry/614958"},{"mim_id":"614957","title":"SCHLAFEN FAMILY, MEMBER 13; SLFN13","url":"https://www.omim.org/entry/614957"},{"mim_id":"614956","title":"SCHLAFEN FAMILY, MEMBER 12-LIKE; SLFN12L","url":"https://www.omim.org/entry/614956"},{"mim_id":"614955","title":"SCHLAFEN FAMILY, MEMBER 12; SLFN12","url":"https://www.omim.org/entry/614955"},{"mim_id":"614953","title":"SCHLAFEN FAMILY, MEMBER 11; SLFN11","url":"https://www.omim.org/entry/614953"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PEX12"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O00623","domains":[{"cath_id":"-","chopping":"16-219","consensus_level":"high","plddt":84.6492,"start":16,"end":219},{"cath_id":"3.30.40.10","chopping":"297-359","consensus_level":"medium","plddt":86.8284,"start":297,"end":359}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00623","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00623-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00623-F1-predicted_aligned_error_v6.png","plddt_mean":81.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PEX12","jax_strain_url":"https://www.jax.org/strain/search?query=PEX12"},"sequence":{"accession":"O00623","fasta_url":"https://rest.uniprot.org/uniprotkb/O00623.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00623/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00623"}},"corpus_meta":[{"pmid":"19687296","id":"PMC_19687296","title":"Pex2 and pex12 function as protein-ubiquitin ligases in peroxisomal protein import.","date":"2009","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19687296","citation_count":164,"is_preprint":false},{"pmid":"9090384","id":"PMC_9090384","title":"Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders.","date":"1997","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9090384","citation_count":123,"is_preprint":false},{"pmid":"10562279","id":"PMC_10562279","title":"PEX12 interacts with PEX5 and PEX10 and acts downstream of receptor docking in peroxisomal matrix protein import.","date":"1999","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10562279","citation_count":117,"is_preprint":false},{"pmid":"9632816","id":"PMC_9632816","title":"PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p.","date":"1998","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9632816","citation_count":93,"is_preprint":false},{"pmid":"20679226","id":"PMC_20679226","title":"Different functions of the C3HC4 zinc RING finger peroxins PEX10, PEX2, and PEX12 in peroxisome formation and matrix protein import.","date":"2010","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/20679226","citation_count":48,"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":"17534573","id":"PMC_17534573","title":"A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 degrees C.","date":"2007","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17534573","citation_count":33,"is_preprint":false},{"pmid":"12456682","id":"PMC_12456682","title":"PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12456682","citation_count":30,"is_preprint":false},{"pmid":"14571262","id":"PMC_14571262","title":"Novel mutations in the PEX12 gene of patients with a peroxisome biogenesis disorder.","date":"2004","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/14571262","citation_count":17,"is_preprint":false},{"pmid":"33123925","id":"PMC_33123925","title":"A founder mutation in PEX12 among Egyptian patients in peroxisomal biogenesis disorder.","date":"2020","source":"Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/33123925","citation_count":5,"is_preprint":false},{"pmid":"26094004","id":"PMC_26094004","title":"A novel mutation in the PEX12 gene causing a peroxisomal biogenesis disorder.","date":"2015","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/26094004","citation_count":5,"is_preprint":false},{"pmid":"26947510","id":"PMC_26947510","title":"Effect of l-Arginine in One Patient with Peroxisome Biogenesis Disorder due to PEX12 Deficiency.","date":"2016","source":"Neuropediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/26947510","citation_count":5,"is_preprint":false},{"pmid":"40718457","id":"PMC_40718457","title":"Evaluation of Antimicrobial Activity of Novel Chimeric M-PEX12 Peptide Against Acinetobacter baumannii.","date":"2025","source":"Iranian journal of pharmaceutical research : IJPR","url":"https://pubmed.ncbi.nlm.nih.gov/40718457","citation_count":2,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.30.673231","title":"Structure of Pex8 in complex with peroxisomal receptor Pex5 reveals its essential role in peroxisomal cargo translocation","date":"2025-09-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.30.673231","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9584,"output_tokens":1786,"usd":0.027771,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8555,"output_tokens":2275,"usd":0.049825,"stage2_stop_reason":"end_turn"},"total_usd":0.077596,"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\": 2009,\n      \"finding\": \"PEX12 (along with PEX2 and PEX10) functions as a RING-domain ubiquitin-protein isopeptide ligase (E3) in peroxisomal protein import. Specifically, PEX12 facilitates the PEX4 (Ubc10)-dependent monoubiquitination of the PTS1 receptor PEX5, targeting it for receptor recycling rather than proteasomal degradation.