{"gene":"VPS36","run_date":"2026-04-28T23:00:23","timeline":{"discoveries":[{"year":2005,"finding":"Mammalian ESCRT-II subunit EAP45 (VPS36) contains a novel GLUE (GRAM-like ubiquitin-binding in Eap45) domain that binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains, and also binds a subset of 3-phosphoinositides; EAP45 colocalizes with ubiquitinated proteins on late endosomes, consistent with a role in endosomal sorting of ubiquitinated cargo.","method":"Biochemical binding assays (ubiquitin and phosphoinositide binding), colocalization by fluorescence microscopy, domain analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (binding assays, colocalization, domain mapping), replicated by subsequent structural study","pmids":["15755741"],"is_preprint":false},{"year":2006,"finding":"Crystallographic analysis reveals that the GLUE domain of human ESCRT-II EAP45/VPS36 is a split pleckstrin-homology (PH) domain that binds ubiquitin along one edge of a beta-sandwich, structurally explaining how ESCRT-II couples recognition of ubiquitinated cargoes with endosomal phospholipid binding during MVB protein sorting.","method":"X-ray crystallography, biochemical binding assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical validation, independently consistent with PMID:15755741","pmids":["17057716"],"is_preprint":false},{"year":2006,"finding":"RILP (Rab7/Rab34 effector) directly interacts with VPS36 (via its C-terminal half) and VPS22 of ESCRT-II to mediate their recruitment to endosomal membranes; overexpression of RILP or its C-terminal fragment retards sorting of internalized EGF at EEA1-positive sorting endosomes, linking the early and late endocytic machineries.","method":"Co-immunoprecipitation, domain mapping, fluorescence colocalization, EGF trafficking assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, Co-IP and colocalization with functional trafficking readout","pmids":["17010938"],"is_preprint":false},{"year":2013,"finding":"Drosophila Vps36 of the ESCRT-II complex specifically recognizes ubiquitin on the seven-transmembrane protein Smoothened (Smo) and regulates its trafficking from the plasma membrane to intracellular compartments in the absence of Hedgehog; loss of Vps36 leads to Smo accumulation at the membrane and enhanced Hedgehog signaling.","method":"Genetic loss-of-function (Drosophila mutants), ubiquitination assays, trafficking assays, epistasis with Hedgehog pathway components","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — ortholog in Drosophila (consistent with mammalian ESCRT-II function), multiple orthogonal methods including genetics, ubiquitination, and trafficking assays","pmids":["23843610"],"is_preprint":false},{"year":2019,"finding":"VPS36 interacts with porcine epidemic diarrhea virus (PEDV) ORF3 protein independent of its GLUE domain; VPS36 overexpression suppresses PEDV replication and reduces ORF3 protein levels (not rescued by lysosomal inhibitors), while siRNA-mediated VPS36 knockdown partially augments PEDV replication.","method":"Immunoprecipitation, mass spectrometry, siRNA knockdown, overexpression, virus replication assay","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, Co-IP plus functional viral replication assay, mechanistic detail on domain requirement included","pmids":["31022991"],"is_preprint":false},{"year":2021,"finding":"EAP45/VPS36 colocalizes with HIV-1 Gag at the plasma membrane during budding; the N-terminal H0 domain of EAP45 is required for association with budding HIV-1, whereas the GLUE domain is more critical for ESCRT recruitment during cytokinesis, indicating distinct domain requirements for viral versus cellular functions.","method":"SNAP-tag fluorescent labelling, fixed and live cell imaging, domain deletion analysis","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 — live imaging with domain mutagenesis, two functional contexts compared, single lab","pmids":["34580994"],"is_preprint":false},{"year":2023,"finding":"Endosomal Arl4A directly binds VPS36 (ESCRT-II), stabilizing VPS36-ESCRT-III association and affecting subsequent CHMP2A-mediated recruitment of the deubiquitinating enzyme USP8; this interaction prolongs EGFR