{"gene":"PEX3","run_date":"2026-04-29T11:37:58","timeline":{"discoveries":[{"year":2004,"finding":"PEX3 functions as the docking factor for PEX19 at the peroxisomal membrane: PEX3 interacts specifically with the docking domain of PEX19, is required for PEX19 to dock at peroxisomes, and is sufficient to dock PEX19 at heterologous organelles. PEX3 binds PEX19 via a conserved motif essential for docking activity, and transient inhibition of PEX3 abrogates class I PMP import without affecting class II PMP or matrix protein import.","method":"Co-immunoprecipitation, heterologous organelle docking assay, transient inhibition/knockdown with PMP import readout, domain mapping","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, functional docking assay, import assay, domain mutants) in a single rigorous study","pmids":["15007061"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the cytosolic domain of human PEX3 in complex with a PEX19-derived peptide reveals that PEX3 adopts a novel helical bundle fold with a hydrophobic groove at its membrane-distal end that engages PEX19 with nanomolar affinity. Mutagenesis identifies phenylalanine 29 of PEX19 as critical for this interaction, and key PEX3 residues are highly conserved across species.","method":"X-ray crystallography, isothermal titration calorimetry, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis validation","pmids":["20554521"],"is_preprint":false},{"year":1999,"finding":"Human PEX3 is an integral peroxisomal membrane protein with its N-terminus inside the peroxisome and C-terminus facing the cytoplasm. The N-terminal 33 amino acids are necessary and sufficient to direct PEX3 to peroxisomes. PEX3 physically interacts with PEX19 as shown by mammalian two-hybrid assay and co-immunoprecipitation of in vitro translated proteins.","method":"Immunofluorescence microscopy with N/C-terminal tags, GFP fusion truncation analysis, mammalian two-hybrid, co-immunoprecipitation of in vitro translated proteins","journal":"European journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing topology, targeting signal, and PEX19 interaction","pmids":["10430017"],"is_preprint":false},{"year":2000,"finding":"Loss-of-function mutations in human PEX3 cause Zellweger syndrome by abrogating peroxisome membrane synthesis, resulting in reduced abundance and/or mislocalization of peroxisomal membrane proteins to mitochondria. PEX3-mediated peroxisome biogenesis is independent of COPI (brefeldin A-insensitive) and COPII (dominant-negative SAR1-insensitive) membrane trafficking pathways.","method":"Patient mutation analysis, PEX3 re-expression rescue, brefeldin A treatment, dominant-negative SAR1 expression, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue plus pharmacological pathway inhibition with functional readouts","pmids":["10871277"],"is_preprint":false},{"year":2003,"finding":"FRET analysis demonstrates that the main intracellular site of PEX3-PEX19 interaction is at the peroxisomal membrane. PEX3 deletion constructs lacking either the N-terminal peroxisomal targeting sequence or the C-terminal PEX19-binding domain (residues 1-140) abolish both peroxisomal localization and PEX19 interaction.","method":"FRET (EYFP/ECFP fusion proteins), donor fluorescence photobleaching, PEX3 deletion mutant analysis in PEX3-deficient fibroblasts","journal":"European journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — FRET in living cells with domain mutants and functional rescue validation","pmids":["12924628"],"is_preprint":false},{"year":2012,"finding":"In S. cerevisiae, Pex3 recruits the pexophagy receptor Atg36 to peroxisomes. Pex3 alleles blocked specifically in pexophagy fail to recruit Atg36. When Pex3 is redirected to mitochondria, Atg36 follows and restores mitophagy in atg32 mutants. Atg36 in turn binds Atg8 and adaptor Atg11 to link peroxisomes to the core autophagy machinery.","method":"Pex3 mutant isolation (pexophagy-specific alleles), co-localization studies, Pex3 mitochondrial retargeting experiment, genetic epistasis (atg32 mutant rescue)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including separation-of-function mutants and organelle retargeting rescue","pmids":["22643220"],"is_preprint":false},{"year":2012,"finding":"Mutagenesis of three conserved surface regions of PEX3 (PEX19-binding region, hydrophobic groove, acidic cluster) shows the PEX19-binding region is critical for PEX19 affinity and PEX3 stability. The hydrophobic groove near the base of PEX3 is required for PMP insertion and maturation of preperoxisomes, while the acidic cluster is not functionally relevant. The PEX3-PEX19 interaction has a dual function: PMP import and de novo peroxisome formation.","method":"Site-directed mutagenesis, biochemical binding assays, functional complementation assays in peroxisome-deficient cells","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with biochemical and functional assays with clear structure-function outcomes","pmids":["22624858"],"is_preprint":false},{"year":2014,"finding":"High-level expression of PEX3 in mammalian cells induces pexophagy via a pathway requiring peroxisome ubiquitination and NBR1. Peroxisome targeting of PEX3 is essential for initiating this degradation. SQSTM1/p62 is required only for peroxisome clustering, not degradation. Ubiquitination of PEX3 itself is dispensable; an endogenous peroxisomal protein is the ubiquitination target.","method":"PEX3 overexpression, siRNA knockdown of NBR1/p62, autophagy inhibitors, PEX3 lysine/cysteine substitution mutant, immunofluorescence","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (siRNA, inhibitors, mutagenesis) with defined pathway dissection","pmids":["25007327"],"is_preprint":false},{"year":2014,"finding":"PEX16 mediates the peroxisomal trafficking of PEX3 (and PMP34) via the ER in mammalian cells. ER-redirected PEX3 (ssPEX3) is continuously imported into pre-existing peroxisomes, demonstrating that the ER constitutively supplies membrane proteins to peroxisomes. This was shown by depletion and overexpression of PEX16 combined with quantitative time-lapse live-cell fluorescence microscopy.","method":"ER signal sequence redirected PEX3, PEX16 siRNA depletion and overexpression, quantitative time-lapse fluorescence microscopy, biochemical fractionation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — live-cell imaging with functional perturbations (depletion and overexpression) and biochemical validation","pmids":["25002403"],"is_preprint":false},{"year":2015,"finding":"In Pichia pastoris, Pex3 not only docks pexophagy receptor Atg30 at the peroxisomal membrane but actively promotes Atg30 phosphorylation and Atg11 recruitment. Specific Pex3 residues define the Atg30-binding site, and Pex3 interaction is required for activation of Atg30-dependent pexophagy signaling.","method":"Binding site mapping by mutagenesis, pexophagy assays, phosphorylation assays, Atg11 recruitment assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple biochemical and functional assays defining Pex3's active role in Atg30 regulation","pmids":["25694426"],"is_preprint":false},{"year":2015,"finding":"Human PEX3 inserts co-translationally into the mammalian ER via the Sec61 translocon. The N-terminal transmembrane segment of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP), targeting the ribosome-nascent chain complex to the translocon. PEX3 then integrates into the ER membrane adjacent to Sec61α and TRAM, and subsequently exits the ER via ATP-dependent budding vesicles.","method":"Photocrosslinking, fluorescence spectroscopy, SRP binding assay, ATP-dependent budding assay, ribosome-nascent chain analysis","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 1 — multiple biochemical reconstitution approaches (photocrosslinking, spectroscopy, budding assay) establishing co-translational ER insertion