{"gene":"RHOBTB3","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2009,"finding":"RhoBTB3 is an atypical Rho GTPase family ATPase that binds and hydrolyzes ATP (not GTP), directly binds Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans-Golgi network. Rab9 binding opens the autoinhibited RhoBTB3 to permit maximal ATP hydrolysis. RhoBTB3 also interacts with TIP47 on membranes, suggesting it releases this cargo selection protein from vesicles to permit efficient docking and fusion at the Golgi.","method":"Biochemical ATPase assay, direct binding assay, gene replacement/domain-swap experiments, co-immunoprecipitation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro biochemical assay for ATPase activity, direct binding reconstitution, domain mutagenesis, and functional rescue experiments; foundational paper","pmids":["19490898"],"is_preprint":false},{"year":2013,"finding":"RhoBTB3 is a Golgi-associated protein that targets cyclin E for ubiquitylation as part of a CUL3-dependent RING-E3 ubiquitin ligase complex (RhoBTB3/CUL3/RBX1) on the Golgi, thereby regulating the S/G2 cell cycle transition. Depletion of RhoBTB3 arrested cells in S phase, caused Golgi fragmentation, and elevated cyclin E levels. Golgi association of the complex was required for its catalytic activity.","method":"Co-immunoprecipitation, RNAi depletion with cell cycle and morphology readouts, ubiquitylation assay, Golgi targeting experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, clean KD with defined cell cycle phenotype, functional localization requirement; multiple orthogonal methods","pmids":["24145166"],"is_preprint":false},{"year":2015,"finding":"RHOBTB3 directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation and also directly interacts with VHL (E3 ubiquitin ligase component) to facilitate HIFα ubiquitination. RHOBTB3 dimerizes with LIMD1 and assembles a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex for maximal HIFα degradation. Hypoxia disrupts this complex, stabilizing HIFα.","method":"Co-immunoprecipitation, ubiquitination assay, in vitro hydroxylation assay, RNAi depletion with HIF-α level readouts, xenograft model","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including in vitro hydroxylation assay, co-IP, ubiquitination assay, and functional in vivo rescue","pmids":["26215701"],"is_preprint":false},{"year":2012,"finding":"RhoBTB3 directly interacts with the 5-HT7a serotonin receptor via both the C-terminal tail and the third intracellular loop of the receptor. This interaction inhibits proteasomal degradation of the 5-HT7a receptor, without recruiting CUL3/ROC1 to the receptor or mediating receptor ubiquitination. RhoBTB3 and 5-HT7a co-localize at the plasma membrane and endoplasmic reticulum.","method":"Yeast two-hybrid screen, co-immunoprecipitation in HEK293T cells, domain mapping, immunofluorescence microscopy, proteasome inhibitor assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP confirmed interaction, domain mapping performed, functional consequence (proteasomal degradation) measured; single lab","pmids":["22245496"],"is_preprint":false},{"year":2020,"finding":"RhoBTB3 depletion affects Golgi morphology and alters the trafficking speeds of carriers at the Golgi-ER interface. RhoBTB3 is present on these carriers, indicating a role in Golgi-ER membrane traffic.","method":"RNAi-based high-content imaging screen, live-cell imaging of carrier dynamics, immunofluorescence","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2-3 — image-based functional screen with quantitative trafficking readout; single lab, systematic approach","pmids":["32354068"],"is_preprint":false},{"year":2024,"finding":"RhoBTB3 forms a complex with SHIP164 and Vps26B (a retromer subunit) at Golgi-endosome contacts to promote the formation of Rab14-positive early endosome buds. Vps26B acts as a novel Rab14 effector. Depletion of RhoBTB3 causes enlarged Rab14+ early endosomes without buds, phenocopying SHIP164 depletion.","method":"Co-immunoprecipitation, RNAi depletion with rescue experiments using wild-type and lipid transfer-defective SHIP164 mutants, live-cell imaging, Rab14 effector pulldown assay","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1-2 — complex reconstitution, domain-function mutant rescue, multiple orthogonal methods; mechanistic model supported by mutant rescue","pmids":["38565878"],"is_preprint":false},{"year":2024,"finding":"RhoBTB3 negatively regulates autophagy by mediating K27-linked