ubiquitinylation and attenuates transport of endocytosed EGFR to lysosomes for degradation.","method":"Co-immunoprecipitation, direct interaction assay, siRNA knockdown, EGFR trafficking/ubiquitination assays, domain mapping","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, multiple orthogonal functional assays (ubiquitination, trafficking, ESCRT assembly), mechanistic epistasis established","pmids":["38030597"],"is_preprint":false}],"current_model":"VPS36/EAP45 is the ubiquitin- and phosphoinositide-binding subunit of the ESCRT-II complex whose split-PH (GLUE) domain couples recognition of ubiquitinated transmembrane cargo with endosomal phospholipids to drive MVB sorting; it is recruited to endosomal membranes partly via RILP interaction, is regulated by Arl4A binding (which stabilizes ESCRT-II/III association and retards EGFR lysosomal delivery), and uses its N-terminal H0 domain (distinct from GLUE) for co-option by HIV-1 during budding."},"narrative":{"teleology":[{"year":2005,"claim":"Identifying how ESCRT-II contacts ubiquitinated cargo: discovery that the GLUE domain of VPS36 binds both ubiquitin and 3-phosphoinositides established VPS36 as the dual-specificity adaptor linking ESCRT-II to endosomal membranes bearing ubiquitinated cargo.","evidence":"Biochemical binding assays (ubiquitin and phosphoinositide pull-downs) and fluorescence colocalization on late endosomes","pmids":["15755741"],"confidence":"High","gaps":["Structural basis of simultaneous ubiquitin and lipid binding was unknown","Functional consequence of disrupting GLUE-ubiquitin binding in vivo was not tested"]},{"year":2006,"claim":"Structural resolution of the GLUE domain as a split PH fold explained how a single domain can bind both ubiquitin and phospholipid, providing the first atomic model for ESCRT-II cargo–membrane coupling.","evidence":"X-ray crystallography of the GLUE domain with biochemical validation","pmids":["17057716"],"confidence":"High","gaps":["No structure of GLUE bound simultaneously to ubiquitin and lipid","Relative contribution of ubiquitin- vs. lipid-binding surfaces in intact ESCRT-II not dissected"]},{"year":2006,"claim":"The question of how ESCRT-II reaches endosomal membranes was partially answered by showing that the Rab7/Rab34 effector RILP directly binds VPS36 and VPS22, linking Rab-dependent membrane identity to ESCRT-II recruitment.","evidence":"Co-immunoprecipitation, domain mapping, and EGF sorting assays in mammalian cells","pmids":["17010938"],"confidence":"Medium","gaps":["Interaction shown by overexpression Co-IP; endogenous reciprocal pull-down not performed","Whether RILP is essential for ESCRT-II recruitment in physiological trafficking is unclear","Relationship between RILP-mediated and PI3P-mediated ESCRT-II recruitment not resolved"]},{"year":2013,"claim":"Demonstrating physiological specificity of VPS36 ubiquitin recognition, Drosophila Vps36 was shown to sort ubiquitinated Smoothened for internalization, with its loss causing membrane Smo accumulation and aberrant Hedgehog signaling — establishing ESCRT-II as a signaling regulator.","evidence":"Drosophila genetic loss-of-function, ubiquitination assays, trafficking and Hedgehog pathway epistasis","pmids":["23843610"],"confidence":"High","gaps":["Whether VPS36 directly contacts Smo ubiquitin moieties via its GLUE domain was not shown biochemically","Mammalian relevance of VPS36-Hedgehog connection not tested"]},{"year":2019,"claim":"Expanding VPS36 function beyond endogenous cargo, interaction with PEDV ORF3 independent of the GLUE domain indicated that VPS36 can restrict viral replication through a non-canonical binding interface.","evidence":"Co-IP/mass spectrometry identification of VPS36–ORF3 interaction; overexpression/knockdown of VPS36 with viral replication assays","pmids":["31022991"],"confidence":"Medium","gaps":["Single-laboratory finding; no reciprocal validation by alternative binding method","Mechanism by which VPS36 reduces ORF3 levels (not lysosomal) is unresolved","Relevance to other coronaviruses not examined"]},{"year":2021,"claim":"Domain-separation experiments revealed that VPS36's N-terminal H0 domain — distinct from GLUE — mediates association with HIV-1 Gag at the plasma membrane, while the GLUE domain is preferentially used during cytokinetic abscission, establishing that ESCRT-II exploits different VPS36 surfaces for viral vs. cellular functions.","evidence":"SNAP-tag live/fixed imaging with domain deletion constructs in HIV-1-producing and dividing cells","pmids":["34580994"],"confidence":"Medium","gaps":["Direct binding between H0 domain and Gag not demonstrated biochemically","Whether H0-mediated recruitment is required (rather than redundant) for HIV-1 budding efficiency is unknown"]},{"year":2023,"claim":"The small GTPase Arl4A was identified as a direct endosomal regulator of VPS36 that stabilizes ESCRT-II/III coupling and modulates downstream USP8 deubiquitinase access, explaining how EGFR ubiquitination dynamics and lysosomal delivery are tuned at the ESCRT-II–III transition.","evidence":"Reciprocal Co-IP, direct binding assays, siRNA epistasis, EGFR ubiquitination and degradation kinetics","pmids":["38030597"],"confidence":"High","gaps":["Structural basis of Arl4A–VPS36 interaction and whether it competes with ubiquitin or lipid binding is unknown","Generality of Arl4A regulation beyond EGFR cargo not tested","Whether Arl4A-GTP cycling controls the duration of ESCRT-II–III association in vivo is unresolved"]},{"year":null,"claim":"A unified structural model of full-length mammalian VPS36 in complex with ESCRT-II, simultaneously engaging ubiquitin, PI3P, and Arl4A on an endosomal membrane, is still lacking; the relative hierarchy among RILP, PI3P, and Arl4A in controlling ESCRT-II membrane recruitment remains undefined.","evidence":"","pmids":[],"confidence":"High","gaps":["No cryo-EM or crystal structure of mammalian ESCRT-II holo-complex with VPS36 GLUE domain in membrane-bound state","Quantitative dissection of RILP vs. Arl4A vs. PI3P contributions to endosomal ESCRT-II recruitment is missing","Role of VPS36 in non-endosomal ESCRT functions (e.g., nuclear envelope sealing, autophagy) is unexplored in primary literature"]}],"mechanism_profile":{"molecular_activity":[{"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":[0,1,6]}],"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":[0,1,2,3,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6]}],"complexes":["ESCRT-II"],"partners":["VPS22","RILP","ARL4A","CHMP2A"],"other_free_text":[]},"mechanistic_narrative":"VPS36 (EAP45) is the ubiquitin- and phosphoinositide-binding subunit of the ESCRT-II complex that couples recognition of ubiquitinated transmembrane cargo to endosomal membranes during multivesicular body (MVB) sorting. Its GLUE domain adopts a split pleckstrin-homology fold that simultaneously engages ubiquitin and 3-phosphoinositides, providing the molecular basis for ESCRT-II cargo selection [PMID:15755741, PMID:17057716]; in Drosophila, Vps36-mediated ubiquitin recognition on Smoothened controls Hedgehog pathway output by directing receptor internalization [PMID:23843610]. VPS36 is recruited to endosomes in part through RILP interaction [PMID:17010938], and its association with the small GTPase Arl4A stabilizes ESCRT-II/III coupling, modulates CHMP2A-dependent USP8 recruitment, and thereby regulates the kinetics of EGFR ubiquitination and lysosomal delivery [PMID:38030597]. Beyond canonical MVB sorting, VPS36 is co-opted during HIV-1 budding through its N-terminal H0 domain, which is functionally separable from the GLUE domain used in cytokinetic abscission [PMID:34580994]."},"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":43,"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":4,"is_preprint":false},{"pmid":"28340324","id":"PMC_28340324","title":"VPS36-Mediated plasma membrane protein turnover is critical for Arabidopsis root gravitropism.","date":"2017","source":"Plant signaling & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/28340324","citation_count":4,"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":5657,"output_tokens":1784,"usd":0.021865},"stage2":{"model":"claude-opus-4-6","input_tokens":5036,"output_tokens":2396,"usd":0.12762},"total_usd":0.149485,"stage1_batch_id":"msgbatch_01BparvgRZ9S2LMidvaoo86K","stage2_batch_id":"msgbatch_01NkBfv9SVZWthhS7j6mU6G3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Mammalian ESCRT-II subunit EAP45 (VPS36) contains a novel GLUE (GRAM-like ubiquitin-binding in Eap45) domain that binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains, and also binds a subset of 3-phosphoinositides; EAP45 colocalizes with ubiquitinated proteins on late endosomes, consistent with a role in endosomal sorting of ubiquitinated cargo.