mechanism","pmids":["26572236"],"is_preprint":false},{"year":2009,"finding":"In the yeast Yarrowia lipolytica, Pex3p and Pex3Bp function as peroxisomal receptors for class V myosin (Myo2p), directly interacting with the myosin globular tail domain to mediate peroxisome inheritance. Loss of Pex3Bp causes peroxisomes to be preferentially retained in the mother cell; overexpression of either Pex3Bp or Pex3p shifts peroxisomes to the bud.","method":"Direct interaction assay (myosin tail binding), genetic deletion and overexpression with peroxisome inheritance readout, fluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — direct interaction plus genetic gain/loss-of-function with quantified inheritance phenotype","pmids":["19822674"],"is_preprint":false},{"year":2013,"finding":"The N-terminal 17-amino acid segment of Pex3 contains two signals sufficient for sorting to the peroxisomal ER (pER) subdomain in S. cerevisiae. The Pex3 transmembrane segment is additionally required for subsequent transport from the pER to peroxisomes. The luminal domain mediates intra-ER sorting whereas the transmembrane segment mediates exit to peroxisomes.","method":"Pex3-Sec66 chimera analysis, GFP fusion truncations, expression in Drosophila S2R+ cells, fluorescence microscopy","journal":"Biology open","confidence":"High","confidence_rationale":"Tier 2 — chimeric protein analysis with functional readout across multiple cell types separating distinct sorting signals","pmids":["23951409"],"is_preprint":false},{"year":2009,"finding":"The cytosolic domain of human PEX3 binds membrane lipids: purified recombinant cytosolic domain precipitates with mild detergents and induces flocculation or partial solubilization of liposomes in lipid-binding assays.","method":"Recombinant protein purification, detergent precipitation assay, liposome binding and flocculation assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 in vitro assay, but single lab, single study with no mutagenesis to identify key residues","pmids":["19715730"],"is_preprint":false},{"year":2006,"finding":"In Hansenula polymorpha, reintroduced Pex3-GFP initially localizes to the ER and nuclear envelope before peroxisome formation occurs, and fractionation experiments confirm a small ER/nuclear envelope-associated Pex3 fraction during early peroxisome reassembly, suggesting the ER/nuclear envelope contributes to peroxisome formation de novo.","method":"Inducible Pex3-GFP expression, live fluorescence microscopy, cell fractionation","journal":"FEMS yeast research","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging plus biochemical fractionation, single lab","pmids":["16487342"],"is_preprint":false},{"year":2018,"finding":"In Pichia pastoris, Pex3 and Atg37 compete for overlapping binding sites in the middle domain of pexophagy receptor Atg30. Pex3 binding to Atg30 negatively regulates Hrr25 kinase-mediated phosphorylation of Atg30, while Atg37 binding positively regulates it. Atg37 localization at the peroxisomal membrane depends on Pex3. The competition between Pex3 and Atg37 for Atg30 controls assembly and activation of the pexophagic receptor protein complex.","method":"Binding site mapping, phosphorylation assays, co-immunoprecipitation, fluorescence microscopy, genetic epistasis","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal biochemical and genetic methods in a single study","pmids":["29260977"],"is_preprint":false},{"year":2020,"finding":"In S. cerevisiae, Pex3 interaction with pexophagy receptor Atg36 is essential for Hrr25 kinase-mediated phosphorylation of Atg36: cells lacking Pex3 or expressing a Pex3 mutant defective in Atg36 binding show abolished Atg36 phosphorylation. In vitro reconstitution shows Pex3 directly promotes Atg36 phosphorylation by Hrr25. Co-immunoprecipitation reveals that Atg36-Hrr25 interaction depends on Pex3. Pex3 binding also protects Atg36 from proteasomal degradation.","method":"In vitro reconstitution with recombinant proteins, co-immunoprecipitation, Pex3 mutant analysis, proteasome inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus co-IP and genetic mutant analysis","pmids":["32958557"],"is_preprint":false},{"year":2020,"finding":"In S. cerevisiae, the Pex3-Inp1 complex tethers peroxisomes to the plasma membrane. Inp1 bridges the peroxisomal membrane (via C-terminal Pex3-binding domain) and the plasma membrane (via N-terminal PI(4,5)P2-binding domain). Expression of artificial PM-PER tethers restores peroxisome retention in inp1Δ cells, and Inp1 meets criteria for a bona fide contact site tether.","method":"Genetic epistasis, domain truncation analysis, PI(4,5)P2 binding assay, artificial tether complementation, fluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — domain analysis, lipid binding assay, and artificial tether rescue establish molecular tethering mechanism","pmids":["32970792"],"is_preprint":false},{"year":2018,"finding":"In Hansenula polymorpha, Pex3 accumulates in patches at peroxisome-vacuole contact sites specifically during conditions of strong peroxisome expansion (methanol medium), and overproduction of Pex3 induces peroxisome-vacuole contact sites even under glucose conditions, indicating a direct role for Pex3 in forming this contact site.","method":"Electron and fluorescence microscopy, Pex3 overproduction, localization studies under different growth conditions","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 3 — localization correlation with functional context, single lab, no biochemical tether reconstitution","pmids":["30595161"],"is_preprint":false},{"year":2014,"finding":"Hydrogen-deuterium exchange mass spectrometry of the PEX3-PEX19 complex shows PEX19 remains intrinsically disordered upon binding, with three specific regions becoming shielded: the N-terminus, a stretch F64-L74, and the C-terminus. PEX3 becomes more protected in its PEX19-binding groove with minor changes elsewhere. PEX3 stabilizes PEX19 and prevents PEX3 aggregation.","method":"Hydrogen-deuterium exchange mass spectrometry (HDX-MS) of purified proteins","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 method (structural) but single lab, and functional consequences are inferred rather than directly tested","pmids":["25062251"],"is_preprint":false},{"year":2025,"finding":"In S. cerevisiae, the binding sites of Pex19, Atg30, and Inp1 on Pex3 overlap, and overexpression of any one partner causes competitive displacement affecting other Pex3-dependent peroxisomal processes (biogenesis, pexophagy, retention). Crystal structure of H. polymorpha Pex3-Pex19 complex and AlphaFold2 predictions confirm overlapping interaction regions.","method":"Overexpression competition assays, peroxisomal process readouts, crystal structure analysis, AlphaFold2 structural prediction","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 — crystal structure combined with functional competition assays, single lab","pmids":["40847803"],"is_preprint":false},{"year":2025,"finding":"High Pex3 levels in S. cerevisiae induce formation of peroxisome clusters surrounded by lipid droplets, mediated by peroxisome-peroxisome and peroxisome-lipid droplet contact sites. This clustering is independent of Pex19, Inp1, and Atg36. The cytosolic domain of Pex3 directly binds peroxisomes, suggesting a role in homotypic contact site formation. The lipid droplet-peroxisome contact sites require triacylglycerol lipase Tgl4. Similar effects are seen upon Pex3 overexpression in Drosophila.","method":"Pex3 overexpression, deletion of known Pex3 partners, GST-pulldown of cytosolic domain with peroxisomes, fluorescence microscopy in yeast and Drosophila","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding assay plus genetic dissection and cross-species validation, but single lab","pmids":["40628847"],"is_preprint":false},{"year":2024,"finding":"Cardiomyocyte-specific PEX3 knockout in mice impairs redox homeostasis