polyubiquitination of AMBRA1 at K45, leading to proteasome-mediated AMBRA1 degradation. AMBRA1 was identified as a RhoBTB3 substrate by immunoprecipitation and mass spectrometry. Knockdown of AMBRA1 blocks RhoBTB3-depletion-induced autophagy.","method":"Immunoprecipitation/mass spectrometry, ubiquitination assay with site-specific mutation (K45), epistasis (double KD), autophagy flux assays","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — MS-based substrate identification, site-specific mutagenesis, epistasis experiment; single lab","pmids":["39404422"],"is_preprint":false},{"year":2019,"finding":"Genetic deletion of RhoBTB3 in mice leads to increased alpha-granule secretion from platelets in response to thrombin, CRP, and U46619/ADP, and reduced platelet accrual on collagen under flow conditions, revealing a role for RhoBTB3 in platelet alpha-granule secretion and adhesion.","method":"Knockout mouse model, platelet aggregation and secretion assays, flow adhesion assay, flow cytometry","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype in primary cells; single lab","pmids":["30754723"],"is_preprint":false},{"year":2025,"finding":"Diosmetin reduces the formation of the RhoBTB3/PHD2 complex, thereby diminishing HIFα hydroxylation and ubiquitination in UVR-exposed keratinocytes; overexpression of RhoBTB3 in mice confirmed that RhoBTB3, PHD2, and HIF-1α are enriched in the epidermis and that the RhoBTB3/PHD2 interaction promotes HIF-1α ubiquitination in this tissue context.","method":"Co-immunoprecipitation, RNA sequencing, rAAV-mediated RhoBTB3 overexpression in vivo, immunohistochemistry","journal":"Phytomedicine","confidence":"Low","confidence_rationale":"Tier 3 — co-IP and in vivo OE; primarily pharmacological study, single lab, limited mechanistic depth beyond confirming the PHD2 interaction","pmids":["40311591"],"is_preprint":false}],"current_model":"RhoBTB3 is a Golgi-associated atypical Rho GTPase that hydrolyzes ATP (not GTP) and functions as a scaffold/adaptor in multiple processes: it acts as part of a CUL3/RBX1-dependent E3 ubiquitin ligase complex on the Golgi to ubiquitylate cyclin E (regulating S/G2 cell cycle progression), organizes a RHOBTB3/LIMD1-PHD2-VHL scaffold to promote HIFα hydroxylation and ubiquitination, mediates AMBRA1 ubiquitination to suppress autophagy, forms a complex with SHIP164 and Vps26B at Golgi-endosome contacts to drive early endosome bud formation, and interacts with Rab9 (opening autoinhibition to enable maximal ATPase activity) to facilitate endosome-to-trans-Golgi-network trafficking."},"narrative":{"teleology":[{"year":2009,"claim":"Establishing that RHOBTB3 is an ATPase rather than a GTPase, and that Rab9-dependent relief of autoinhibition couples its enzymatic activity to endosome-to-TGN trafficking, provided the first mechanistic framework for this atypical Rho family member.","evidence":"In vitro ATPase assays, direct binding reconstitution with Rab9, domain-swap rescue experiments, and co-immunoprecipitation in mammalian cells","pmids":["19490898"],"confidence":"High","gaps":["Structural basis for autoinhibition and its relief by Rab9 is unknown","Role of ATP hydrolysis versus ATP binding in trafficking has not been dissected","TIP47 release model lacks direct reconstitution of vesicle docking/fusion"]},{"year":2012,"claim":"Discovery that RHOBTB3 binds the 5-HT7a serotonin receptor and inhibits its proteasomal degradation — independent of CUL3 recruitment — revealed a non-ubiquitin-ligase protective function for RHOBTB3 at the ER/plasma membrane.","evidence":"Yeast two-hybrid screen, co-IP with domain mapping, immunofluorescence co-localization, proteasome inhibitor experiments in HEK293T cells","pmids":["22245496"],"confidence":"Medium","gaps":["Mechanism by which RHOBTB3 inhibits receptor degradation without mediating ubiquitination is unresolved","Interaction has not been confirmed by an independent lab","Physiological significance in serotonergic signaling is untested"]},{"year":2013,"claim":"Identification of RHOBTB3 as a Golgi-localized CUL3/RBX1 E3 ligase adaptor that ubiquitylates cyclin E established its role as a cell cycle regulator linking Golgi integrity to S/G2 transition.","evidence":"Reciprocal co-IP, RNAi depletion with cell cycle arrest and Golgi fragmentation phenotypes, in vivo ubiquitylation assays, Golgi-targeting experiments","pmids":["24145166"],"confidence":"High","gaps":["How Golgi localization is required for ligase activity is mechanistically