{"gene":"AUP1","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2010,"finding":"AUP1 localizes to lipid droplets where it integrates into the LD surface in a monotopic fashion with both termini facing the cytosol, and its C-terminal G2BR domain binds the E2 ubiquitin conjugase UBE2G2; deletion or mutation of the G2BR domain abolishes UBE2G2 binding without affecting LD localization.","method":"Fluorescence microscopy, domain deletion/mutation analysis, co-immunoprecipitation, in vitro binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (localization, pulldown, mutagenesis) in a single study, replicated by subsequent papers","pmids":["21127063"],"is_preprint":false},{"year":2011,"finding":"AUP1 physically associates with the mammalian HRD1-SEL1L ERAD complex and recruits the E2 ubiquitin-conjugating enzyme UBE2G2 to the ER; AUP1 depletion impairs degradation of misfolded ER proteins, and its CUE domain regulates polyubiquitylation and facilitates interaction with the HRD1 complex and dislocation substrates.","method":"Co-immunoprecipitation, siRNA knockdown, degradation assays, domain mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, functional knockdown with defined ERAD phenotype, multiple orthogonal methods","pmids":["21857022"],"is_preprint":false},{"year":2011,"finding":"AUP1 expression level affects the abundance of cellular lipid droplets, linking it to LD regulatory activity; AUP1 localizes to both the ER and lipid droplets.","method":"siRNA knockdown, fluorescence microscopy, LD quantification","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype (LD abundance), single lab","pmids":["21857022"],"is_preprint":false},{"year":2018,"finding":"AUP1 associates with dengue virus NS4A protein; upon DENV infection, AUP1 relocalizes from lipid droplets to autophagosomes; AUP1's acyltransferase domain activity is required for lipophagy and virus production; mono-ubiquitylation of AUP1 disrupts the AUP1-NS4A interaction, inhibiting acyltransferase activity and blocking lipophagy.","method":"Functional proteomics (ubiquitylation screen), CRISPR knockout, co-immunoprecipitation, acyltransferase domain mutant analysis, live imaging/confocal microscopy, virus production assay","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including KO, domain mutant, co-IP, and live imaging with direct phenotypic readouts; highly cited","pmids":["29902443"],"is_preprint":false},{"year":2017,"finding":"AUP1 directly interacts with apolipoprotein B-100 (apoB100) on the ER; AUP1 knockdown reduces apoB100 ubiquitination, decreases its intracellular proteasomal degradation, and enhances VLDL secretion, establishing AUP1 as a regulator of apoB100 quality control and hepatic VLDL assembly.","method":"siRNA knockdown in HepG2 cells, co-immunoprecipitation, metabolic labeling, VLDL secretion assay, ubiquitination assay","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, functional KD with defined lipoprotein secretion phenotype, multiple orthogonal methods","pmids":["28183703"],"is_preprint":false},{"year":2021,"finding":"The 27-amino acid G2BR domain of AUP1 binds with nanomolar affinity to the backside of UBE2G2; crystal structure of the G2BR-UBE2G2 complex revealed a salt bridge/hydrogen bond/hydrophobic interface; G2BR allosterically activates ubiquitination in vitro with ERAD E3s; in cells, G2BR binding prevents rapid UBE2G2 degradation, recruits UBE2G2 to the ER membrane, and activates it there.","method":"Crystal structure determination, isothermal titration calorimetry, in vitro ubiquitination assay, mutagenesis, cell-based ERAD assay, immunofluorescence","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional mutagenesis, in vitro reconstitution, and cell-based validation","pmids":["34879065"],"is_preprint":false},{"year":2024,"finding":"AUP1 interacts with the ERAD lectin OS9 and directly binds the ER-resident form of NKCC2 cotransporter; AUP1 promotes polyubiquitination and ERAD of NKCC2 via a proteasome- and α-mannosidase-dependent pathway; AUP1 knockdown or expression of a dominant-negative form strikingly decreases NKCC2 polyubiquitination and increases NKCC2 protein levels.","method":"Co-immunoprecipitation, siRNA knockdown, dominant-negative overexpression, proteasome inhibitor treatment, Western blot","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional KD with defined substrate degradation phenotype, single lab","pmids":["38474353"],"is_preprint":false},{"year":2025,"finding":"AUP1 forms a complex with UBE2G2 to interact with STING and retains STING in the ER membrane, preventing its translocation to the Golgi and limiting STING signaling in the resting state; AUP1 deficiency causes spontaneous STING activation and enhanced type I interferon