and disrupts myocardial regenerative repair. Lipid metabolomics reveals that PEX3 promotes plasmalogen metabolism, and PEX3-regulated plasmalogens activate the AKT/GSK3β signaling pathway via plasma membrane localization of ITGB3.","method":"Cardiomyocyte-specific knockout mice, lipid metabolomics, AKT/GSK3β pathway analysis, ITGB3 plasma membrane localization assay, myocardial injury model","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO with metabolomics and signaling pathway mechanistic follow-up, single lab","pmids":["38951640"],"is_preprint":false},{"year":2025,"finding":"In yeast, newly synthesized Pex15 (a tail-anchored peroxisomal membrane protein) is targeted to peroxisomes primarily via the Pex19- and Pex3-dependent pathway. Mistargeted Pex15 on mitochondria is extracted by Msp1 and re-routed to peroxisomes via Pex19-Pex3. Even endogenous peroxisomal Pex15 is extracted by peroxisomal Msp1 but returns via Pex19-Pex3, demonstrating a constitutive recycling role for the Pex3-Pex19 pathway.","method":"Genetic analysis (msp1, pex19, pex3 mutants), fluorescence microscopy, co-immunoprecipitation, in vivo targeting assays","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with multiple mutants and biochemical support, single lab","pmids":["40344504"],"is_preprint":false},{"year":2023,"finding":"Germ cell-specific knockout of Pex3 in mice causes male sterility, with destruction of intercellular bridges between spermatids and formation of multinucleated giant cells. Sertoli cell-specific Pex3 deletion has no effect. Proteomics of Pex3-deleted spermatids reveals defective expression of peroxisomal and spermiogenesis-related proteins.","method":"Conditional germ cell-specific and Sertoli cell-specific Pex3 knockout mice, histology, proteomics","journal":"Journal of biomedical research","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific KO with defined cellular phenotype and proteomics, single lab","pmids":["38062668"],"is_preprint":false}],"current_model":"PEX3 is an integral peroxisomal membrane protein with a cytosol-facing helical bundle domain that serves as the docking receptor for PEX19 (via a hydrophobic groove engaging PEX19-Phe29), is co-translationally inserted into the ER via SRP/Sec61 and exits in ATP-dependent budding vesicles (with PEX16 mediating ER-to-peroxisome trafficking in mammals), nucleates peroxisome membrane biogenesis by enabling PEX19-dependent class I PMP import, recruits process-specific effectors (Atg36/Atg30 for pexophagy, Inp1 for plasma membrane tethering, myosin for inheritance) to peroxisomes through overlapping binding sites on its cytosolic domain, actively promotes phosphorylation and stabilization of pexophagy receptors by facilitating their interaction with the Hrr25 kinase, and in mammalian cells regulates plasmalogen metabolism to support AKT/GSK3β signaling via ITGB3 membrane localization."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing that PEX3 is an integral peroxisomal membrane protein with a defined topology (N-in/C-out) and identifying its N-terminal targeting signal and physical interaction with PEX19 laid the foundation for understanding its role in peroxisome biogenesis.","evidence":"GFP truncation analysis, immunofluorescence topology mapping, mammalian two-hybrid, and co-immunoprecipitation in human cells","pmids":["10430017"],"confidence":"High","gaps":["No information yet on whether PEX3 is a receptor or merely a cargo of PEX19","PEX3 function in biogenesis not yet directly tested"]},{"year":2000,"claim":"Linking PEX3 loss-of-function to Zellweger syndrome and demonstrating that PEX3-mediated peroxisome biogenesis is independent of COPI/COPII trafficking defined PEX3 as essential for peroxisome membrane assembly through a non-classical vesicular route.","evidence":"Patient mutation analysis, PEX3 re-expression rescue, brefeldin A and dominant-negative SAR1 insensitivity in human fibroblasts","pmids":["10871277"],"confidence":"High","gaps":["The actual membrane trafficking route for PEX3 remained unknown","How PEX3 absence leads to PMP mislocalization to mitochondria was not resolved"]},{"year":2004,"claim":"Demonstrating that PEX3 is the docking factor for PEX19 at the peroxisomal membrane—sufficient to dock PEX19 at heterologous organelles and required specifically for class I PMP import—resolved the directionality of the PEX3-PEX19 interaction.","evidence":"Co-immunoprecipitation, heterologous organelle docking assay, transient PEX3 inhibition with PMP import readout, domain mapping in human cells","pmids":["15007061"],"confidence":"High","gaps":["Structural basis of the PEX3-PEX19 interaction unknown","Whether PEX3 has additional roles beyond PEX19 docking not explored"]},{"year":2006,"claim":"Observing that reintroduced Pex3 first localizes to the ER/nuclear envelope before peroxisomes form provided early evidence that the ER contributes membrane material for de novo peroxisome biogenesis.","evidence":"Inducible Pex3-GFP expression with live imaging and fractionation in Hansenula polymorpha","pmids":["16487342"],"confidence":"Medium","gaps":["No direct demonstration of ER-to-peroxisome vesicular transport","Mechanism of Pex3 exit from the ER was undefined"]},{"year":2009,"claim":"Discovery that Pex3 directly binds the myosin V motor (Myo2p) to mediate peroxisome inheritance expanded PEX3's role from biogenesis factor to a multi-functional organelle scaffold.","evidence":"Direct interaction assay with myosin tail, genetic deletion/overexpression with inheritance readout in Yarrowia lipolytica","pmids":["19822674"],"confidence":"High","gaps":["Whether Pex3-myosin interaction is conserved in mammals unknown","How multiple effector interactions are coordinated on one Pex3 molecule unclear"]},{"year":2010,"claim":"The crystal structure of the PEX3 cytosolic domain bound to a PEX19 peptide revealed a novel helical bundle fold with a conserved hydrophobic groove, and mutagenesis pinpointed PEX19-Phe29 as critical, providing the atomic-level mechanism of docking.","evidence":"X-ray crystallography, isothermal titration calorimetry, and site-directed mutagenesis of human proteins","pmids":["20554521"],"confidence":"High","gaps":["Structure of full-length membrane-embedded PEX3 not determined","How PEX3 facilitates PMP insertion after docking remained unknown"]},{"year":2012,"claim":"Two parallel advances revealed that PEX3 operates as a dual-function scaffold: mutagenesis of surface regions showed the hydrophobic groove is required for PMP insertion distinct from PEX19 docking, while identification of pexophagy-specific Pex3 alleles demonstrated that Pex3 recruits the autophagy receptor Atg36 through a separable surface.","evidence":"Site-directed mutagenesis with PMP import and de novo biogenesis assays in human cells; pexophagy-specific mutant isolation and mitochondrial retargeting in S. cerevisiae","pmids":["22624858","22643220"],"confidence":"High","gaps":["Structural details of Atg36 binding site on Pex3 not resolved","Whether PMP insertion and pexophagy receptor docking are mutually exclusive was unknown"]},{"year":2014,"claim":"Three studies collectively established the ER-to-peroxisome trafficking route of PEX3: PEX16 was shown to mediate ER-to-peroxisome transport of PEX3, HDX-MS revealed that PEX19 remains disordered upon PEX3 binding, and overexpressed PEX3 was found to trigger ubiquitin- and NBR1-dependent pexophagy in mammalian cells.","evidence":"ER-redirected PEX3 with PEX16 depletion and live-cell imaging; HDX-MS of purified PEX3-PEX19 complex; PEX3 overexpression with siRNA knockdown of NBR1/p62 in human cells","pmids":["25002403","25062251","25007327"],"confidence":"High","gaps":["Identity of the ubiquitinated peroxisomal substrate in mammalian pexophagy not determined","Whether HDX-MS disorder of PEX19 reflects the cargo-loaded state