unclear","Whether cyclin E is ubiquitylated directly on the Golgi membrane or after release is unresolved","Relationship between Golgi fragmentation and cyclin E accumulation is correlative"]},{"year":2015,"claim":"Assembly of a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes both HIFα hydroxylation and ubiquitination demonstrated that RHOBTB3 coordinates an oxygen-sensing degradation complex disrupted by hypoxia.","evidence":"Co-IP, in vitro hydroxylation assay, ubiquitination assay, RNAi with HIFα readouts, xenograft model","pmids":["26215701"],"confidence":"High","gaps":["Whether scaffold assembly is constitutive or regulated by signals other than hypoxia is unknown","Subcellular site of RHOBTB3–PHD2–VHL complex assembly has not been determined","Stoichiometry and structural organization of the pentameric complex are uncharacterized"]},{"year":2019,"claim":"Genetic knockout in mice revealed that RHOBTB3 restrains platelet alpha-granule secretion and supports adhesion under flow, extending its functional repertoire to hemostasis.","evidence":"RhoBTB3-knockout mouse, platelet aggregation/secretion assays, flow adhesion assay, flow cytometry","pmids":["30754723"],"confidence":"Medium","gaps":["Molecular mechanism linking RHOBTB3 to granule secretion machinery is unknown","Whether the phenotype depends on CUL3-dependent ubiquitination or ATPase activity is untested","In vivo thrombosis or bleeding phenotypes were not reported"]},{"year":2020,"claim":"High-content screening confirmed that RHOBTB3 depletion alters Golgi morphology and carrier dynamics at the Golgi–ER interface, broadening its trafficking role beyond the endosome–TGN axis.","evidence":"RNAi-based imaging screen, live-cell carrier tracking, immunofluorescence","pmids":["32054068"],"confidence":"Medium","gaps":["Whether Golgi–ER carrier phenotype is a direct consequence of RHOBTB3 ATPase or ubiquitin ligase activity is unknown","Molecular cargo affected in this route is not identified","Single-lab observation awaiting independent replication"]},{"year":2024,"claim":"Identification of a RHOBTB3–SHIP164–Vps26B complex at Golgi–endosome contacts that drives Rab14-positive endosome bud formation established a new contact-site-based membrane remodeling function.","evidence":"Co-IP, RNAi depletion with rescue using wild-type and lipid-transfer-defective SHIP164 mutants, live-cell imaging, Rab14 effector pulldown","pmids":["38565878"],"confidence":"High","gaps":["Whether RHOBTB3 ATPase activity is required for bud formation is untested","Lipid species transferred by SHIP164 at these contacts are not identified","Structural basis for RHOBTB3–SHIP164–Vps26B tricomplex is unknown"]},{"year":2024,"claim":"Demonstrating that RHOBTB3 mediates K27-linked polyubiquitination of AMBRA1 at K45 to suppress autophagy added autophagy regulation to its CUL3-dependent substrates.","evidence":"IP/mass spectrometry substrate identification, site-specific K45 mutagenesis, ubiquitination assay, epistasis by double knockdown, autophagy flux assays","pmids":["39404422"],"confidence":"Medium","gaps":["Single-lab finding; independent validation of K27-linked ubiquitination specificity is needed","Whether this regulation occurs at the Golgi like cyclin E ubiquitylation is unknown","Physiological contexts triggering RHOBTB3-dependent AMBRA1 degradation are uncharacterized"]},{"year":null,"claim":"How RHOBTB3's dual enzymatic activities — ATP hydrolysis and CUL3-dependent E3 ligase adaptor function — are coordinated on the Golgi membrane, and whether they serve the same or independent trafficking and signaling pathways, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of full-length RHOBTB3 or its CUL3-bound form exists","Whether ATPase and ubiquitin ligase activities are mutually exclusive or cooperative is unknown","In vivo phenotyping beyond platelets (e.g., conditional tissue-specific knockouts) has not been reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,5]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,1,4,5]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,5]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,2,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6]}],"complexes":["CUL3/RBX1/RHOBTB3 E3 ligase","RHOBTB3/LIMD1-PHD2-VHL scaffold","RHOBTB3/SHIP164/Vps26B complex"],"partners":["RAB9A","CUL3","RBX1","PHD2","VHL","LIMD1","SHIP164","VPS26B"],"other_free_text":[]},"mechanistic_narrative":"RHOBTB3 is an atypical Rho family GTPase that functions as