expression.","method":"Co-immunoprecipitation, AUP1 knockout (in vitro and in vivo), STING translocation assay, interferon reporter assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus KO with defined STING localization and innate immune phenotype, single lab","pmids":["40237449"],"is_preprint":false},{"year":2002,"finding":"AUP1 binds adenovirus E4ORF3 protein via the central part of E4ORF3 and the C-terminal region of AUP1, and also binds Ad5 E1A via the N-terminal segment of AUP1, as demonstrated by GST pulldown assays.","method":"Yeast two-hybrid screen, GST pulldown, in vitro transcription/translation","journal":"Tsitologiia","confidence":"Low","confidence_rationale":"Tier 3 — single pulldown assay, in vitro only, no cellular confirmation","pmids":["12534236"],"is_preprint":false}],"current_model":"AUP1 is a lipid droplet- and ER-resident type-III membrane protein that acts as a scaffold in ERAD by recruiting the E2 ubiquitin-conjugating enzyme UBE2G2 via its nanomolar-affinity G2BR domain (structurally characterized), engaging the HRD1-SEL1L E3 complex to drive polyubiquitination and proteasomal degradation of misfolded ER substrates (including apoB100 and NKCC2); its acyltransferase activity on lipid droplets is exploited by flavivirus NS4A to trigger lipophagy, a process regulated by mono-ubiquitylation of AUP1 itself; additionally, the AUP1–UBE2G2 complex retains STING in the ER to suppress basal innate immune signaling."},"narrative":{"teleology":[{"year":2010,"claim":"Establishing AUP1 as a lipid droplet-resident scaffold for UBE2G2 answered how this E2 enzyme is recruited to a specific membrane compartment, revealing that AUP1's monotopic membrane insertion and its C-terminal G2BR domain are functionally separable.","evidence":"Fluorescence microscopy, domain deletion/mutation, co-IP, and in vitro binding in mammalian cells","pmids":["21127063"],"confidence":"High","gaps":["Whether AUP1-UBE2G2 recruitment to LDs has a distinct function from ER recruitment was unclear","No structural detail of the G2BR–UBE2G2 interface","The E3 ligase partner was not identified"]},{"year":2011,"claim":"Demonstrating that AUP1 physically associates with the HRD1–SEL1L ERAD complex and is required for misfolded ER protein degradation established AUP1 as a functional ERAD component bridging E2 recruitment to E3 ligase activity, while also linking it to lipid droplet abundance regulation.","evidence":"Reciprocal co-IP, siRNA knockdown with ERAD substrate degradation assays, CUE domain mutant analysis, LD quantification","pmids":["21857022"],"confidence":"High","gaps":["Identity of specific physiological ERAD substrates channeled through AUP1 was unknown","Whether the CUE domain binds ubiquitin on substrates or on AUP1 itself was unresolved","Mechanism linking AUP1 to LD abundance was not defined"]},{"year":2017,"claim":"Identifying apoB100 as a direct AUP1-dependent ERAD substrate demonstrated a physiological role for AUP1 in hepatic lipoprotein quality control and VLDL secretion, extending the ERAD scaffold function to a clinically relevant substrate.","evidence":"siRNA knockdown in HepG2 cells, co-IP, metabolic labeling, ubiquitination and VLDL secretion assays","pmids":["28183703"],"confidence":"High","gaps":["Whether AUP1 acts on apoB100 via HRD1 or another E3 was not shown","In vivo relevance in animal models was not tested"]},{"year":2018,"claim":"Revealing that dengue virus NS4A hijacks AUP1's acyltransferase activity to trigger lipophagy — and that mono-ubiquitylation of AUP1 blocks this — uncovered a dual enzymatic function for AUP1 beyond ERAD scaffolding and identified a host–pathogen regulatory switch.","evidence":"CRISPR knockout, functional proteomics/ubiquitylation screen, acyltransferase domain mutants, live imaging, virus production assay","pmids":["29902443"],"confidence":"High","gaps":["The E3 ligase responsible for AUP1 mono-ubiquitylation was not identified","Whether AUP1 acyltransferase activity functions independently of viral exploitation was not determined","Lipid substrate specificity of the acyltransferase domain was not characterized"]},{"year":2021,"claim":"The crystal structure of the G2BR–UBE2G2 complex revealed nanomolar-affinity binding via a salt bridge/hydrophobic interface on UBE2G2's backside, explaining how AUP1 allosterically activates ubiquitination and stabilizes UBE2G2 at the ER membrane.","evidence":"X-ray crystallography, isothermal titration calorimetry, in vitro ubiquitination reconstitution, mutagenesis, cell-based ERAD assays","pmids":["34879065"],"confidence":"High","gaps":["Full-length AUP1 structure including the transmembrane and CUE domains is lacking","How AUP1's allosteric activation of UBE2G2 is coordinated with E3 engagement