unclear"]},{"year":2015,"claim":"Reconstitution of PEX3's co-translational insertion into the ER via SRP/Sec61 and demonstration that it exits in ATP-dependent budding vesicles defined the complete biogenetic itinerary of PEX3 from ribosome to peroxisome; concurrently, Pex3 was shown to actively promote Atg30 phosphorylation and Atg11 recruitment in P. pastoris pexophagy.","evidence":"Photocrosslinking and SRP binding assay with ribosome-nascent chains, ATP-dependent budding reconstitution; Atg30 phosphorylation and binding site mapping in P. pastoris","pmids":["26572236","25694426"],"confidence":"High","gaps":["Whether ER exit vesicles require specific coat proteins remains unresolved","Kinase identity for Atg30 phosphorylation not fully confirmed in this study"]},{"year":2018,"claim":"Competition between Pex3 and Atg37 for overlapping binding sites on Atg30 was shown to control pexophagic receptor activation via Hrr25 kinase, revealing a regulatory switch on Pex3's cytosolic domain that balances biogenesis and degradation.","evidence":"Binding site mapping, phosphorylation assays, co-immunoprecipitation, and genetic epistasis in P. pastoris","pmids":["29260977"],"confidence":"High","gaps":["Whether a similar competitive mechanism operates in S. cerevisiae or mammals is unknown","Structural basis of the Pex3-Atg30-Atg37 switch not resolved"]},{"year":2020,"claim":"In vitro reconstitution demonstrated that Pex3 directly promotes Hrr25-mediated phosphorylation of Atg36 and protects Atg36 from proteasomal degradation, while Pex3-Inp1 was established as a bona fide peroxisome–plasma membrane tether via PI(4,5)P2 binding, consolidating PEX3's role as a multi-effector scaffold.","evidence":"Reconstitution with recombinant Pex3/Atg36/Hrr25, proteasome inhibitor experiments in S. cerevisiae; Inp1 domain truncation, PI(4,5)P2 binding assay, and artificial tether rescue in S. cerevisiae","pmids":["32958557","32970792"],"confidence":"High","gaps":["Structural mechanism by which Pex3 facilitates Hrr25 access to Atg36 is unknown","Whether Pex3-Inp1 tethering is regulated remains unexplored"]},{"year":2023,"claim":"Germ cell-specific Pex3 knockout in mice demonstrated that Pex3-dependent peroxisome biogenesis is essential for spermatid intercellular bridge integrity and male fertility, extending functional relevance beyond Zellweger syndrome.","evidence":"Conditional germ cell-specific Pex3 knockout mice, histology, proteomics","pmids":["38062668"],"confidence":"Medium","gaps":["Specific peroxisomal metabolites underlying bridge destruction not identified","Whether PEX3 has non-peroxisomal roles in germ cells is unclear"]},{"year":2024,"claim":"Cardiomyocyte-specific PEX3 knockout revealed that PEX3-dependent plasmalogen metabolism activates AKT/GSK3β signaling through ITGB3 membrane localization, linking peroxisome biogenesis to cardiac regenerative repair.","evidence":"Conditional cardiomyocyte PEX3 knockout mice, lipid metabolomics, AKT/GSK3β pathway analysis, myocardial injury model","pmids":["38951640"],"confidence":"Medium","gaps":["Whether AKT activation is a direct consequence of plasmalogen loss or secondary is unresolved","Mechanism of plasmalogen-dependent ITGB3 membrane retention not defined"]},{"year":2025,"claim":"Structural and competition studies confirmed that PEX19, Atg36/Atg30, and Inp1 bind overlapping sites on PEX3, creating a competitive regulatory network; additionally, PEX3 was shown to participate in constitutive Pex15 recycling and in homotypic peroxisome–peroxisome and peroxisome–lipid droplet contact site formation.","evidence":"Crystal structure of H. polymorpha Pex3-Pex19, AlphaFold2 predictions, overexpression competition assays in yeast; genetic epistasis of Msp1/Pex3/Pex19 for Pex15 recycling; Pex3 overexpression with GST-pulldown in yeast and Drosophila","pmids":["40847803","40344504","40628847"],"confidence":"Medium","gaps":["Quantitative binding affinities for the competing partners not fully determined","Physiological relevance of Pex3-mediated peroxisome-lipid droplet contacts unclear","Whether constitutive Pex15 recycling occurs in mammals not tested"]},{"year":null,"claim":"How PEX3 mechanistically facilitates the membrane insertion step of PMPs after PEX19 docking, and whether the competitive binding of biogenesis versus quality-control effectors is regulated by post-translational modifications or signaling inputs, remain central unresolved questions.","evidence":"","pmids":[],"confidence":"High","gaps":["No reconstituted PMP insertion assay with defined lipid bilayers exists","Post-translational regulation of PEX3 effector switching is unexplored","Full-length membrane-embedded PEX3 structure not available"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,9,16,17]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[10,14,12,8]},{"term_id":"GO:0043226","term_label":"organelle","supporting_discovery_ids":[2,0,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,3,6,10,8]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[5,7,9,16]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[8,10,12,23]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[22]}],"complexes":["PEX3-PEX19 import receptor complex","Pex3-Inp1 tethering complex"],"partners":["PEX19","PEX16","ATG36","ATG30","INP1","HRR25","NBR1","MYO2"],"other_free_text":[]},"mechanistic_narrative":"PEX3 is an integral peroxisomal membrane protein that serves as a central platform for peroxisome membrane biogenesis, organelle quality control, and inter-organelle communication. Its cytosolic helical bundle domain contains a hydrophobic groove that docks PEX19 with nanomolar affinity (engaging PEX19-Phe29), enabling PEX19-dependent import of class I peroxisomal membrane proteins and de novo peroxisome formation; the same groove region is also required for PMP insertion and preperoxisome maturation [PMID:20554521, PMID:15007061, PMID:22624858]. PEX3 is co-translationally inserted into the ER via the SRP/Sec61 pathway and traffics to peroxisomes through PEX16-dependent and ATP-dependent budding vesicles, with overlapping binding sites on its cytosolic domain recruiting process-specific effectors—Atg36/Atg30 for pexophagy (where PEX3 actively promotes Hrr25-kinase-mediated phosphorylation and stabilization of these receptors), Inp1 for plasma-membrane tethering, and myosin for organelle inheritance [PMID:26572236, PMID:25002403, PMID:22643220, PMID:32958557, PMID:32970792, PMID:19822674, PMID:40847803]. Loss-of-function mutations in human PEX3 cause Zellweger syndrome by abolishing peroxisome membrane assembly [PMID:10871277]."},"prefetch_data":{"uniprot":{"accession":"P56589","full_name":"Peroxisomal biogenesis factor 3","aliases":["Peroxin-3","Peroxisomal assembly protein PEX3"],"length_aa":373,"mass_kda":42.1,"function":"Involved in peroxisome biosynthesis and integrity. Assembles membrane vesicles before the matrix proteins are translocated. As a docking factor for PEX19, is necessary for the import of peroxisomal membrane proteins in the peroxisomes","subcellular_location":"Peroxisome membrane","url":"https://www.uniprot.org/uniprotkb/P56589/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PEX3","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000034693","cell_line_id":"CID001586","localizations":[{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"PEX19","stoichiometry":10.0},{"gene":"ABCD3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001586","total_profiled":1310},"omim":[{"mim_id":"617370","title":"PEROXISOME BIOGENESIS DISORDER 10B; PBD10B","url":"https://www.omim.org/entry/617370"},{"mim_id":"614882","title":"PEROXISOME BIOGENESIS DISORDER 10A (ZELLWEGER); 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membrane: PEX3 interacts specifically with the docking domain of PEX19, is required for PEX19 to dock at peroxisomes, and is sufficient to dock PEX19 at heterologous organelles. PEX3 binds PEX19 via a conserved motif essential for docking activity, and transient inhibition of PEX3 abrogates class I PMP import without affecting class II PMP or matrix protein import.