a Golgi-associated ATPase and scaffolding protein, coordinating membrane trafficking, ubiquitin-dependent protein degradation, and cell cycle control. It hydrolyzes ATP rather than GTP, with maximal ATPase activity achieved upon Rab9 binding that relieves autoinhibition, and cooperates with Rab9 and TIP47 in endosome-to-trans-Golgi network transport [PMID:19490898]; at Golgi–endosome contacts it additionally assembles a complex with SHIP164 and Vps26B to drive Rab14-positive early endosome bud formation [PMID:38565878]. RHOBTB3 serves as a substrate adaptor for CUL3/RBX1 E3 ubiquitin ligase complexes on the Golgi, targeting cyclin E for ubiquitylation to regulate S/G2 cell cycle progression [PMID:24145166], and mediating K27-linked polyubiquitination of AMBRA1 to suppress autophagy [PMID:39404422]. RHOBTB3 also organizes a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes HIFα hydroxylation and subsequent ubiquitination under normoxia; hypoxia disrupts this scaffold, stabilizing HIFα [PMID:26215701]."},"prefetch_data":{"uniprot":{"accession":"O94955","full_name":"Rho-related BTB domain-containing protein 3","aliases":[],"length_aa":611,"mass_kda":69.4,"function":"Rab9-regulated ATPase required for endosome to Golgi transport. Involved in transport vesicle docking at the Golgi complex, possibly by participating in release M6PRBP1/TIP47 from vesicles to permit their efficient docking and fusion at the Golgi. Specifically binds Rab9, but not other Rab proteins. Has low intrinsic ATPase activity due to autoinhibition, which is relieved by Rab9","subcellular_location":"Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/O94955/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RHOBTB3","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RHOBTB3","total_profiled":1310},"omim":[{"mim_id":"618753","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 41; LRRC41","url":"https://www.omim.org/entry/618753"},{"mim_id":"607353","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 3; RHOBTB3","url":"https://www.omim.org/entry/607353"},{"mim_id":"607352","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 2; RHOBTB2","url":"https://www.omim.org/entry/607352"},{"mim_id":"607351","title":"RHO-RELATED BTB DOMAIN-CONTAINING PROTEIN 1; RHOBTB1","url":"https://www.omim.org/entry/607351"},{"mim_id":"603575","title":"NME/NM23 FAMILY, MEMBER 5; NME5","url":"https://www.omim.org/entry/603575"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Vesicles","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RHOBTB3"},"hgnc":{"alias_symbol":["KIAA0878"],"prev_symbol":[]},"alphafold":{"accession":"O94955","domains":[{"cath_id":"3.40.50.300","chopping":"3-43_50-140_155-206","consensus_level":"high","plddt":73.3604,"start":3,"end":206},{"cath_id":"3.30.710.10","chopping":"240-313_324-388","consensus_level":"high","plddt":89.6767,"start":240,"end":388},{"cath_id":"3.30.710.10","chopping":"412-592","consensus_level":"medium","plddt":91.3071,"start":412,"end":592}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O94955","model_url":"https://alphafold.ebi.ac.uk/files/AF-O94955-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O94955-F1-predicted_aligned_error_v6.png","plddt_mean":78.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RHOBTB3","jax_strain_url":"https://www.jax.org/strain/search?query=RHOBTB3"},"sequence":{"accession":"O94955","fasta_url":"https://rest.uniprot.org/uniprotkb/O94955.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O94955/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O94955"}},"corpus_meta":[{"pmid":"19490898","id":"PMC_19490898","title":"RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport.","date":"2009","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/19490898","citation_count":79,"is_preprint":false},{"pmid":"26215701","id":"PMC_26215701","title":"RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.","date":"2015","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/26215701","citation_count":48,"is_preprint":false},{"pmid":"24145166","id":"PMC_24145166","title":"Golgi-associated RhoBTB3 targets cyclin E for ubiquitylation and promotes cell cycle progression.","date":"2013","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24145166","citation_count":39,"is_preprint":false},{"pmid":"35698915","id":"PMC_35698915","title":"RhoBTB3 Regulates Proliferation and Invasion of Breast Cancer Cells via