{"gene":"VPS36","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2005,"finding":"Mammalian Eap45 (VPS36) contains a novel ubiquitin-binding domain called GLUE (GRAM-like ubiquitin-binding in Eap45) that binds ubiquitin with affinity and specificity comparable to other ubiquitin-binding domains. The GLUE domain also binds a subset of 3-phosphoinositides, suggesting coordinated ubiquitin and phosphoinositide recognition. Eap45 colocalizes with ubiquitinated proteins on late endosomes.","method":"Biochemical ubiquitin-binding assays, phosphoinositide-binding assays, colocalization by immunofluorescence, sequence/structural analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding assays, localization), replicated by subsequent structural study (PMID:17057716)","pmids":["15755741"],"is_preprint":false},{"year":2006,"finding":"Crystal structure of the human ESCRT-II EAP45 (VPS36) GLUE domain reveals it is a split pleckstrin-homology (PH) domain that binds ubiquitin along one edge of the beta-sandwich, structurally explaining how ESCRT-II couples recognition of ubiquitinated cargo with endosomal phospholipids during MVB sorting.","method":"X-ray crystallography, biochemical binding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with biochemical validation, independently corroborated by PMID:15755741","pmids":["17057716"],"is_preprint":false},{"year":2006,"finding":"RILP interacts directly with VPS36 (via RILP's C-terminal half) and with VPS22 of ESCRT-II to mediate ESCRT-II membrane recruitment. Overexpression of RILP or its C-terminal fragment retards EGF sorting to degradation at EEA1-positive sorting endosomes, indicating that RILP-ESCRT-II interaction integrates early and late endocytic machinery.","method":"Co-immunoprecipitation, domain mapping, overexpression with endosome marker analysis, EGF degradation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with domain mapping plus functional readout, single lab, multiple methods","pmids":["17010938"],"is_preprint":false},{"year":2013,"finding":"Drosophila Vps36 of ESCRT-II specifically recognizes ubiquitin on the seven-transmembrane protein Smoothened (Smo) in the absence of Hedgehog signal, directing Smo trafficking away from the plasma membrane. Loss of Vps36 leads to Smo accumulation at the cell surface and elevated Hh pathway activity, placing Vps36 as a negative regulator of Hh signaling via ubiquitin-dependent Smo trafficking.","method":"Genetic loss-of-function (Drosophila mutants), ubiquitination assays, epistasis with Hh pathway components, cell surface trafficking assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in Drosophila with cellular trafficking readout, single lab","pmids":["23843610"],"is_preprint":false},{"year":2019,"finding":"PEDV ORF3 protein interacts with cellular VPS36, independently of the VPS36 GLUE domain. VPS36 overexpression suppresses PEDV replication and reduces ORF3 protein levels through a lysosome-independent mechanism; conversely, siRNA knockdown of endogenous VPS36 partially augments PEDV replication.","method":"Immunoprecipitation/mass spectrometry, co-IP domain mapping, VPS36 overexpression and siRNA knockdown with viral replication assays, lysosomal inhibitor treatment","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal interaction confirmed, functional readout with OE and KD, single lab","pmids":["31022991"],"is_preprint":false},{"year":2021,"finding":"The ESCRT-II component EAP45 (VPS36) colocalizes with HIV-1 Gag at the plasma membrane during viral budding; a proportion of EAP45 may be packaged into virions. The N-terminal H0 domain of EAP45 is required for association with budding HIV-1, whereas the GLUE domain is more critical for EAP45 function during cytokinesis, indicating domain-specific functional contexts.","method":"SNAP-tag fluorescent labeling, fixed and live-cell imaging, domain truncation/mutation analysis","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with domain mapping, single lab, two orthogonal imaging approaches","pmids":["34580994"],"is_preprint":false},{"year":2023,"finding":"Arl4A directly binds VPS36 at endosomal compartments. This Arl4A-VPS36 interaction stabilizes VPS36-ESCRT-III association and attenuates subsequent recruitment of the deubiquitinating enzyme USP8 by CHMP2A, thereby prolonging EGFR ubiquitination and deterring endocytosed EGFR transport from endosomes to lysosomes. Disruption of the Arl4A-VPS36 interaction enhances EGFR degradation and accelerates clearance of EGFR ubiquitination.","method":"Co-IP, pulldown, EGFR degradation assay, USP8 recruitment assay, mutagenesis to disrupt Arl4A-VPS36 interaction, endosomal fractionation, live-cell imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding confirmed by pulldown, mechanistic pathway mapped with multiple orthogonal functional assays and mutagenesis in single rigorous study","pmids":["38030597"],"is_preprint":false}],"current_model":"VPS36 (EAP45) is the ubiquitin-binding subunit of the mammalian ESCRT-II complex, whose split-PH GLUE domain simultaneously binds ubiquitinated cargo and endosomal 3-phosphoinositides to direct MVB sorting; it is recruited to endosomal membranes in part via RILP, regulates Smoothened trafficking to control Hedgehog signaling, and engages Arl4A to stabilize ESCRT-II/III association and modulate USP8-dependent deubiquitination, thereby attenuating EGFR lysosomal degradation."