\",\n      \"method\": \"Co-immunoprecipitation, heterologous organelle docking assay, transient inhibition/knockdown with PMP import readout, domain mapping\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, functional docking assay, import assay, domain mutants) in a single rigorous study\",\n      \"pmids\": [\"15007061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the cytosolic domain of human PEX3 in complex with a PEX19-derived peptide reveals that PEX3 adopts a novel helical bundle fold with a hydrophobic groove at its membrane-distal end that engages PEX19 with nanomolar affinity. Mutagenesis identifies phenylalanine 29 of PEX19 as critical for this interaction, and key PEX3 residues are highly conserved across species.\",\n      \"method\": \"X-ray crystallography, isothermal titration calorimetry, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis validation\",\n      \"pmids\": [\"20554521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human PEX3 is an integral peroxisomal membrane protein with its N-terminus inside the peroxisome and C-terminus facing the cytoplasm. The N-terminal 33 amino acids are necessary and sufficient to direct PEX3 to peroxisomes. PEX3 physically interacts with PEX19 as shown by mammalian two-hybrid assay and co-immunoprecipitation of in vitro translated proteins.\",\n      \"method\": \"Immunofluorescence microscopy with N/C-terminal tags, GFP fusion truncation analysis, mammalian two-hybrid, co-immunoprecipitation of in vitro translated proteins\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing topology, targeting signal, and PEX19 interaction\",\n      \"pmids\": [\"10430017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Loss-of-function mutations in human PEX3 cause Zellweger syndrome by abrogating peroxisome membrane synthesis, resulting in reduced abundance and/or mislocalization of peroxisomal membrane proteins to mitochondria. PEX3-mediated peroxisome biogenesis is independent of COPI (brefeldin A-insensitive) and COPII (dominant-negative SAR1-insensitive) membrane trafficking pathways.\",\n      \"method\": \"Patient mutation analysis, PEX3 re-expression rescue, brefeldin A treatment, dominant-negative SAR1 expression, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue plus pharmacological pathway inhibition with functional readouts\",\n      \"pmids\": [\"10871277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"FRET analysis demonstrates that the main intracellular site of PEX3-PEX19 interaction is at the peroxisomal membrane. PEX3 deletion constructs lacking either the N-terminal peroxisomal targeting sequence or the C-terminal PEX19-binding domain (residues 1-140) abolish both peroxisomal localization and PEX19 interaction.\",\n      \"method\": \"FRET (EYFP/ECFP fusion proteins), donor fluorescence photobleaching, PEX3 deletion mutant analysis in PEX3-deficient fibroblasts\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — FRET in living cells with domain mutants and functional rescue validation\",\n      \"pmids\": [\"12924628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In S. cerevisiae, Pex3 recruits the pexophagy receptor Atg36 to peroxisomes. Pex3 alleles blocked specifically in pexophagy fail to recruit Atg36. When Pex3 is redirected to mitochondria, Atg36 follows and restores mitophagy in atg32 mutants. Atg36 in turn binds Atg8 and adaptor Atg11 to link peroxisomes to the core autophagy machinery.\",\n      \"method\": \"Pex3 mutant isolation (pexophagy-specific alleles), co-localization studies, Pex3 mitochondrial retargeting experiment, genetic epistasis (atg32 mutant rescue)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including separation-of-function mutants and organelle retargeting rescue\",\n      \"pmids\": [\"22643220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Mutagenesis of three conserved surface regions of PEX3 (PEX19-binding region, hydrophobic groove, acidic cluster) shows the PEX19-binding region is critical for PEX19 affinity and PEX3 stability. The hydrophobic groove near the base of PEX3 is required for PMP insertion and maturation of preperoxisomes, while the acidic cluster is not functionally relevant. The PEX3-PEX19 interaction has a dual function: PMP import and de novo peroxisome formation.\",\n      \"method\": \"Site-directed mutagenesis, biochemical binding assays, functional complementation assays in peroxisome-deficient cells\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with biochemical and functional assays with clear structure-function outcomes\",\n      \"pmids\": [\"22624858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"High-level expression of PEX3 in mammalian cells induces pexophagy via a pathway requiring peroxisome ubiquitination and NBR1. Peroxisome targeting of PEX3 is essential for initiating this degradation. SQSTM1/p62 is required only for peroxisome clustering, not degradation. Ubiquitination of PEX3 itself is dispensable; an endogenous peroxisomal protein is the ubiquitination target.\",\n      \"method\": \"PEX3 overexpression, siRNA knockdown of NBR1/p62, autophagy inhibitors, PEX3 lysine/cysteine substitution mutant, immunofluorescence\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (siRNA, inhibitors, mutagenesis) with defined pathway dissection\",\n      \"pmids\": [\"25007327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PEX16 mediates the peroxisomal trafficking of PEX3 (and PMP34) via the ER in mammalian cells. ER-redirected PEX3 (ssPEX3) is continuously imported into pre-existing peroxisomes, demonstrating that the ER constitutively supplies membrane proteins to peroxisomes. This was shown by depletion and overexpression of PEX16 combined with quantitative time-lapse live-cell fluorescence microscopy.\",\n      \"method\": \"ER signal sequence redirected PEX3, PEX16 siRNA depletion and overexpression, quantitative time-lapse fluorescence microscopy, biochemical fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging with functional perturbations (depletion and overexpression) and biochemical validation\",\n      \"pmids\": [\"25002403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Pichia pastoris, Pex3 not only docks pexophagy receptor Atg30 at the peroxisomal membrane but actively promotes Atg30 phosphorylation and Atg11 recruitment. Specific Pex3 residues define the Atg30-binding site, and Pex3 interaction is required for activation of Atg30-dependent pexophagy signaling.\",\n      \"method\": \"Binding site mapping by mutagenesis, pexophagy assays, phosphorylation assays, Atg11 recruitment assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical and functional assays defining Pex3's active role in Atg30 regulation\",\n      \"pmids\": [\"25694426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Human PEX3 inserts co-translationally into the mammalian ER via the Sec61 translocon. The N-terminal transmembrane segment of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP), targeting the ribosome-nascent chain complex to the translocon. PEX3 then integrates into the ER membrane adjacent to Sec61α and TRAM, and subsequently exits the ER via ATP-dependent budding vesicles.\",\n      \"method\": \"Photocrosslinking, fluorescence spectroscopy, SRP binding assay, ATP-dependent budding assay, ribosome-nascent chain analysis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biochemical reconstitution approaches (photocrosslinking, spectroscopy, budding assay) establishing co-translational ER insertion mechanism\",\n      \"pmids\": [\"26572236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In the yeast Yarrowia lipolytica, Pex3p and Pex3Bp function as peroxisomal receptors for class V myosin (Myo2p), directly interacting with the myosin globular tail domain to mediate peroxisome inheritance. Loss of Pex3Bp causes peroxisomes to be preferentially retained in the mother cell; overexpression of either Pex3Bp or Pex3p shifts peroxisomes to the bud.