Col1a1.","date":"2022","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/35698915","citation_count":19,"is_preprint":false},{"pmid":"22245496","id":"PMC_22245496","title":"RhoBTB3 interacts with the 5-HT7a receptor and inhibits its proteasomal degradation.","date":"2012","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/22245496","citation_count":14,"is_preprint":false},{"pmid":"24923387","id":"PMC_24923387","title":"Expression analysis of mouse Rhobtb3 using a LacZ reporter and preliminary characterization of a knockout strain.","date":"2014","source":"Histochemistry and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24923387","citation_count":10,"is_preprint":false},{"pmid":"37045834","id":"PMC_37045834","title":"MicroRNA-142-3p promotes renal cell carcinoma progression by targeting RhoBTB3 to regulate HIF-1 signaling and GGT/GSH pathways.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/37045834","citation_count":8,"is_preprint":false},{"pmid":"32354068","id":"PMC_32354068","title":"RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32354068","citation_count":7,"is_preprint":false},{"pmid":"34093823","id":"PMC_34093823","title":"High Expression of RhoBTB3 Predicts Favorable Chemothrapy Outcomes in non-M3 Acute Myeloid Leukemia.","date":"2021","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/34093823","citation_count":7,"is_preprint":false},{"pmid":"38565878","id":"PMC_38565878","title":"Biogenesis of Rab14-positive endosome buds at Golgi-endosome contacts by the RhoBTB3-SHIP164-Vps26B complex.","date":"2024","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38565878","citation_count":4,"is_preprint":false},{"pmid":"30754723","id":"PMC_30754723","title":"Alterations in Platelet Alpha-Granule Secretion and Adhesion on Collagen under Flow in Mice Lacking the Atypical Rho GTPase RhoBTB3.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/30754723","citation_count":4,"is_preprint":false},{"pmid":"39404422","id":"PMC_39404422","title":"RhoBTB3 Functions as a Novel Regulator of Autophagy by Suppressing AMBRA1 Stability.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/39404422","citation_count":2,"is_preprint":false},{"pmid":"40311591","id":"PMC_40311591","title":"Diosmetin attenuates the ubiquitination of epidermal hypoxia-inducible factor 1 alpha by diminishing the formation of RhoBTB3/PHD2 complex in ultraviolet radiation-induced sunburn in mice.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40311591","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7924,"output_tokens":2548,"usd":0.030996},"stage2":{"model":"claude-opus-4-6","input_tokens":5888,"output_tokens":2839,"usd":0.150622},"total_usd":0.181618,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"RhoBTB3 is an atypical Rho GTPase family ATPase that binds and hydrolyzes ATP (not GTP), directly binds Rab9 GTPase, and functions with Rab9 in protein transport from endosomes to the trans-Golgi network. Rab9 binding opens the autoinhibited RhoBTB3 to permit maximal ATP hydrolysis. RhoBTB3 also interacts with TIP47 on membranes, suggesting it releases this cargo selection protein from vesicles to permit efficient docking and fusion at the Golgi.\",\n      \"method\": \"Biochemical ATPase assay, direct binding assay, gene replacement/domain-swap experiments, co-immunoprecipitation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro biochemical assay for ATPase activity, direct binding reconstitution, domain mutagenesis, and functional rescue experiments; foundational paper\",\n      \"pmids\": [\"19490898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RhoBTB3 is a Golgi-associated protein that targets cyclin E for ubiquitylation as part of a CUL3-dependent RING-E3 ubiquitin ligase complex (RhoBTB3/CUL3/RBX1) on the Golgi, thereby regulating the S/G2 cell cycle transition. Depletion of RhoBTB3 arrested cells in S phase, caused Golgi fragmentation, and elevated cyclin E levels. Golgi association of the complex was required for its catalytic activity.\",\n      \"method\": \"Co-immunoprecipitation, RNAi depletion with cell cycle and morphology readouts, ubiquitylation assay, Golgi targeting experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, clean KD with defined cell cycle phenotype, functional localization requirement; multiple orthogonal methods\",\n      \"pmids\": [\"24145166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RHOBTB3 directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation and also directly interacts with VHL (E3 ubiquitin ligase component) to facilitate HIFα ubiquitination. RHOBTB3 dimerizes with LIMD1 and assembles a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex for maximal HIFα degradation. Hypoxia disrupts this complex, stabilizing HIFα.