\",\n      \"method\": \"Biochemical binding assays (ubiquitin and phosphoinositide binding), colocalization by fluorescence microscopy, domain analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (binding assays, colocalization, domain mapping), replicated by subsequent structural study\",\n      \"pmids\": [\"15755741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystallographic analysis reveals that the GLUE domain of human ESCRT-II EAP45/VPS36 is a split pleckstrin-homology (PH) domain that binds ubiquitin along one edge of a beta-sandwich, structurally explaining how ESCRT-II couples recognition of ubiquitinated cargoes with endosomal phospholipid binding during MVB protein sorting.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical validation, independently consistent with PMID:15755741\",\n      \"pmids\": [\"17057716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RILP (Rab7/Rab34 effector) directly interacts with VPS36 (via its C-terminal half) and VPS22 of ESCRT-II to mediate their recruitment to endosomal membranes; overexpression of RILP or its C-terminal fragment retards sorting of internalized EGF at EEA1-positive sorting endosomes, linking the early and late endocytic machineries.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, fluorescence colocalization, EGF trafficking assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, Co-IP and colocalization with functional trafficking readout\",\n      \"pmids\": [\"17010938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Drosophila Vps36 of the ESCRT-II complex specifically recognizes ubiquitin on the seven-transmembrane protein Smoothened (Smo) and regulates its trafficking from the plasma membrane to intracellular compartments in the absence of Hedgehog; loss of Vps36 leads to Smo accumulation at the membrane and enhanced Hedgehog signaling.\",\n      \"method\": \"Genetic loss-of-function (Drosophila mutants), ubiquitination assays, trafficking assays, epistasis with Hedgehog pathway components\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ortholog in Drosophila (consistent with mammalian ESCRT-II function), multiple orthogonal methods including genetics, ubiquitination, and trafficking assays\",\n      \"pmids\": [\"23843610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"VPS36 interacts with porcine epidemic diarrhea virus (PEDV) ORF3 protein independent of its GLUE domain; VPS36 overexpression suppresses PEDV replication and reduces ORF3 protein levels (not rescued by lysosomal inhibitors), while siRNA-mediated VPS36 knockdown partially augments PEDV replication.\",\n      \"method\": \"Immunoprecipitation, mass spectrometry, siRNA knockdown, overexpression, virus replication assay\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, Co-IP plus functional viral replication assay, mechanistic detail on domain requirement included\",\n      \"pmids\": [\"31022991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EAP45/VPS36 colocalizes with HIV-1 Gag at the plasma membrane during budding; the N-terminal H0 domain of EAP45 is required for association with budding HIV-1, whereas the GLUE domain is more critical for ESCRT recruitment during cytokinesis, indicating distinct domain requirements for viral versus cellular functions.\",\n      \"method\": \"SNAP-tag fluorescent labelling, fixed and live cell imaging, domain deletion analysis\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with domain mutagenesis, two functional contexts compared, single lab\",\n      \"pmids\": [\"34580994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Endosomal Arl4A directly binds VPS36 (ESCRT-II), stabilizing VPS36-ESCRT-III association and affecting subsequent CHMP2A-mediated recruitment of the deubiquitinating enzyme USP8; this interaction prolongs EGFR ubiquitinylation and attenuates transport of endocytosed EGFR to lysosomes for degradation.