was not resolved"]},{"year":2024,"claim":"Demonstrating that AUP1 interacts with the ERAD lectin OS9 and promotes polyubiquitination-dependent degradation of the renal cotransporter NKCC2 expanded the roster of physiological ERAD substrates handled by AUP1 to include an ion transporter.","evidence":"Co-IP, siRNA knockdown, dominant-negative overexpression, proteasome inhibitor treatment","pmids":["38474353"],"confidence":"Medium","gaps":["Single-lab study; independent confirmation needed","Whether AUP1–OS9 interaction is direct or mediated through other ERAD factors was not resolved","In vivo renal phenotype of AUP1 deficiency was not tested"]},{"year":2025,"claim":"Showing that the AUP1–UBE2G2 complex retains STING in the ER and prevents its spontaneous activation revealed an unexpected role for the ERAD scaffold in restraining basal innate immune signaling.","evidence":"Co-IP, AUP1 knockout in vitro and in vivo, STING translocation and interferon reporter assays","pmids":["40237449"],"confidence":"Medium","gaps":["Single-lab study; independent confirmation needed","Whether AUP1 promotes STING degradation or purely retains it via physical interaction was not fully distinguished","Relevance to autoinflammatory disease in humans is untested"]},{"year":null,"claim":"Key unresolved questions include the full-length structure of AUP1, the identity of the E3 ligase(s) that mono-ubiquitylate AUP1 to regulate its acyltransferase activity, the lipid substrate specificity of the acyltransferase domain, and the in vivo physiological consequences of AUP1 loss in mammalian models.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length AUP1 structure available","E3 ligase for AUP1 mono-ubiquitylation unidentified","Acyltransferase substrate specificity uncharacterized","No mammalian genetic model phenotypically characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,2,5,7]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[0,2,3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,4,5,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7]}],"complexes":["HRD1-SEL1L ERAD complex"],"partners":["UBE2G2","HRD1","SEL1L","OS9","STING","APOB","SLC12A1"],"other_free_text":[]},"mechanistic_narrative":"AUP1 is a type-III membrane protein that resides on both the endoplasmic reticulum and lipid droplets, functioning as a central scaffold in ER-associated degradation (ERAD) and lipid droplet homeostasis. Its C-terminal G2BR domain binds the E2 ubiquitin-conjugating enzyme UBE2G2 with nanomolar affinity, allosterically activating UBE2G2 and recruiting it to the ER membrane where AUP1 engages the HRD1–SEL1L E3 ligase complex via its CUE domain to drive polyubiquitination and proteasomal degradation of misfolded substrates including apoB100 and NKCC2 [PMID:21127063, PMID:21857022, PMID:34879065, PMID:28183703, PMID:38474353]. The AUP1–UBE2G2 complex also retains STING in the ER to suppress basal type I interferon signaling, with AUP1 deficiency causing spontaneous STING activation [PMID:40237449]. AUP1 possesses acyltransferase activity on lipid droplets that is exploited by flavivirus NS4A to trigger lipophagy, a process negatively regulated by mono-ubiquitylation of AUP1 itself [PMID:29902443]."},"prefetch_data":{"uniprot":{"accession":"Q9Y679","full_name":"Lipid droplet-regulating VLDL assembly factor AUP1","aliases":["Ancient ubiquitous protein 1"],"length_aa":410,"mass_kda":45.8,"function":"Plays a role in the translocation of terminally misfolded proteins from the endoplasmic reticulum lumen to the cytoplasm and their degradation by the proteasome (PubMed:18711132, PubMed:21857022). Plays a role in lipid droplet formation (PubMed:21857022). Induces lipid droplet clustering (PubMed:24039768). Recruits ubiquitin-conjugating enzyme UBE2G2 to lipid droplets which facilitates its interaction with ubiquitin ligases AMFR/gp78 and RNF139/TRC8, leading to sterol-induced ubiquitination of HMGCR and its subsequent proteasomal degradation (PubMed:21127063, PubMed:23223569). Also required for the degradation of INSIG1, SREBF1 and SREBF2 (PubMed:23223569). Plays a role in regulating assembly and secretion of very low density lipoprotein particles and stability of apolipoprotein APOB (PubMed:28183703) (Microbial infection) Following Dengue virus infection, required for induction of lipophagy which facilitates production of virus progeny particles","subcellular_location":"Cytoplasmic vesicle, autophagosome","url":"https://www.uniprot.org/uniprotkb/Q9Y679/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AUP1","classification":"Not Classified","n_dependent_lines":60,"n_total_lines":1208,"dependency_fraction":0.04966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CANX","stoichiometry":0.2},{"gene":"VCP","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AUP1","total_profiled":1310},"omim":[{"mim_id":"616175","title":"UBIQUITIN-CONJUGATING