},"narrative":{"mechanistic_narrative":"VPS36 (EAP45) is the ubiquitin-binding subunit of the mammalian ESCRT-II complex that couples recognition of ubiquitinated cargo to endosomal membrane lipids during multivesicular body sorting [PMID:15755741, PMID:17057716]. Cargo recognition is mediated by its GLUE domain, a split pleckstrin-homology fold that binds ubiquitin along one edge of its beta-sandwich while simultaneously engaging a subset of 3-phosphoinositides, structurally integrating ubiquitin and phosphoinositide sensing on late endosomes [PMID:15755741, PMID:17057716]. Membrane recruitment of ESCRT-II is reinforced by direct interaction of VPS36 (and VPS22) with RILP, which couples the early and late endocytic machinery and influences EGF cargo sorting toward degradation [PMID:17010938]. VPS36 sets the kinetics of receptor downregulation: Arl4A binding to VPS36 stabilizes the VPS36–ESCRT-III association and attenuates CHMP2A-dependent recruitment of the deubiquitinase USP8, prolonging EGFR ubiquitination and deterring its transport from endosomes to lysosomes [PMID:38030597]. Through ubiquitin-dependent cargo sorting VPS36 also negatively regulates Hedgehog signaling by directing Smoothened away from the cell surface in the absence of Hh signal [PMID:23843610]. Beyond endosomal sorting, VPS36 participates in cytokinesis and is co-opted at the plasma membrane during HIV-1 budding through domain-specific contributions, with the N-terminal H0 domain mediating association with budding Gag and the GLUE domain being more critical for cytokinesis [PMID:34580994].","teleology":[{"year":2005,"claim":"Establishing how ESCRT-II reads cargo, the discovery that VPS36 harbors the GLUE domain showed a single module can recognize both ubiquitin and endosomal lipids, explaining how ubiquitinated cargo is captured at the membrane.","evidence":"Biochemical ubiquitin- and phosphoinositide-binding assays with immunofluorescence colocalization on late endosomes","pmids":["15755741"],"confidence":"High","gaps":["Does not resolve the structural basis of simultaneous ubiquitin/lipid binding","Selectivity among 3-phosphoinositides not fully defined"]},{"year":2006,"claim":"The crystal structure of the GLUE domain settled how dual recognition is achieved, revealing a split-PH fold that binds ubiquitin along one edge of its beta-sandwich while accommodating phospholipid recognition.","evidence":"X-ray crystallography with biochemical binding validation of the human EAP45 GLUE domain","pmids":["17057716"],"confidence":"High","gaps":["Structure of GLUE within the assembled ESCRT-II complex not determined","Membrane-bound conformation not captured"]},{"year":2006,"claim":"Identifying RILP as a direct VPS36/VPS22 partner answered how ESCRT-II is recruited to membranes, linking early sorting endosome machinery to ESCRT-II-dependent cargo degradation.","evidence":"Co-immunoprecipitation, domain mapping, endosome marker analysis and EGF degradation assay (single lab)","pmids":["17010938"],"confidence":"Medium","gaps":["Recruitment mechanism shown largely via overexpression","Direct contribution to physiological ESCRT-II assembly not isolated"]},{"year":2013,"claim":"Genetic work in Drosophila connected VPS36 cargo sorting to signaling control, showing Vps36 recognizes ubiquitinated Smoothened to restrain Hedgehog pathway activity.","evidence":"Drosophila loss-of-function genetics with ubiquitination, surface trafficking, and Hh epistasis assays (single lab)","pmids":["23843610"],"confidence":"Medium","gaps":["Conservation of Smo regulation in mammalian VPS36 not established","Ubiquitin ligase directing Smo not identified here"]},{"year":2019,"claim":"A virus interaction screen revealed a non-canonical, GLUE-independent VPS36 binding and antiviral role, with VPS36 suppressing PEDV replication by lowering ORF3 levels through a lysosome-independent route.","evidence":"IP/mass spectrometry, co-IP domain mapping, overexpression/knockdown viral replication assays with lysosomal inhibitors (single lab)","pmids":["31022991"],"confidence":"Medium","gaps":["Mechanism of ORF3 reduction not defined","Domain mediating the