\",\n      \"method\": \"Direct interaction assay (myosin tail binding), genetic deletion and overexpression with peroxisome inheritance readout, fluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction plus genetic gain/loss-of-function with quantified inheritance phenotype\",\n      \"pmids\": [\"19822674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal 17-amino acid segment of Pex3 contains two signals sufficient for sorting to the peroxisomal ER (pER) subdomain in S. cerevisiae. The Pex3 transmembrane segment is additionally required for subsequent transport from the pER to peroxisomes. The luminal domain mediates intra-ER sorting whereas the transmembrane segment mediates exit to peroxisomes.\",\n      \"method\": \"Pex3-Sec66 chimera analysis, GFP fusion truncations, expression in Drosophila S2R+ cells, fluorescence microscopy\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chimeric protein analysis with functional readout across multiple cell types separating distinct sorting signals\",\n      \"pmids\": [\"23951409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The cytosolic domain of human PEX3 binds membrane lipids: purified recombinant cytosolic domain precipitates with mild detergents and induces flocculation or partial solubilization of liposomes in lipid-binding assays.\",\n      \"method\": \"Recombinant protein purification, detergent precipitation assay, liposome binding and flocculation assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 in vitro assay, but single lab, single study with no mutagenesis to identify key residues\",\n      \"pmids\": [\"19715730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In Hansenula polymorpha, reintroduced Pex3-GFP initially localizes to the ER and nuclear envelope before peroxisome formation occurs, and fractionation experiments confirm a small ER/nuclear envelope-associated Pex3 fraction during early peroxisome reassembly, suggesting the ER/nuclear envelope contributes to peroxisome formation de novo.\",\n      \"method\": \"Inducible Pex3-GFP expression, live fluorescence microscopy, cell fractionation\",\n      \"journal\": \"FEMS yeast research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging plus biochemical fractionation, single lab\",\n      \"pmids\": [\"16487342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Pichia pastoris, Pex3 and Atg37 compete for overlapping binding sites in the middle domain of pexophagy receptor Atg30. Pex3 binding to Atg30 negatively regulates Hrr25 kinase-mediated phosphorylation of Atg30, while Atg37 binding positively regulates it. Atg37 localization at the peroxisomal membrane depends on Pex3. The competition between Pex3 and Atg37 for Atg30 controls assembly and activation of the pexophagic receptor protein complex.\",\n      \"method\": \"Binding site mapping, phosphorylation assays, co-immunoprecipitation, fluorescence microscopy, genetic epistasis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical and genetic methods in a single study\",\n      \"pmids\": [\"29260977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In S. cerevisiae, Pex3 interaction with pexophagy receptor Atg36 is essential for Hrr25 kinase-mediated phosphorylation of Atg36: cells lacking Pex3 or expressing a Pex3 mutant defective in Atg36 binding show abolished Atg36 phosphorylation. In vitro reconstitution shows Pex3 directly promotes Atg36 phosphorylation by Hrr25. Co-immunoprecipitation reveals that Atg36-Hrr25 interaction depends on Pex3. Pex3 binding also protects Atg36 from proteasomal degradation.\",\n      \"method\": \"In vitro reconstitution with recombinant proteins, co-immunoprecipitation, Pex3 mutant analysis, proteasome inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus co-IP and genetic mutant analysis\",\n      \"pmids\": [\"32958557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In S. cerevisiae, the Pex3-Inp1 complex tethers peroxisomes to the plasma membrane. Inp1 bridges the peroxisomal membrane (via C-terminal Pex3-binding domain) and the plasma membrane (via N-terminal PI(4,5)P2-binding domain). Expression of artificial PM-PER tethers restores peroxisome retention in inp1Δ cells, and Inp1 meets criteria for a bona fide contact site tether.\",\n      \"method\": \"Genetic epistasis, domain truncation analysis, PI(4,5)P2 binding assay, artificial tether complementation, fluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain analysis, lipid binding assay, and artificial tether rescue establish molecular tethering mechanism\",\n      \"pmids\": [\"32970792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Hansenula polymorpha, Pex3 accumulates in patches at peroxisome-vacuole contact sites specifically during conditions of strong peroxisome expansion (methanol medium), and overproduction of Pex3 induces peroxisome-vacuole contact sites even under glucose conditions, indicating a direct role for Pex3 in forming this contact site.\",\n      \"method\": \"Electron and fluorescence microscopy, Pex3 overproduction, localization studies under different growth conditions\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization correlation with functional context, single lab, no biochemical tether reconstitution\",\n      \"pmids\": [\"30595161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Hydrogen-deuterium exchange mass spectrometry of the PEX3-PEX19 complex shows PEX19 remains intrinsically disordered upon binding, with three specific regions becoming shielded: the N-terminus, a stretch F64-L74, and the C-terminus. PEX3 becomes more protected in its PEX19-binding groove with minor changes elsewhere. PEX3 stabilizes PEX19 and prevents PEX3 aggregation.\",\n      \"method\": \"Hydrogen-deuterium exchange mass spectrometry (HDX-MS) of purified proteins\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 method (structural) but single lab, and functional consequences are inferred rather than directly tested\",\n      \"pmids\": [\"25062251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In S. cerevisiae, the binding sites of Pex19, Atg30, and Inp1 on Pex3 overlap, and overexpression of any one partner causes competitive displacement affecting other Pex3-dependent peroxisomal processes (biogenesis, pexophagy, retention). Crystal structure of H. polymorpha Pex3-Pex19 complex and AlphaFold2 predictions confirm overlapping interaction regions.\",\n      \"method\": \"Overexpression competition assays, peroxisomal process readouts, crystal structure analysis, AlphaFold2 structural prediction\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — crystal structure combined with functional competition assays, single lab\",\n      \"pmids\": [\"40847803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"High Pex3 levels in S. cerevisiae induce formation of peroxisome clusters surrounded by lipid droplets, mediated by peroxisome-peroxisome and peroxisome-lipid droplet contact sites. This clustering is independent of Pex19, Inp1, and Atg36. The cytosolic domain of Pex3 directly binds peroxisomes, suggesting a role in homotypic contact site formation. The lipid droplet-peroxisome contact sites require triacylglycerol lipase Tgl4. Similar effects are seen upon Pex3 overexpression in Drosophila.