ENZYME E2 J1; UBE2J1","url":"https://www.omim.org/entry/616175"},{"mim_id":"610304","title":"DER1-LIKE DOMAIN FAMILY, MEMBER 2; DERL2","url":"https://www.omim.org/entry/610304"},{"mim_id":"609677","title":"OS9 ENDOPLASMIC RETICULUM LECTIN; OS9","url":"https://www.omim.org/entry/609677"},{"mim_id":"608046","title":"SYNOVIAL APOPTOSIS INHIBITOR 1; SYVN1","url":"https://www.omim.org/entry/608046"},{"mim_id":"602434","title":"ANCIENT UBIQUITOUS PROTEIN 1; AUP1","url":"https://www.omim.org/entry/602434"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AUP1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9Y679","domains":[{"cath_id":"-","chopping":"10-260","consensus_level":"high","plddt":87.4403,"start":10,"end":260},{"cath_id":"1.10.8.10","chopping":"294-336","consensus_level":"high","plddt":77.4967,"start":294,"end":336}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y679","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y679-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y679-F1-predicted_aligned_error_v6.png","plddt_mean":79.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AUP1","jax_strain_url":"https://www.jax.org/strain/search?query=AUP1"},"sequence":{"accession":"Q9Y679","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y679.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y679/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y679"}},"corpus_meta":[{"pmid":"29902443","id":"PMC_29902443","title":"Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production.","date":"2018","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/29902443","citation_count":153,"is_preprint":false},{"pmid":"21857022","id":"PMC_21857022","title":"Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21857022","citation_count":105,"is_preprint":false},{"pmid":"21127063","id":"PMC_21127063","title":"Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21127063","citation_count":103,"is_preprint":false},{"pmid":"19840933","id":"PMC_19840933","title":"Aup1-mediated regulation of Rtg3 during mitophagy.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19840933","citation_count":75,"is_preprint":false},{"pmid":"35633317","id":"PMC_35633317","title":"AUP1 regulates lipid metabolism and induces lipid accumulation to accelerate the progression of renal clear cell carcinoma.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/35633317","citation_count":37,"is_preprint":false},{"pmid":"28183703","id":"PMC_28183703","title":"AUP1 (Ancient Ubiquitous Protein 1) Is a Key Determinant of Hepatic Very-Low-Density Lipoprotein Assembly and Secretion.","date":"2017","source":"Arteriosclerosis, thrombosis, and vascular biology","url":"https://pubmed.ncbi.nlm.nih.gov/28183703","citation_count":17,"is_preprint":false},{"pmid":"34879065","id":"PMC_34879065","title":"A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.","date":"2021","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/34879065","citation_count":14,"is_preprint":false},{"pmid":"8812468","id":"PMC_8812468","title":"Aup1, a novel gene on mouse chromosome 6 and human chromosome 2p13.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8812468","citation_count":11,"is_preprint":false},{"pmid":"38474353","id":"PMC_38474353","title":"AUP1 Regulates the Endoplasmic Reticulum-Associated Degradation and Polyubiquitination of NKCC2.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/38474353","citation_count":4,"is_preprint":false},{"pmid":"39329209","id":"PMC_39329209","title":"AUP1 transcriptionally activated by KDM5B reprograms lipid metabolism to promote the malignant progression of cervical cancer.","date":"2024","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39329209","citation_count":4,"is_preprint":false},{"pmid":"12534238","id":"PMC_12534238","title":"[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534238","citation_count":1,"is_preprint":false},{"pmid":"12534237","id":"PMC_12534237","title":"[A new human cellular protein AUP1. II. cDNA cloning, genomic organization of Aup1 gene ans preliminary characterization of human AUP1 protein].