VPS36-ORF3 interaction unmapped"]},{"year":2021,"claim":"Imaging during HIV-1 budding assigned domain-specific roles to VPS36, separating an H0-dependent association with budding Gag from a GLUE-dependent role in cytokinesis.","evidence":"SNAP-tag fixed and live-cell imaging with domain truncation/mutation (single lab)","pmids":["34580994"],"confidence":"Medium","gaps":["Functional necessity of EAP45 for HIV-1 release not quantified","Mechanism of virion packaging unresolved"]},{"year":2023,"claim":"Defining a regulatory input on receptor downregulation, Arl4A binding to VPS36 was shown to stabilize ESCRT-III association and block USP8 recruitment, thereby slowing EGFR deubiquitination and lysosomal delivery.","evidence":"Co-IP, pulldown, mutagenesis, endosomal fractionation, EGFR degradation and USP8 recruitment assays, live-cell imaging","pmids":["38030597"],"confidence":"High","gaps":["Generality of Arl4A control beyond EGFR cargo not established","Structural basis of Arl4A-VPS36 contact not resolved"]},{"year":null,"claim":"How VPS36-dependent cargo selection and ESCRT-II/III dynamics are integrated across diverse cargoes and signaling contexts in mammalian cells remains incompletely defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Mammalian Smoothened regulation by VPS36 untested","Cargo-specific deubiquitination control beyond EGFR unknown","In situ ESCRT-II assembly structure lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,2,6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,2,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6]}],"complexes":["ESCRT-II"],"partners":["RILP","VPS22","ARL4A","USP8","CHMP2A","SMOOTHENED"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86VN1","full_name":"Vacuolar protein-sorting-associated protein 36","aliases":["ELL-associated protein of 45 kDa","ESCRT-II complex subunit VPS36"],"length_aa":386,"mass_kda":43.8,"function":"Component of the ESCRT-II complex (endosomal sorting complex required for transport II), which is required for multivesicular body (MVB) formation and sorting of endosomal cargo proteins into MVBs. The MVB pathway mediates delivery of transmembrane proteins into the lumen of the lysosome for degradation. The ESCRT-II complex is probably involved in the recruitment of the ESCRT-III complex. Its ability to bind ubiquitin probably plays a role in endosomal sorting of ubiquitinated cargo proteins by ESCRT complexes. The ESCRT-II complex may also play a role in transcription regulation, possibly via its interaction with ELL. Binds phosphoinosides such as PtdIns(3,4,5)P3","subcellular_location":"Cytoplasm; Endosome; Late endosome; Membrane; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86VN1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VPS36","classification":"Not Classified","n_dependent_lines":226,"n_total_lines":1208,"dependency_fraction":0.1870860927152318},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000136100","cell_line_id":"CID000790","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"vesicles","grade":1}],"interactors":[{"gene":"SNF8","stoichiometry":10.0},{"gene":"VPS25","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000790","total_profiled":1310},"omim":[{"mim_id":"620784","title":"NEURODEVELOPMENTAL DISORDER PLUS OPTIC ATROPHY; NEDOA","url":"https://www.omim.org/entry/620784"},{"mim_id":"620783","title":"DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHY 115; DEE115","url":"https://www.omim.org/entry/620783"},{"mim_id":"610907","title":"VACUOLAR PROTEIN SORTING 25 HOMOLOG; VPS25","url":"https://www.omim.org/entry/610907"},{"mim_id":"610904","title":"SNF8 SUBUNIT OF ESCRIT-II; SNF8","url":"https://www.omim.org/entry/610904"},{"mim_id":"610903","title":"VACUOLAR PROTEIN SORTING 36 HOMOLOG; VPS36","url":"https://www.omim.org/entry/610903"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Lysosomes","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Cell Junctions","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/VPS36"},"hgnc":{"alias_symbol":["CGI-145","Eap45"],"prev_symbol":["C13orf9"]},"alphafold":{"accession":"Q86VN1","domains":[{"cath_id":"2.30.29.30","chopping":"4-129","consensus_level":"high","plddt":91.7379,"start":4,"end":129},{"cath_id":"1.10.10.10","chopping":"238-314","consensus_level":"medium","plddt":89.0713,"start":238,"end":314},{"cath_id":"1.10.10.10","chopping":"319-382","consensus_level":"high","plddt":94.9133,"start":319,"end":382},{"cath_id":"1.10.287","chopping":"175-228","consensus_level":"medium","plddt":66.1863,"start":175,"end":228}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86VN1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86VN1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86VN1-F1-predicted_aligned_error_v6.png","plddt_mean":82.