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, in vitro hydroxylation assay, RNAi depletion with HIF-α level readouts, xenograft model\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including in vitro hydroxylation assay, co-IP, ubiquitination assay, and functional in vivo rescue\",\n      \"pmids\": [\"26215701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RhoBTB3 directly interacts with the 5-HT7a serotonin receptor via both the C-terminal tail and the third intracellular loop of the receptor. This interaction inhibits proteasomal degradation of the 5-HT7a receptor, without recruiting CUL3/ROC1 to the receptor or mediating receptor ubiquitination. RhoBTB3 and 5-HT7a co-localize at the plasma membrane and endoplasmic reticulum.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation in HEK293T cells, domain mapping, immunofluorescence microscopy, proteasome inhibitor assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP confirmed interaction, domain mapping performed, functional consequence (proteasomal degradation) measured; single lab\",\n      \"pmids\": [\"22245496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RhoBTB3 depletion affects Golgi morphology and alters the trafficking speeds of carriers at the Golgi-ER interface. RhoBTB3 is present on these carriers, indicating a role in Golgi-ER membrane traffic.\",\n      \"method\": \"RNAi-based high-content imaging screen, live-cell imaging of carrier dynamics, immunofluorescence\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — image-based functional screen with quantitative trafficking readout; single lab, systematic approach\",\n      \"pmids\": [\"32354068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RhoBTB3 forms a complex with SHIP164 and Vps26B (a retromer subunit) at Golgi-endosome contacts to promote the formation of Rab14-positive early endosome buds. Vps26B acts as a novel Rab14 effector. Depletion of RhoBTB3 causes enlarged Rab14+ early endosomes without buds, phenocopying SHIP164 depletion.\",\n      \"method\": \"Co-immunoprecipitation, RNAi depletion with rescue experiments using wild-type and lipid transfer-defective SHIP164 mutants, live-cell imaging, Rab14 effector pulldown assay\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — complex reconstitution, domain-function mutant rescue, multiple orthogonal methods; mechanistic model supported by mutant rescue\",\n      \"pmids\": [\"38565878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RhoBTB3 negatively regulates autophagy by mediating K27-linked polyubiquitination of AMBRA1 at K45, leading to proteasome-mediated AMBRA1 degradation. AMBRA1 was identified as a RhoBTB3 substrate by immunoprecipitation and mass spectrometry. Knockdown of AMBRA1 blocks RhoBTB3-depletion-induced autophagy.\",\n      \"method\": \"Immunoprecipitation/mass spectrometry, ubiquitination assay with site-specific mutation (K45), epistasis (double KD), autophagy flux assays\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-based substrate identification, site-specific mutagenesis, epistasis experiment; single lab\",\n      \"pmids\": [\"39404422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Genetic deletion of RhoBTB3 in mice leads to increased alpha-granule secretion from platelets in response to thrombin, CRP, and U46619/ADP, and reduced platelet accrual on collagen under flow conditions, revealing a role for RhoBTB3 in platelet alpha-granule secretion and adhesion.\",\n      \"method\": \"Knockout mouse model, platelet aggregation and secretion assays, flow adhesion assay, flow cytometry\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype in primary cells; single lab\",\n      \"pmids\": [\"30754723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Diosmetin reduces the formation of the RhoBTB3/PHD2 complex, thereby diminishing HIFα hydroxylation and ubiquitination in UVR-exposed keratinocytes; overexpression of RhoBTB3 in mice confirmed that RhoBTB3, PHD2, and HIF-1α are enriched in the epidermis and that the RhoBTB3/PHD2 interaction promotes HIF-1α ubiquitination in this tissue context.