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534237","citation_count":1,"is_preprint":false},{"pmid":"12534236","id":"PMC_12534236","title":"[A new human cellular protein AUP1. I. In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A].","date":"2002","source":"Tsitologiia","url":"https://pubmed.ncbi.nlm.nih.gov/12534236","citation_count":1,"is_preprint":false},{"pmid":"40237449","id":"PMC_40237449","title":"AUP1 and UBE2G2 complex targets STING signaling and regulates virus-induced innate immunity.","date":"2025","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/40237449","citation_count":0,"is_preprint":false},{"pmid":"40484276","id":"PMC_40484276","title":"The role of ancient ubiquitous protein 1 (Aup1) in regulating hepatic lipid droplet levels, endoplasmic reticulum stress, and inflammation in zebrafish (Danio rerio).","date":"2025","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/40484276","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9141,"output_tokens":2311,"usd":0.031044},"stage2":{"model":"claude-opus-4-6","input_tokens":5609,"output_tokens":2492,"usd":0.135518},"total_usd":0.166562,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"AUP1 localizes to lipid droplets where it integrates into the LD surface in a monotopic fashion with both termini facing the cytosol, and its C-terminal G2BR domain binds the E2 ubiquitin conjugase UBE2G2; deletion or mutation of the G2BR domain abolishes UBE2G2 binding without affecting LD localization.\",\n      \"method\": \"Fluorescence microscopy, domain deletion/mutation analysis, co-immunoprecipitation, in vitro binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (localization, pulldown, mutagenesis) in a single study, replicated by subsequent papers\",\n      \"pmids\": [\"21127063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AUP1 physically associates with the mammalian HRD1-SEL1L ERAD complex and recruits the E2 ubiquitin-conjugating enzyme UBE2G2 to the ER; AUP1 depletion impairs degradation of misfolded ER proteins, and its CUE domain regulates polyubiquitylation and facilitates interaction with the HRD1 complex and dislocation substrates.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, degradation assays, domain mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, functional knockdown with defined ERAD phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"21857022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AUP1 expression level affects the abundance of cellular lipid droplets, linking it to LD regulatory activity; AUP1 localizes to both the ER and lipid droplets.\",\n      \"method\": \"siRNA knockdown, fluorescence microscopy, LD quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype (LD abundance), single lab\",\n      \"pmids\": [\"21857022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AUP1 associates with dengue virus NS4A protein; upon DENV infection, AUP1 relocalizes from lipid droplets to autophagosomes; AUP1's acyltransferase domain activity is required for lipophagy and virus production; mono-ubiquitylation of AUP1 disrupts the AUP1-NS4A interaction, inhibiting acyltransferase activity and blocking lipophagy.\",\n      \"method\": \"Functional proteomics (ubiquitylation screen), CRISPR knockout, co-immunoprecipitation, acyltransferase domain mutant analysis, live imaging/confocal microscopy, virus production assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including KO, domain mutant, co-IP, and live imaging with direct phenotypic readouts; highly cited\",\n      \"pmids\": [\"29902443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AUP1 directly interacts with apolipoprotein B-100 (apoB100) on the ER; AUP1 knockdown reduces apoB100 ubiquitination, decreases its intracellular proteasomal degradation, and enhances VLDL secretion, establishing AUP1 as a regulator of apoB100 quality control and hepatic VLDL assembly.\",\n      \"method\": \"siRNA knockdown in HepG2 cells, co-immunoprecipitation, metabolic labeling, VLDL secretion assay, ubiquitination assay\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, functional KD with defined lipoprotein secretion phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"28183703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The 27-amino acid G2BR domain of AUP1 binds with nanomolar affinity to the backside of UBE2G2; crystal structure of the G2BR-UBE2G2 complex revealed a salt bridge/hydrogen bond/hydrophobic interface; G2BR allosterically activates ubiquitination in vitro with ERAD E3s; in cells, G2BR binding prevents rapid UBE2G2 degradation, recruits UBE2G2 to the ER membrane, and activates it there.\",\n      \"method\": \"Crystal structure determination, isothermal titration calorimetry, in vitro ubiquitination assay, mutagenesis, cell-based ERAD assay, immunofluorescence\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional mutagenesis, in vitro reconstitution, and cell-based validation\",\n      \"pmids\": [\"34879065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AUP1 interacts with the ERAD lectin OS9 and directly binds the ER-resident form of NKCC2 cotransporter; AUP1 promotes polyubiquitination and ERAD of NKCC2 via a proteasome- and α-mannosidase-dependent pathway; AUP1 knockdown or expression of a dominant-negative form strikingly decreases NKCC2 polyubiquitination and increases NKCC2 protein levels.