\",\n      \"method\": \"Co-immunoprecipitation, direct interaction assay, siRNA knockdown, EGFR trafficking/ubiquitination assays, domain mapping\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, multiple orthogonal functional assays (ubiquitination, trafficking, ESCRT assembly), mechanistic epistasis established\",\n      \"pmids\": [\"38030597\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPS36/EAP45 is the ubiquitin- and phosphoinositide-binding subunit of the ESCRT-II complex whose split-PH (GLUE) domain couples recognition of ubiquitinated transmembrane cargo with endosomal phospholipids to drive MVB sorting; it is recruited to endosomal membranes partly via RILP interaction, is regulated by Arl4A binding (which stabilizes ESCRT-II/III association and retards EGFR lysosomal delivery), and uses its N-terminal H0 domain (distinct from GLUE) for co-option by HIV-1 during budding.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"VPS36 (EAP45) is the ubiquitin- and phosphoinositide-binding subunit of the ESCRT-II complex that couples recognition of ubiquitinated transmembrane cargo to endosomal membranes during multivesicular body (MVB) sorting. Its GLUE domain adopts a split pleckstrin-homology fold that simultaneously engages ubiquitin and 3-phosphoinositides, providing the molecular basis for ESCRT-II cargo selection [PMID:15755741, PMID:17057716]; in Drosophila, Vps36-mediated ubiquitin recognition on Smoothened controls Hedgehog pathway output by directing receptor internalization [PMID:23843610]. VPS36 is recruited to endosomes in part through RILP interaction [PMID:17010938], and its association with the small GTPase Arl4A stabilizes ESCRT-II/III coupling, modulates CHMP2A-dependent USP8 recruitment, and thereby regulates the kinetics of EGFR ubiquitination and lysosomal delivery [PMID:38030597]. Beyond canonical MVB sorting, VPS36 is co-opted during HIV-1 budding through its N-terminal H0 domain, which is functionally separable from the GLUE domain used in cytokinetic abscission [PMID:34580994].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying how ESCRT-II contacts ubiquitinated cargo: discovery that the GLUE domain of VPS36 binds both ubiquitin and 3-phosphoinositides established VPS36 as the dual-specificity adaptor linking ESCRT-II to endosomal membranes bearing ubiquitinated cargo.\",\n      \"evidence\": \"Biochemical binding assays (ubiquitin and phosphoinositide pull-downs) and fluorescence colocalization on late endosomes\",\n      \"pmids\": [\"15755741\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of simultaneous ubiquitin and lipid binding was unknown\",\n        \"Functional consequence of disrupting GLUE-ubiquitin binding in vivo was not tested\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Structural resolution of the GLUE domain as a split PH fold explained how a single domain can bind both ubiquitin and phospholipid, providing the first atomic model for ESCRT-II cargo–membrane coupling.\",\n      \"evidence\": \"X-ray crystallography of the GLUE domain with biochemical validation\",\n      \"pmids\": [\"17057716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of GLUE bound simultaneously to ubiquitin and lipid\",\n        \"Relative contribution of ubiquitin- vs. lipid-binding surfaces in intact ESCRT-II not dissected\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The question of how ESCRT-II reaches endosomal membranes was partially answered by showing that the Rab7/Rab34 effector RILP directly binds VPS36 and VPS22, linking Rab-dependent membrane identity to ESCRT-II recruitment.\",\n      \"evidence\": \"Co-immunoprecipitation, domain mapping, and EGF sorting assays in mammalian cells\",\n      \"pmids\": [\"17010938\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Interaction shown by overexpression Co-IP; endogenous reciprocal pull-down not performed\",\n        \"Whether RILP is essential for ESCRT-II recruitment in physiological trafficking is unclear\",\n        \"Relationship between RILP-mediated and PI3P-mediated ESCRT-II recruitment not resolved\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating physiological specificity of VPS36 ubiquitin recognition, Drosophila Vps36 was shown to sort ubiquitinated Smoothened for internalization, with its loss causing membrane Smo accumulation and aberrant Hedgehog signaling — establishing ESCRT-II as a signaling regulator.