\",\n      \"method\": \"In vitro ubiquitin ligase activity assays; genetic and biochemical dissection of ubiquitin-conjugating enzyme specificity (Ubc4 vs. Pex4/Ubc10); yeast molecular and cellular biology\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic activity demonstrated, E3 ligase activity confirmed biochemically, enzyme-substrate specificity (Pex4-dependent monoubiquitination) established with multiple orthogonal approaches in a focused study\",\n      \"pmids\": [\"19687296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The zinc RING domain of PEX12 physically interacts with PEX5 (the PTS1 receptor) and PEX10 (another integral peroxisomal membrane protein). A patient missense mutation S320F in the zinc-binding domain reduces binding to both PEX5 and PEX10. Overexpression of PEX5 or PEX10 suppresses this PEX12 mutation. Loss of PEX12 does not reduce PEX5 docking to peroxisomes, placing PEX12 function downstream of receptor docking in the import pathway.\",\n      \"method\": \"Two-hybrid studies, blot overlay assays, co-immunoprecipitation, patient mutation analysis, genetic suppression by overexpression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, two-hybrid, blot overlay (three orthogonal binding methods), patient mutation corroborating functional residue, genetic epistasis via suppressor overexpression, all in one focused study\",\n      \"pmids\": [\"10562279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human PEX12 localizes to the peroxisome membrane and is essential for peroxisomal matrix protein import. Expression of PEX12 restores peroxisomal protein import in fibroblasts from PBD complement group 3 patients, and frameshift mutations in PEX12 are causative for this group.\",\n      \"method\": \"Subcellular fractionation/immunofluorescence for localization; functional complementation of PBD patient fibroblasts; patient mutation detection\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct complementation rescue with defined localization, replicated in patient cells and confirmed by independent group (PMID:9632816), establishing PEX12 as essential for matrix protein import\",\n      \"pmids\": [\"9090384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PEX12 is an integral peroxisomal membrane protein with two transmembrane segments and a cytoplasm-facing C-terminal RING (zinc finger) motif. Both the N- and C-terminal cytoplasmic regions are essential for PEX12 biological function. The protein exposes both termini to the cytosol.\",\n      \"method\": \"Functional complementation of CHO mutant cells; topology mapping by expression of epitope-tagged and truncation mutants; GFP-based peroxisome restoration assay; patient mutation analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — topology established by systematic epitope-tag/truncation mapping with functional readout, complemented by patient mutations confirming essential domains, replicated across labs\",\n      \"pmids\": [\"9632816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The PEX12 zinc RING domain does not detectably affect the kinetics of PEX5 binding to PTS1 peptide, demonstrating that PEX12 interaction with PEX5 does not regulate the initial cargo recognition step.\",\n      \"method\": \"Fluorescence anisotropy-based in vitro binding assay with purified recombinant PEX5 and PEX12 zinc RING domain added as competitor/modulator\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstituted binding assay with purified components and rigorous controls, but single lab and single method; reports a negative result (no effect on PEX5-PTS1 binding kinetics)\",\n      \"pmids\": [\"12456682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PEX12 is part of the peroxisomal PEX2/PEX10/PEX12 E3-ubiquitin ligase complex. Structural and functional data show that the Pex5/Pex8 complex assembles with this E3 complex to initiate receptor recycling after cargo release, placing PEX12 in the translocation-to-recycling transition step.\",\n      \"method\": \"Cryo-EM/X-ray structure of Pex8-Pex5 complex; functional translocation assays in yeast; mutagenesis disrupting Pex8-Pex5 interaction; biochemical assembly assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural and functional evidence from a single preprint study; the specific PEX12 mechanistic placement is contextual within a broader Pex8-Pex5 study and not independently replicated yet\",\n      \"pmids\": [\"bio_10.1101_2025.08.30.673231\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PEX12 is an integral peroxisomal membrane protein with two transmembrane segments and cytoplasm-facing N- and C-terminal regions; its C-terminal RING (zinc finger) domain acts as an E3 ubiquitin ligase that, in partnership with the E2 enzyme PEX4/Ubc10, monoubiquitinates the PTS1 import receptor PEX5 to drive receptor recycling after cargo release—a step that occurs downstream of PEX5 docking and is coordinated through direct RING-domain interactions with both PEX5 and PEX10 within the PEX2/PEX10/PEX12 E3 complex.