\",\n      \"method\": \"Co-immunoprecipitation, RNA sequencing, rAAV-mediated RhoBTB3 overexpression in vivo, immunohistochemistry\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and in vivo OE; primarily pharmacological study, single lab, limited mechanistic depth beyond confirming the PHD2 interaction\",\n      \"pmids\": [\"40311591\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RhoBTB3 is a Golgi-associated atypical Rho GTPase that hydrolyzes ATP (not GTP) and functions as a scaffold/adaptor in multiple processes: it acts as part of a CUL3/RBX1-dependent E3 ubiquitin ligase complex on the Golgi to ubiquitylate cyclin E (regulating S/G2 cell cycle progression), organizes a RHOBTB3/LIMD1-PHD2-VHL scaffold to promote HIFα hydroxylation and ubiquitination, mediates AMBRA1 ubiquitination to suppress autophagy, forms a complex with SHIP164 and Vps26B at Golgi-endosome contacts to drive early endosome bud formation, and interacts with Rab9 (opening autoinhibition to enable maximal ATPase activity) to facilitate endosome-to-trans-Golgi-network trafficking.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RHOBTB3 is an atypical Rho family GTPase that functions as a Golgi-associated ATPase and scaffolding protein, coordinating membrane trafficking, ubiquitin-dependent protein degradation, and cell cycle control. It hydrolyzes ATP rather than GTP, with maximal ATPase activity achieved upon Rab9 binding that relieves autoinhibition, and cooperates with Rab9 and TIP47 in endosome-to-trans-Golgi network transport [PMID:19490898]; at Golgi–endosome contacts it additionally assembles a complex with SHIP164 and Vps26B to drive Rab14-positive early endosome bud formation [PMID:38565878]. RHOBTB3 serves as a substrate adaptor for CUL3/RBX1 E3 ubiquitin ligase complexes on the Golgi, targeting cyclin E for ubiquitylation to regulate S/G2 cell cycle progression [PMID:24145166], and mediating K27-linked polyubiquitination of AMBRA1 to suppress autophagy [PMID:39404422]. RHOBTB3 also organizes a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes HIFα hydroxylation and subsequent ubiquitination under normoxia; hypoxia disrupts this scaffold, stabilizing HIFα [PMID:26215701].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that RHOBTB3 is an ATPase rather than a GTPase, and that Rab9-dependent relief of autoinhibition couples its enzymatic activity to endosome-to-TGN trafficking, provided the first mechanistic framework for this atypical Rho family member.\",\n      \"evidence\": \"In vitro ATPase assays, direct binding reconstitution with Rab9, domain-swap rescue experiments, and co-immunoprecipitation in mammalian cells\",\n      \"pmids\": [\"19490898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for autoinhibition and its relief by Rab9 is unknown\",\n        \"Role of ATP hydrolysis versus ATP binding in trafficking has not been dissected\",\n        \"TIP47 release model lacks direct reconstitution of vesicle docking/fusion\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that RHOBTB3 binds the 5-HT7a serotonin receptor and inhibits its proteasomal degradation — independent of CUL3 recruitment — revealed a non-ubiquitin-ligase protective function for RHOBTB3 at the ER/plasma membrane.\",\n      \"evidence\": \"Yeast two-hybrid screen, co-IP with domain mapping, immunofluorescence co-localization, proteasome inhibitor experiments in HEK293T cells\",\n      \"pmids\": [\"22245496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which RHOBTB3 inhibits receptor degradation without mediating ubiquitination is unresolved\",\n        \"Interaction has not been confirmed by an independent lab\",\n        \"Physiological significance in serotonergic signaling is untested\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of RHOBTB3 as a Golgi-localized CUL3/RBX1 E3 ligase adaptor that ubiquitylates cyclin E established its role as a cell cycle regulator linking Golgi integrity to S/G2 transition.\",\n      \"evidence\": \"Reciprocal co-IP, RNAi depletion with cell cycle arrest and Golgi fragmentation phenotypes, in vivo ubiquitylation assays, Golgi-targeting experiments\",\n      \"pmids\": [\"24145166\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How Golgi localization is required for ligase activity is mechanistically unclear\",\n        \"Whether cyclin E is ubiquitylated directly on the Golgi membrane or after release is unresolved\",\n        \"Relationship between Golgi fragmentation and cyclin E accumulation is correlative\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Assembly of a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes both HIFα hydroxylation and ubiquitination demonstrated that RHOBTB3 coordinates an oxygen-sensing degradation complex disrupted by hypoxia.