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, dominant-negative overexpression, proteasome inhibitor treatment, Western blot\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional KD with defined substrate degradation phenotype, single lab\",\n      \"pmids\": [\"38474353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AUP1 forms a complex with UBE2G2 to interact with STING and retains STING in the ER membrane, preventing its translocation to the Golgi and limiting STING signaling in the resting state; AUP1 deficiency causes spontaneous STING activation and enhanced type I interferon expression.\",\n      \"method\": \"Co-immunoprecipitation, AUP1 knockout (in vitro and in vivo), STING translocation assay, interferon reporter assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus KO with defined STING localization and innate immune phenotype, single lab\",\n      \"pmids\": [\"40237449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AUP1 binds adenovirus E4ORF3 protein via the central part of E4ORF3 and the C-terminal region of AUP1, and also binds Ad5 E1A via the N-terminal segment of AUP1, as demonstrated by GST pulldown assays.\",\n      \"method\": \"Yeast two-hybrid screen, GST pulldown, in vitro transcription/translation\",\n      \"journal\": \"Tsitologiia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown assay, in vitro only, no cellular confirmation\",\n      \"pmids\": [\"12534236\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AUP1 is a lipid droplet- and ER-resident type-III membrane protein that acts as a scaffold in ERAD by recruiting the E2 ubiquitin-conjugating enzyme UBE2G2 via its nanomolar-affinity G2BR domain (structurally characterized), engaging the HRD1-SEL1L E3 complex to drive polyubiquitination and proteasomal degradation of misfolded ER substrates (including apoB100 and NKCC2); its acyltransferase activity on lipid droplets is exploited by flavivirus NS4A to trigger lipophagy, a process regulated by mono-ubiquitylation of AUP1 itself; additionally, the AUP1–UBE2G2 complex retains STING in the ER to suppress basal innate immune signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"AUP1 is a type-III membrane protein that resides on both the endoplasmic reticulum and lipid droplets, functioning as a central scaffold in ER-associated degradation (ERAD) and lipid droplet homeostasis. Its C-terminal G2BR domain binds the E2 ubiquitin-conjugating enzyme UBE2G2 with nanomolar affinity, allosterically activating UBE2G2 and recruiting it to the ER membrane where AUP1 engages the HRD1–SEL1L E3 ligase complex via its CUE domain to drive polyubiquitination and proteasomal degradation of misfolded substrates including apoB100 and NKCC2 [PMID:21127063, PMID:21857022, PMID:34879065, PMID:28183703, PMID:38474353]. The AUP1–UBE2G2 complex also retains STING in the ER to suppress basal type I interferon signaling, with AUP1 deficiency causing spontaneous STING activation [PMID:40237449]. AUP1 possesses acyltransferase activity on lipid droplets that is exploited by flavivirus NS4A to trigger lipophagy, a process negatively regulated by mono-ubiquitylation of AUP1 itself [PMID:29902443].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing AUP1 as a lipid droplet-resident scaffold for UBE2G2 answered how this E2 enzyme is recruited to a specific membrane compartment, revealing that AUP1's monotopic membrane insertion and its C-terminal G2BR domain are functionally separable.\",\n      \"evidence\": \"Fluorescence microscopy, domain deletion/mutation, co-IP, and in vitro binding in mammalian cells\",\n      \"pmids\": [\"21127063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether AUP1-UBE2G2 recruitment to LDs has a distinct function from ER recruitment was unclear\",\n        \"No structural detail of the G2BR–UBE2G2 interface\",\n        \"The E3 ligase partner was not identified\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that AUP1 physically associates with the HRD1–SEL1L ERAD complex and is required for misfolded ER protein degradation established AUP1 as a functional ERAD component bridging E2 recruitment to E3 ligase activity, while also linking it to lipid droplet abundance regulation.\",\n      \"evidence\": \"Reciprocal co-IP, siRNA knockdown with ERAD substrate degradation assays, CUE domain mutant analysis, LD quantification\",\n      \"pmids\": [\"21857022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of specific physiological ERAD substrates channeled through AUP1 was unknown\",\n        \"Whether the CUE domain binds ubiquitin on substrates or on AUP1 itself was unresolved\",\n        \"Mechanism linking AUP1 to LD abundance was not defined\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying apoB100 as a direct AUP1-dependent ERAD substrate demonstrated a physiological role for AUP1 in hepatic lipoprotein quality control and VLDL secretion, extending the ERAD scaffold function to a clinically relevant substrate.