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VPS36","jax_strain_url":"https://www.jax.org/strain/search?query=VPS36"},"sequence":{"accession":"Q86VN1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86VN1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86VN1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86VN1"}},"corpus_meta":[{"pmid":"15755741","id":"PMC_15755741","title":"Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15755741","citation_count":144,"is_preprint":false},{"pmid":"17057716","id":"PMC_17057716","title":"Structural basis for ubiquitin recognition by the human ESCRT-II EAP45 GLUE domain.","date":"2006","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17057716","citation_count":59,"is_preprint":false},{"pmid":"17010938","id":"PMC_17010938","title":"RILP interacts with VPS22 and VPS36 of ESCRT-II and regulates their membrane recruitment.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17010938","citation_count":49,"is_preprint":false},{"pmid":"27879389","id":"PMC_27879389","title":"VPS36-Dependent Multivesicular Bodies Are Critical for Plasmamembrane Protein Turnover and Vacuolar Biogenesis.","date":"2016","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27879389","citation_count":44,"is_preprint":false},{"pmid":"23843610","id":"PMC_23843610","title":"Drosophila Vps36 regulates Smo trafficking in Hedgehog signaling.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23843610","citation_count":32,"is_preprint":false},{"pmid":"28197629","id":"PMC_28197629","title":"RAB27A, RAB27B and VPS36 are downregulated in advanced prostate cancer and show functional relevance in prostate cancer cells.","date":"2017","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/28197629","citation_count":30,"is_preprint":false},{"pmid":"31022991","id":"PMC_31022991","title":"Porcine Epidemic Diarrhea Virus (PEDV) ORF3 Interactome Reveals Inhibition of Virus Replication by Cellular VPS36 Protein.","date":"2019","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/31022991","citation_count":18,"is_preprint":false},{"pmid":"38030597","id":"PMC_38030597","title":"Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38030597","citation_count":5,"is_preprint":false},{"pmid":"34580994","id":"PMC_34580994","title":"EAP45 association with budding HIV-1: Kinetics and domain requirements.","date":"2021","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/34580994","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6098,"output_tokens":1916,"usd":0.023517,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8817,"output_tokens":2792,"usd":0.056943,"stage2_stop_reason":"end_turn"},"total_usd":0.08046,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Mammalian Eap45 (VPS36) contains a novel ubiquitin-binding domain called GLUE (GRAM-like ubiquitin-binding in Eap45) that binds ubiquitin with affinity and specificity comparable to other ubiquitin-binding domains. The GLUE domain also binds a subset of 3-phosphoinositides, suggesting coordinated ubiquitin and phosphoinositide recognition. Eap45 colocalizes with ubiquitinated proteins on late endosomes.\",\n      \"method\": \"Biochemical ubiquitin-binding assays, phosphoinositide-binding assays, colocalization by immunofluorescence, sequence/structural analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding assays, localization), replicated by subsequent structural study (PMID:17057716)\",\n      \"pmids\": [\"15755741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of the human ESCRT-II EAP45 (VPS36) GLUE domain reveals it is a split pleckstrin-homology (PH) domain that binds ubiquitin along one edge of the beta-sandwich, structurally explaining how ESCRT-II couples recognition of ubiquitinated cargo with endosomal phospholipids during MVB sorting.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with biochemical validation, independently corroborated by PMID:15755741\",\n      \"pmids\": [\"17057716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RILP interacts directly with VPS36 (via RILP's C-terminal half) and with VPS22 of ESCRT-II to mediate ESCRT-II membrane recruitment. Overexpression of RILP or its C-terminal fragment retards EGF sorting to degradation at EEA1-positive sorting endosomes, indicating that RILP-ESCRT-II interaction integrates early and late endocytic machinery.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, overexpression with endosome marker analysis, EGF degradation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with domain mapping plus functional readout, single lab, multiple methods\",\n      \"pmids\": [\"17010938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Drosophila Vps36 of ESCRT-II specifically recognizes ubiquitin on the seven-transmembrane protein Smoothened (Smo) in the absence of Hedgehog signal, directing Smo trafficking away from the plasma membrane. Loss of Vps36 leads to Smo accumulation at the cell surface and elevated Hh pathway activity, placing Vps36 as a negative regulator of Hh signaling via ubiquitin-dependent Smo trafficking.