\",\n      \"evidence\": \"Drosophila genetic loss-of-function, ubiquitination assays, trafficking and Hedgehog pathway epistasis\",\n      \"pmids\": [\"23843610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether VPS36 directly contacts Smo ubiquitin moieties via its GLUE domain was not shown biochemically\",\n        \"Mammalian relevance of VPS36-Hedgehog connection not tested\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Expanding VPS36 function beyond endogenous cargo, interaction with PEDV ORF3 independent of the GLUE domain indicated that VPS36 can restrict viral replication through a non-canonical binding interface.\",\n      \"evidence\": \"Co-IP/mass spectrometry identification of VPS36–ORF3 interaction; overexpression/knockdown of VPS36 with viral replication assays\",\n      \"pmids\": [\"31022991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-laboratory finding; no reciprocal validation by alternative binding method\",\n        \"Mechanism by which VPS36 reduces ORF3 levels (not lysosomal) is unresolved\",\n        \"Relevance to other coronaviruses not examined\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Domain-separation experiments revealed that VPS36's N-terminal H0 domain — distinct from GLUE — mediates association with HIV-1 Gag at the plasma membrane, while the GLUE domain is preferentially used during cytokinetic abscission, establishing that ESCRT-II exploits different VPS36 surfaces for viral vs. cellular functions.\",\n      \"evidence\": \"SNAP-tag live/fixed imaging with domain deletion constructs in HIV-1-producing and dividing cells\",\n      \"pmids\": [\"34580994\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct binding between H0 domain and Gag not demonstrated biochemically\",\n        \"Whether H0-mediated recruitment is required (rather than redundant) for HIV-1 budding efficiency is unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The small GTPase Arl4A was identified as a direct endosomal regulator of VPS36 that stabilizes ESCRT-II/III coupling and modulates downstream USP8 deubiquitinase access, explaining how EGFR ubiquitination dynamics and lysosomal delivery are tuned at the ESCRT-II–III transition.\",\n      \"evidence\": \"Reciprocal Co-IP, direct binding assays, siRNA epistasis, EGFR ubiquitination and degradation kinetics\",\n      \"pmids\": [\"38030597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of Arl4A–VPS36 interaction and whether it competes with ubiquitin or lipid binding is unknown\",\n        \"Generality of Arl4A regulation beyond EGFR cargo not tested\",\n        \"Whether Arl4A-GTP cycling controls the duration of ESCRT-II–III association in vivo is unresolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unified structural model of full-length mammalian VPS36 in complex with ESCRT-II, simultaneously engaging ubiquitin, PI3P, and Arl4A on an endosomal membrane, is still lacking; the relative hierarchy among RILP, PI3P, and Arl4A in controlling ESCRT-II membrane recruitment remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No cryo-EM or crystal structure of mammalian ESCRT-II holo-complex with VPS36 GLUE domain in membrane-bound state\",\n        \"Quantitative dissection of RILP vs. Arl4A vs. PI3P contributions to endosomal ESCRT-II recruitment is missing\",\n        \"Role of VPS36 in non-endosomal ESCRT functions (e.g., nuclear envelope sealing, autophagy) is unexplored in primary literature\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 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, 1, 2, 3, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [\n      \"ESCRT-II\"\n    ],\n    \"partners\": [\n      \"VPS22\",\n      \"RILP\",\n      \"ARL4A\",\n      \"CHMP2A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}