\",\n      \"method\": \"Pex3 overexpression, deletion of known Pex3 partners, GST-pulldown of cytosolic domain with peroxisomes, fluorescence microscopy in yeast and Drosophila\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding assay plus genetic dissection and cross-species validation, but single lab\",\n      \"pmids\": [\"40628847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cardiomyocyte-specific PEX3 knockout in mice impairs redox homeostasis and disrupts myocardial regenerative repair. Lipid metabolomics reveals that PEX3 promotes plasmalogen metabolism, and PEX3-regulated plasmalogens activate the AKT/GSK3β signaling pathway via plasma membrane localization of ITGB3.\",\n      \"method\": \"Cardiomyocyte-specific knockout mice, lipid metabolomics, AKT/GSK3β pathway analysis, ITGB3 plasma membrane localization assay, myocardial injury model\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with metabolomics and signaling pathway mechanistic follow-up, single lab\",\n      \"pmids\": [\"38951640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In yeast, newly synthesized Pex15 (a tail-anchored peroxisomal membrane protein) is targeted to peroxisomes primarily via the Pex19- and Pex3-dependent pathway. Mistargeted Pex15 on mitochondria is extracted by Msp1 and re-routed to peroxisomes via Pex19-Pex3. Even endogenous peroxisomal Pex15 is extracted by peroxisomal Msp1 but returns via Pex19-Pex3, demonstrating a constitutive recycling role for the Pex3-Pex19 pathway.\",\n      \"method\": \"Genetic analysis (msp1, pex19, pex3 mutants), fluorescence microscopy, co-immunoprecipitation, in vivo targeting assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple mutants and biochemical support, single lab\",\n      \"pmids\": [\"40344504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Germ cell-specific knockout of Pex3 in mice causes male sterility, with destruction of intercellular bridges between spermatids and formation of multinucleated giant cells. Sertoli cell-specific Pex3 deletion has no effect. Proteomics of Pex3-deleted spermatids reveals defective expression of peroxisomal and spermiogenesis-related proteins.\",\n      \"method\": \"Conditional germ cell-specific and Sertoli cell-specific Pex3 knockout mice, histology, proteomics\",\n      \"journal\": \"Journal of biomedical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO with defined cellular phenotype and proteomics, single lab\",\n      \"pmids\": [\"38062668\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PEX3 is an integral peroxisomal membrane protein with a cytosol-facing helical bundle domain that serves as the docking receptor for PEX19 (via a hydrophobic groove engaging PEX19-Phe29), is co-translationally inserted into the ER via SRP/Sec61 and exits in ATP-dependent budding vesicles (with PEX16 mediating ER-to-peroxisome trafficking in mammals), nucleates peroxisome membrane biogenesis by enabling PEX19-dependent class I PMP import, recruits process-specific effectors (Atg36/Atg30 for pexophagy, Inp1 for plasma membrane tethering, myosin for inheritance) to peroxisomes through overlapping binding sites on its cytosolic domain, actively promotes phosphorylation and stabilization of pexophagy receptors by facilitating their interaction with the Hrr25 kinase, and in mammalian cells regulates plasmalogen metabolism to support AKT/GSK3β signaling via ITGB3 membrane localization.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PEX3 is an integral peroxisomal membrane protein that serves as a central platform for peroxisome membrane biogenesis, organelle quality control, and inter-organelle communication. Its cytosolic helical bundle domain contains a hydrophobic groove that docks PEX19 with nanomolar affinity (engaging PEX19-Phe29), enabling PEX19-dependent import of class I peroxisomal membrane proteins and de novo peroxisome formation; the same groove region is also required for PMP insertion and preperoxisome maturation [PMID:20554521, PMID:15007061, PMID:22624858]. PEX3 is co-translationally inserted into the ER via the SRP/Sec61 pathway and traffics to peroxisomes through PEX16-dependent and ATP-dependent budding vesicles, with overlapping binding sites on its cytosolic domain recruiting process-specific effectors—Atg36/Atg30 for pexophagy (where PEX3 actively promotes Hrr25-kinase-mediated phosphorylation and stabilization of these receptors), Inp1 for plasma-membrane tethering, and myosin for organelle inheritance [PMID:26572236, PMID:25002403, PMID:22643220, PMID:32958557, PMID:32970792, PMID:19822674, PMID:40847803]. Loss-of-function mutations in human PEX3 cause Zellweger syndrome by abolishing peroxisome membrane assembly [PMID:10871277].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that PEX3 is an integral peroxisomal membrane protein with a defined topology (N-in/C-out) and identifying its N-terminal targeting signal and physical interaction with PEX19 laid the foundation for understanding its role in peroxisome biogenesis.\",\n      \"evidence\": \"GFP truncation analysis, immunofluorescence topology mapping, mammalian two-hybrid, and co-immunoprecipitation in human cells\",\n      \"pmids\": [\"10430017\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No information yet on whether PEX3 is a receptor or merely a cargo of PEX19\", \"PEX3 function in biogenesis not yet directly tested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linking PEX3 loss-of-function to Zellweger syndrome and demonstrating that PEX3-mediated peroxisome biogenesis is independent of COPI/COPII trafficking defined PEX3 as essential for peroxisome membrane assembly through a non-classical vesicular route.\",\n      \"evidence\": \"Patient mutation analysis, PEX3 re-expression rescue, brefeldin A and dominant-negative SAR1 insensitivity in human fibroblasts\",\n      \"pmids\": [\"10871277\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The actual membrane trafficking route for PEX3 remained unknown\", \"How PEX3 absence leads to PMP mislocalization to mitochondria was not resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating that PEX3 is the docking factor for PEX19 at the peroxisomal membrane—sufficient to dock PEX19 at heterologous organelles and required specifically for class I PMP import—resolved the directionality of the PEX3-PEX19 interaction.\",\n      \"evidence\": \"Co-immunoprecipitation, heterologous organelle docking assay, transient PEX3 inhibition with PMP import readout, domain mapping in human cells\",\n      \"pmids\": [\"15007061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the PEX3-PEX19 interaction unknown\", \"Whether PEX3 has additional roles beyond PEX19 docking not explored\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Observing that reintroduced Pex3 first localizes to the ER/nuclear envelope before peroxisomes form provided early evidence that the ER contributes membrane material for de novo peroxisome biogenesis.\",\n      \"evidence\": \"Inducible Pex3-GFP expression with live imaging and fractionation in Hansenula polymorpha\",\n      \"pmids\": [\"16487342\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct demonstration of ER-to-peroxisome vesicular transport\", \"Mechanism of Pex3 exit from the ER was undefined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that Pex3 directly binds the myosin V motor (Myo2p) to mediate peroxisome inheritance expanded PEX3's role from biogenesis factor to a multi-functional organelle scaffold.\",\n      \"evidence\": \"Direct interaction assay with myosin tail, genetic deletion/overexpression with inheritance readout in Yarrowia lipolytica\",\n      \"pmids\": [\"19822674\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Pex3-myosin interaction is conserved in mammals unknown\", \"How multiple effector interactions are coordinated on one Pex3 molecule unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The crystal structure of the PEX3 cytosolic domain bound to a PEX19 peptide revealed a novel helical bundle fold with a conserved hydrophobic groove, and mutagenesis pinpointed PEX19-Phe29 as critical, providing the atomic-level mechanism of docking.