\",\n      \"method\": \"Genetic loss-of-function (Drosophila mutants), ubiquitination assays, epistasis with Hh pathway components, cell surface trafficking assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in Drosophila with cellular trafficking readout, single lab\",\n      \"pmids\": [\"23843610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PEDV ORF3 protein interacts with cellular VPS36, independently of the VPS36 GLUE domain. VPS36 overexpression suppresses PEDV replication and reduces ORF3 protein levels through a lysosome-independent mechanism; conversely, siRNA knockdown of endogenous VPS36 partially augments PEDV replication.\",\n      \"method\": \"Immunoprecipitation/mass spectrometry, co-IP domain mapping, VPS36 overexpression and siRNA knockdown with viral replication assays, lysosomal inhibitor treatment\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal interaction confirmed, functional readout with OE and KD, single lab\",\n      \"pmids\": [\"31022991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The ESCRT-II component EAP45 (VPS36) colocalizes with HIV-1 Gag at the plasma membrane during viral budding; a proportion of EAP45 may be packaged into virions. The N-terminal H0 domain of EAP45 is required for association with budding HIV-1, whereas the GLUE domain is more critical for EAP45 function during cytokinesis, indicating domain-specific functional contexts.\",\n      \"method\": \"SNAP-tag fluorescent labeling, fixed and live-cell imaging, domain truncation/mutation analysis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with domain mapping, single lab, two orthogonal imaging approaches\",\n      \"pmids\": [\"34580994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Arl4A directly binds VPS36 at endosomal compartments. This Arl4A-VPS36 interaction stabilizes VPS36-ESCRT-III association and attenuates subsequent recruitment of the deubiquitinating enzyme USP8 by CHMP2A, thereby prolonging EGFR ubiquitination and deterring endocytosed EGFR transport from endosomes to lysosomes. Disruption of the Arl4A-VPS36 interaction enhances EGFR degradation and accelerates clearance of EGFR ubiquitination.\",\n      \"method\": \"Co-IP, pulldown, EGFR degradation assay, USP8 recruitment assay, mutagenesis to disrupt Arl4A-VPS36 interaction, endosomal fractionation, live-cell imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding confirmed by pulldown, mechanistic pathway mapped with multiple orthogonal functional assays and mutagenesis in single rigorous study\",\n      \"pmids\": [\"38030597\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPS36 (EAP45) is the ubiquitin-binding subunit of the mammalian ESCRT-II complex, whose split-PH GLUE domain simultaneously binds ubiquitinated cargo and endosomal 3-phosphoinositides to direct MVB sorting; it is recruited to endosomal membranes in part via RILP, regulates Smoothened trafficking to control Hedgehog signaling, and engages Arl4A to stabilize ESCRT-II/III association and modulate USP8-dependent deubiquitination, thereby attenuating EGFR lysosomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VPS36 (EAP45) is the ubiquitin-binding subunit of the mammalian ESCRT-II complex that couples recognition of ubiquitinated cargo to endosomal membrane lipids during multivesicular body sorting [#0, #1]. Cargo recognition is mediated by its GLUE domain, a split pleckstrin-homology fold that binds ubiquitin along one edge of its beta-sandwich while simultaneously engaging a subset of 3-phosphoinositides, structurally integrating ubiquitin and phosphoinositide sensing on late endosomes [#0, #1]. Membrane recruitment of ESCRT-II is reinforced by direct interaction of VPS36 (and VPS22) with RILP, which couples the early and late endocytic machinery and influences EGF cargo sorting toward degradation [#2]. VPS36 sets the kinetics of receptor downregulation: Arl4A binding to VPS36 stabilizes the VPS36–ESCRT-III association and attenuates CHMP2A-dependent recruitment of the deubiquitinase USP8, prolonging EGFR ubiquitination and deterring its transport from endosomes to lysosomes [#6]. Through ubiquitin-dependent cargo sorting VPS36 also negatively regulates Hedgehog signaling by directing Smoothened away from the cell surface in the absence of Hh signal [#3]. Beyond endosomal sorting, VPS36 participates in cytokinesis and is co-opted at the plasma membrane during HIV-1 budding through domain-specific contributions, with the N-terminal H0 domain mediating association with budding Gag and the GLUE domain being more critical for cytokinesis [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing how ESCRT-II reads cargo, the discovery that VPS36 harbors the GLUE domain showed a single module can recognize both ubiquitin and endosomal lipids, explaining how ubiquitinated cargo is captured at the membrane.