\",\n      \"evidence\": \"Co-IP, in vitro hydroxylation assay, ubiquitination assay, RNAi with HIFα readouts, xenograft model\",\n      \"pmids\": [\"26215701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether scaffold assembly is constitutive or regulated by signals other than hypoxia is unknown\",\n        \"Subcellular site of RHOBTB3–PHD2–VHL complex assembly has not been determined\",\n        \"Stoichiometry and structural organization of the pentameric complex are uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic knockout in mice revealed that RHOBTB3 restrains platelet alpha-granule secretion and supports adhesion under flow, extending its functional repertoire to hemostasis.\",\n      \"evidence\": \"RhoBTB3-knockout mouse, platelet aggregation/secretion assays, flow adhesion assay, flow cytometry\",\n      \"pmids\": [\"30754723\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular mechanism linking RHOBTB3 to granule secretion machinery is unknown\",\n        \"Whether the phenotype depends on CUL3-dependent ubiquitination or ATPase activity is untested\",\n        \"In vivo thrombosis or bleeding phenotypes were not reported\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"High-content screening confirmed that RHOBTB3 depletion alters Golgi morphology and carrier dynamics at the Golgi–ER interface, broadening its trafficking role beyond the endosome–TGN axis.\",\n      \"evidence\": \"RNAi-based imaging screen, live-cell carrier tracking, immunofluorescence\",\n      \"pmids\": [\"32054068\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether Golgi–ER carrier phenotype is a direct consequence of RHOBTB3 ATPase or ubiquitin ligase activity is unknown\",\n        \"Molecular cargo affected in this route is not identified\",\n        \"Single-lab observation awaiting independent replication\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of a RHOBTB3–SHIP164–Vps26B complex at Golgi–endosome contacts that drives Rab14-positive endosome bud formation established a new contact-site-based membrane remodeling function.\",\n      \"evidence\": \"Co-IP, RNAi depletion with rescue using wild-type and lipid-transfer-defective SHIP164 mutants, live-cell imaging, Rab14 effector pulldown\",\n      \"pmids\": [\"38565878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether RHOBTB3 ATPase activity is required for bud formation is untested\",\n        \"Lipid species transferred by SHIP164 at these contacts are not identified\",\n        \"Structural basis for RHOBTB3–SHIP164–Vps26B tricomplex is unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that RHOBTB3 mediates K27-linked polyubiquitination of AMBRA1 at K45 to suppress autophagy added autophagy regulation to its CUL3-dependent substrates.\",\n      \"evidence\": \"IP/mass spectrometry substrate identification, site-specific K45 mutagenesis, ubiquitination assay, epistasis by double knockdown, autophagy flux assays\",\n      \"pmids\": [\"39404422\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab finding; independent validation of K27-linked ubiquitination specificity is needed\",\n        \"Whether this regulation occurs at the Golgi like cyclin E ubiquitylation is unknown\",\n        \"Physiological contexts triggering RHOBTB3-dependent AMBRA1 degradation are uncharacterized\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RHOBTB3's dual enzymatic activities — ATP hydrolysis and CUL3-dependent E3 ligase adaptor function — are coordinated on the Golgi membrane, and whether they serve the same or independent trafficking and signaling pathways, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural model of full-length RHOBTB3 or its CUL3-bound form exists\",\n        \"Whether ATPase and ubiquitin ligase activities are mutually exclusive or cooperative is unknown\",\n        \"In vivo phenotyping beyond platelets (e.g., conditional tissue-specific knockouts) has not been reported\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 1, 4, 5]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"CUL3/RBX1/RHOBTB3 E3 ligase\",\n      \"RHOBTB3/LIMD1-PHD2-VHL scaffold\",\n      \"RHOBTB3/SHIP164/Vps26B complex\"\n    ],\n    \"partners\": [\n      \"RAB9A\",\n      \"CUL3\",\n      \"RBX1\",\n      \"PHD2\",\n      \"VHL\",\n      \"LIMD1\",\n      \"SHIP164\",\n      \"VPS26B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}