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PEX12 is an integral peroxisomal membrane protein essential for peroxisomal matrix protein import, and its loss is causative for the complementation group 3 form of peroxisome biogenesis disorder, with import restored by complementation in patient fibroblasts [#2]. The protein spans the membrane twice and exposes both its N- and C-terminal cytoplasmic regions—each required for function—with the C-terminus carrying a RING (zinc finger) motif [#3]. Through this RING domain PEX12 directly binds the PTS1 import receptor PEX5 and the partner membrane protein PEX10, and a patient S320F mutation in the zinc-binding domain weakens both interactions; because loss of PEX12 does not impair PEX5 docking, PEX12 acts downstream of receptor docking [#1]. Mechanistically, PEX12 functions as a RING-domain E3 ubiquitin ligase that, together with PEX2 and PEX10, drives PEX4 (Ubc10)-dependent monoubiquitination of PEX5 to target the receptor for recycling rather than proteasomal degradation [#0]. This activity is confined to the recycling step rather than cargo recognition, as the PEX12 RING domain does not alter PEX5 binding to PTS1 peptide [#4].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that PEX12 is a peroxisomal membrane protein required for matrix protein import and the gene defective in a defined human disease group, answering whether PEX12 is essential for import at all.\",\n      \"evidence\": \"Functional complementation of PBD complementation group 3 patient fibroblasts with localization by fractionation/immunofluorescence and patient mutation detection\",\n      \"pmids\": [\"9090384\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define membrane topology\", \"Did not assign a biochemical activity to the protein\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined PEX12 topology, showing two transmembrane segments with both termini cytosolic and a C-terminal RING motif, establishing where its functional domains face and act.\",\n      \"evidence\": \"Topology mapping via epitope-tagged and truncation mutants with GFP-based peroxisome restoration in CHO mutant cells, plus patient mutation analysis\",\n      \"pmids\": [\"9632816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the molecular activity of the RING domain\", \"Binding partners not identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified PEX5 and PEX10 as direct RING-domain binding partners and placed PEX12 downstream of receptor docking, clarifying its position in the import pathway.\",\n      \"evidence\": \"Two-hybrid, blot overlay, co-immunoprecipitation, patient S320F mutation analysis, and genetic suppression by PEX5/PEX10 overexpression\",\n      \"pmids\": [\"10562279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate enzymatic activity\", \"Functional consequence of the interactions not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed the PEX12 RING domain does not affect PEX5–PTS1 binding kinetics, excluding a role in initial cargo recognition and localizing PEX12 function to a later step.\",\n      \"evidence\": \"Fluorescence anisotropy in vitro binding assay with purified recombinant PEX5 and PEX12 RING domain\",\n      \"pmids\": [\"12456682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab and single method\", \"Negative result; positive function not assigned here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established PEX12 as a RING E3 ubiquitin ligase driving PEX4/Ubc10-dependent monoubiquitination of PEX5 for receptor recycling, defining its catalytic role in import.\",\n      \"evidence\": \"In vitro ubiquitin ligase assays and genetic/biochemical dissection of E2 specificity in yeast\",\n      \"pmids\": [\"19687296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise PEX5 ubiquitination site/structural basis not resolved\", \"Stoichiometry within the E3 complex not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed PEX12 within a PEX2/PEX10/PEX12 E3 complex that engages the Pex5/Pex8 module at the translocation-to-recycling transition, structurally framing the assembly that initiates recycling.\",\n      \"evidence\": \"Cryo-EM/X-ray structure of Pex8–Pex5, yeast translocation assays, and mutagenesis disrupting Pex8–Pex5 (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.08.30.673231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PEX12-specific placement is contextual within a broader study\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the PEX2/PEX10/PEX12 E3 complex is structurally organized in the membrane and how it coordinates E2 charging, substrate positioning, and the retrotranslocation machinery remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of the intact human E3 complex in the timeline\", \"Mechanism linking monoubiquitination to receptor extraction unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"PEX2/PEX10/PEX12 E3 ubiquitin ligase complex\"],\n    \"partners\": [\"PEX5\", \"PEX10\", \"PEX2\", \"PEX4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}