\",\n      \"evidence\": \"Biochemical ubiquitin- and phosphoinositide-binding assays with immunofluorescence colocalization on late endosomes\",\n      \"pmids\": [\"15755741\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the structural basis of simultaneous ubiquitin/lipid binding\", \"Selectivity among 3-phosphoinositides not fully defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The crystal structure of the GLUE domain settled how dual recognition is achieved, revealing a split-PH fold that binds ubiquitin along one edge of its beta-sandwich while accommodating phospholipid recognition.\",\n      \"evidence\": \"X-ray crystallography with biochemical binding validation of the human EAP45 GLUE domain\",\n      \"pmids\": [\"17057716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of GLUE within the assembled ESCRT-II complex not determined\", \"Membrane-bound conformation not captured\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying RILP as a direct VPS36/VPS22 partner answered how ESCRT-II is recruited to membranes, linking early sorting endosome machinery to ESCRT-II-dependent cargo degradation.\",\n      \"evidence\": \"Co-immunoprecipitation, domain mapping, endosome marker analysis and EGF degradation assay (single lab)\",\n      \"pmids\": [\"17010938\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recruitment mechanism shown largely via overexpression\", \"Direct contribution to physiological ESCRT-II assembly not isolated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Genetic work in Drosophila connected VPS36 cargo sorting to signaling control, showing Vps36 recognizes ubiquitinated Smoothened to restrain Hedgehog pathway activity.\",\n      \"evidence\": \"Drosophila loss-of-function genetics with ubiquitination, surface trafficking, and Hh epistasis assays (single lab)\",\n      \"pmids\": [\"23843610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conservation of Smo regulation in mammalian VPS36 not established\", \"Ubiquitin ligase directing Smo not identified here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A virus interaction screen revealed a non-canonical, GLUE-independent VPS36 binding and antiviral role, with VPS36 suppressing PEDV replication by lowering ORF3 levels through a lysosome-independent route.\",\n      \"evidence\": \"IP/mass spectrometry, co-IP domain mapping, overexpression/knockdown viral replication assays with lysosomal inhibitors (single lab)\",\n      \"pmids\": [\"31022991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of ORF3 reduction not defined\", \"Domain mediating the VPS36-ORF3 interaction unmapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Imaging during HIV-1 budding assigned domain-specific roles to VPS36, separating an H0-dependent association with budding Gag from a GLUE-dependent role in cytokinesis.\",\n      \"evidence\": \"SNAP-tag fixed and live-cell imaging with domain truncation/mutation (single lab)\",\n      \"pmids\": [\"34580994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional necessity of EAP45 for HIV-1 release not quantified\", \"Mechanism of virion packaging unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defining a regulatory input on receptor downregulation, Arl4A binding to VPS36 was shown to stabilize ESCRT-III association and block USP8 recruitment, thereby slowing EGFR deubiquitination and lysosomal delivery.\",\n      \"evidence\": \"Co-IP, pulldown, mutagenesis, endosomal fractionation, EGFR degradation and USP8 recruitment assays, live-cell imaging\",\n      \"pmids\": [\"38030597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of Arl4A control beyond EGFR cargo not established\", \"Structural basis of Arl4A-VPS36 contact not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How VPS36-dependent cargo selection and ESCRT-II/III dynamics are integrated across diverse cargoes and signaling contexts in mammalian cells remains incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian Smoothened regulation by VPS36 untested\", \"Cargo-specific deubiquitination control beyond EGFR unknown\", \"In situ ESCRT-II assembly structure lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 2, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [\"ESCRT-II\"],\n    \"partners\": [\"RILP\", \"VPS22\", \"Arl4A\", \"USP8\", \"CHMP2A\", \"Smoothened\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}