\",\n      \"evidence\": \"siRNA knockdown in HepG2 cells, co-IP, metabolic labeling, ubiquitination and VLDL secretion assays\",\n      \"pmids\": [\"28183703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether AUP1 acts on apoB100 via HRD1 or another E3 was not shown\",\n        \"In vivo relevance in animal models was not tested\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealing that dengue virus NS4A hijacks AUP1's acyltransferase activity to trigger lipophagy — and that mono-ubiquitylation of AUP1 blocks this — uncovered a dual enzymatic function for AUP1 beyond ERAD scaffolding and identified a host–pathogen regulatory switch.\",\n      \"evidence\": \"CRISPR knockout, functional proteomics/ubiquitylation screen, acyltransferase domain mutants, live imaging, virus production assay\",\n      \"pmids\": [\"29902443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The E3 ligase responsible for AUP1 mono-ubiquitylation was not identified\",\n        \"Whether AUP1 acyltransferase activity functions independently of viral exploitation was not determined\",\n        \"Lipid substrate specificity of the acyltransferase domain was not characterized\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The crystal structure of the G2BR–UBE2G2 complex revealed nanomolar-affinity binding via a salt bridge/hydrophobic interface on UBE2G2's backside, explaining how AUP1 allosterically activates ubiquitination and stabilizes UBE2G2 at the ER membrane.\",\n      \"evidence\": \"X-ray crystallography, isothermal titration calorimetry, in vitro ubiquitination reconstitution, mutagenesis, cell-based ERAD assays\",\n      \"pmids\": [\"34879065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full-length AUP1 structure including the transmembrane and CUE domains is lacking\",\n        \"How AUP1's allosteric activation of UBE2G2 is coordinated with E3 engagement was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that AUP1 interacts with the ERAD lectin OS9 and promotes polyubiquitination-dependent degradation of the renal cotransporter NKCC2 expanded the roster of physiological ERAD substrates handled by AUP1 to include an ion transporter.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, dominant-negative overexpression, proteasome inhibitor treatment\",\n      \"pmids\": [\"38474353\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; independent confirmation needed\",\n        \"Whether AUP1–OS9 interaction is direct or mediated through other ERAD factors was not resolved\",\n        \"In vivo renal phenotype of AUP1 deficiency was not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showing that the AUP1–UBE2G2 complex retains STING in the ER and prevents its spontaneous activation revealed an unexpected role for the ERAD scaffold in restraining basal innate immune signaling.\",\n      \"evidence\": \"Co-IP, AUP1 knockout in vitro and in vivo, STING translocation and interferon reporter assays\",\n      \"pmids\": [\"40237449\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; independent confirmation needed\",\n        \"Whether AUP1 promotes STING degradation or purely retains it via physical interaction was not fully distinguished\",\n        \"Relevance to autoinflammatory disease in humans is untested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full-length structure of AUP1, the identity of the E3 ligase(s) that mono-ubiquitylate AUP1 to regulate its acyltransferase activity, the lipid substrate specificity of the acyltransferase domain, and the in vivo physiological consequences of AUP1 loss in mammalian models.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No full-length AUP1 structure available\",\n        \"E3 ligase for AUP1 mono-ubiquitylation unidentified\",\n        \"Acyltransferase substrate specificity uncharacterized\",\n        \"No mammalian genetic model phenotypically characterized\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 2, 5, 7]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [0, 2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 4, 5, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\n      \"HRD1-SEL1L ERAD complex\"\n    ],\n    \"partners\": [\n      \"UBE2G2\",\n      \"HRD1\",\n      \"SEL1L\",\n      \"OS9\",\n      \"STING\",\n      \"APOB\",\n      \"SLC12A1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}