\",\n      \"evidence\": \"X-ray crystallography, isothermal titration calorimetry, and site-directed mutagenesis of human proteins\",\n      \"pmids\": [\"20554521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length membrane-embedded PEX3 not determined\", \"How PEX3 facilitates PMP insertion after docking remained unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Two parallel advances revealed that PEX3 operates as a dual-function scaffold: mutagenesis of surface regions showed the hydrophobic groove is required for PMP insertion distinct from PEX19 docking, while identification of pexophagy-specific Pex3 alleles demonstrated that Pex3 recruits the autophagy receptor Atg36 through a separable surface.\",\n      \"evidence\": \"Site-directed mutagenesis with PMP import and de novo biogenesis assays in human cells; pexophagy-specific mutant isolation and mitochondrial retargeting in S. cerevisiae\",\n      \"pmids\": [\"22624858\", \"22643220\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural details of Atg36 binding site on Pex3 not resolved\", \"Whether PMP insertion and pexophagy receptor docking are mutually exclusive was unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Three studies collectively established the ER-to-peroxisome trafficking route of PEX3: PEX16 was shown to mediate ER-to-peroxisome transport of PEX3, HDX-MS revealed that PEX19 remains disordered upon PEX3 binding, and overexpressed PEX3 was found to trigger ubiquitin- and NBR1-dependent pexophagy in mammalian cells.\",\n      \"evidence\": \"ER-redirected PEX3 with PEX16 depletion and live-cell imaging; HDX-MS of purified PEX3-PEX19 complex; PEX3 overexpression with siRNA knockdown of NBR1/p62 in human cells\",\n      \"pmids\": [\"25002403\", \"25062251\", \"25007327\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ubiquitinated peroxisomal substrate in mammalian pexophagy not determined\", \"Whether HDX-MS disorder of PEX19 reflects the cargo-loaded state unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Reconstitution of PEX3's co-translational insertion into the ER via SRP/Sec61 and demonstration that it exits in ATP-dependent budding vesicles defined the complete biogenetic itinerary of PEX3 from ribosome to peroxisome; concurrently, Pex3 was shown to actively promote Atg30 phosphorylation and Atg11 recruitment in P. pastoris pexophagy.\",\n      \"evidence\": \"Photocrosslinking and SRP binding assay with ribosome-nascent chains, ATP-dependent budding reconstitution; Atg30 phosphorylation and binding site mapping in P. pastoris\",\n      \"pmids\": [\"26572236\", \"25694426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ER exit vesicles require specific coat proteins remains unresolved\", \"Kinase identity for Atg30 phosphorylation not fully confirmed in this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Competition between Pex3 and Atg37 for overlapping binding sites on Atg30 was shown to control pexophagic receptor activation via Hrr25 kinase, revealing a regulatory switch on Pex3's cytosolic domain that balances biogenesis and degradation.\",\n      \"evidence\": \"Binding site mapping, phosphorylation assays, co-immunoprecipitation, and genetic epistasis in P. pastoris\",\n      \"pmids\": [\"29260977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether a similar competitive mechanism operates in S. cerevisiae or mammals is unknown\", \"Structural basis of the Pex3-Atg30-Atg37 switch not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"In vitro reconstitution demonstrated that Pex3 directly promotes Hrr25-mediated phosphorylation of Atg36 and protects Atg36 from proteasomal degradation, while Pex3-Inp1 was established as a bona fide peroxisome–plasma membrane tether via PI(4,5)P2 binding, consolidating PEX3's role as a multi-effector scaffold.\",\n      \"evidence\": \"Reconstitution with recombinant Pex3/Atg36/Hrr25, proteasome inhibitor experiments in S. cerevisiae; Inp1 domain truncation, PI(4,5)P2 binding assay, and artificial tether rescue in S. cerevisiae\",\n      \"pmids\": [\"32958557\", \"32970792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism by which Pex3 facilitates Hrr25 access to Atg36 is unknown\", \"Whether Pex3-Inp1 tethering is regulated remains unexplored\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Germ cell-specific Pex3 knockout in mice demonstrated that Pex3-dependent peroxisome biogenesis is essential for spermatid intercellular bridge integrity and male fertility, extending functional relevance beyond Zellweger syndrome.\",\n      \"evidence\": \"Conditional germ cell-specific Pex3 knockout mice, histology, proteomics\",\n      \"pmids\": [\"38062668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific peroxisomal metabolites underlying bridge destruction not identified\", \"Whether PEX3 has non-peroxisomal roles in germ cells is unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cardiomyocyte-specific PEX3 knockout revealed that PEX3-dependent plasmalogen metabolism activates AKT/GSK3β signaling through ITGB3 membrane localization, linking peroxisome biogenesis to cardiac regenerative repair.\",\n      \"evidence\": \"Conditional cardiomyocyte PEX3 knockout mice, lipid metabolomics, AKT/GSK3β pathway analysis, myocardial injury model\",\n      \"pmids\": [\"38951640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AKT activation is a direct consequence of plasmalogen loss or secondary is unresolved\", \"Mechanism of plasmalogen-dependent ITGB3 membrane retention not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structural and competition studies confirmed that PEX19, Atg36/Atg30, and Inp1 bind overlapping sites on PEX3, creating a competitive regulatory network; additionally, PEX3 was shown to participate in constitutive Pex15 recycling and in homotypic peroxisome–peroxisome and peroxisome–lipid droplet contact site formation.\",\n      \"evidence\": \"Crystal structure of H. polymorpha Pex3-Pex19, AlphaFold2 predictions, overexpression competition assays in yeast; genetic epistasis of Msp1/Pex3/Pex19 for Pex15 recycling; Pex3 overexpression with GST-pulldown in yeast and Drosophila\",\n      \"pmids\": [\"40847803\", \"40344504\", \"40628847\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative binding affinities for the competing partners not fully determined\", \"Physiological relevance of Pex3-mediated peroxisome-lipid droplet contacts unclear\", \"Whether constitutive Pex15 recycling occurs in mammals not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PEX3 mechanistically facilitates the membrane insertion step of PMPs after PEX19 docking, and whether the competitive binding of biogenesis versus quality-control effectors is regulated by post-translational modifications or signaling inputs, remain central unresolved questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No reconstituted PMP insertion assay with defined lipid bilayers exists\", \"Post-translational regulation of PEX3 effector switching is unexplored\", \"Full-length membrane-embedded PEX3 structure not available\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 9, 16, 17]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [10, 14, 12, 8]},\n      {\"term_id\": \"GO:0043226\", \"supporting_discovery_ids\": [2, 0, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 3, 6, 10, 8]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [5, 7, 9, 16]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [8, 10, 12, 23]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"complexes\": [\n      \"PEX3-PEX19 import receptor complex\",\n      \"Pex3-Inp1 tethering complex\"\n    ],\n    \"partners\": [\n      \"PEX19\",\n      \"PEX16\",\n      \"ATG36\",\n      \"ATG30\",\n      \"INP1\",\n      \"HRR25\",\n      \"NBR1\",\n      \"MYO2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}