{"gene":"RUFY1","run_date":"2026-06-10T07:46:28","timeline":{"discoveries":[{"year":2001,"finding":"RUFY1 (Rabip4) was identified as a Rab4 effector that specifically binds the GTP-bound form of Rab4 in yeast two-hybrid and mammalian cell assays. It localizes to early endosomes (co-localizing with EEA1) but not recycling (Rab11) or late (Rab7) endosomes. Co-expression with active Rab4 enlarges early endosomes and promotes colocalization of Rab5 and Rab11 markers with Rab4. RUFY1 expression causes intracellular retention of the recycling molecule GLUT1, suggesting it controls a backward transport step from recycling to sorting endosomes.","method":"Yeast two-hybrid, co-localization in CHO cells, dominant-active/inactive Rab4 co-expression, subcellular fractionation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal yeast two-hybrid and mammalian cell binding assays, multiple orthogonal approaches (localization, functional assay with active/inactive mutants, GLUT1 trafficking), replicated in subsequent papers","pmids":["11172003"],"is_preprint":false},{"year":2001,"finding":"The subcellular localization of RUFY1 (Rabip4) is primarily driven by its N-terminal RUN domain to Triton X-100-insoluble endosomal microdomains; the FYVE domain binds phosphatidylinositol 3-phosphate (PI3P) specifically and is necessary but not sufficient for membrane association. A construct containing the RUN domain (residues 1–212) was membrane-associated and localized to a filamentous/honeycomb network, while the FYVE domain alone was cytosolic. The PI3P-binding activity of the FYVE domain further stabilizes RUFY1 in PI3P-enriched lipid microdomains.","method":"Domain deletion constructs, subcellular fractionation, Triton X-100 solubility assay, wortmannin treatment, confocal microscopy in CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple deletion constructs tested with orthogonal biochemical (fractionation) and imaging methods, internally consistent results","pmids":["11509568"],"is_preprint":false},{"year":2002,"finding":"RUFY1 interacts with the tyrosine kinase Etk/BMX through Etk's SH3 and SH2 domains, and RUFY1 is tyrosine-phosphorylated by Etk in cells. A RUFY1 mutant lacking the phosphorylation sites failed to localize to endosomes. The FYVE domain of RUFY1, via its proline-rich motif, is targeted to the plasma membrane through interaction with the SH3 domain of Etk (lipid-binding activity of the FYVE domain is not required for this). Overexpression of Etk increased EGFR plasma membrane localization and delayed EGF-induced EGFR endocytosis; these effects were blocked by the FYVE domain of RUFY1.","method":"Yeast two-hybrid, co-immunoprecipitation, in-cell tyrosine phosphorylation assay, site-directed mutagenesis, confocal microscopy in COS-1 and B82L cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, mutagenesis, and functional EGFR endocytosis assay in single study; not independently replicated","pmids":["11877430"],"is_preprint":false},{"year":2003,"finding":"RUFY1 isoform Rabip4' is a peripheral membrane protein that localizes to early endosomes (co-localizing with EEA1 and internalized transferrin). Membrane association requires the FYVE domain and is disrupted by the PI3K inhibitor wortmannin. Rabip4' binds specifically and simultaneously to the GTP-bound forms of both Rab4 and Rab5. Expression of a dominant-negative Rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, indicating functional coordination of Rab4 and Rab5 activities.","method":"Co-localization, wortmannin treatment, GST pulldown with Rab4-GTP and Rab5-GTP, dominant-negative overexpression, transferrin recycling assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct GTP-form-specific binding assays, dominant-negative functional readout, multiple orthogonal methods in single study, independently consistent with prior Rab4 data","pmids":["14617813"],"is_preprint":false},{"year":2006,"finding":"RUFY1 (Rabip4) controls GLUT4 trafficking in adipocytes. Expression of Rabip4 increased glucose uptake and GLUT4 translocation in response to insulin by approximately 30%. Insulin redistributes Rabip4 to the plasma membrane. Rabip4 defines a subdomain of early endosomes distinct from EEA1, Rab11, transferrin receptor, and GLUT4 sequestration compartments but reachable by internalized transferrin and GLUT4. A Rabip4 mutant unable to bind Rab4 caused accumulation of non-functional GLUT4 at the plasma membrane and perturbed trafficking from endosomes to the GLUT4 sequestration compartment.","method":"Overexpression and Rab4-binding mutant in 3T3-L1 adipocytes, glucose uptake assay, confocal co-localization, insulin stimulation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional glucose uptake assay plus Rab4-binding mutant with co-localization, single lab","pmids":["16522682"],"is_preprint":false},{"year":2006,"finding":"RUFY1 (Rabip4/Rabip4') regulates cell migration. PDGF treatment redistributes endogenous Rabip4/4' toward the cell periphery where they co-localize with F-actin in membrane ruffles. GFP-Rabip4 overexpression induces constitutive localization at leading edges with increased cortical F-actin and increased migration. A Rab4-binding-deficient mutant of Rabip4 abolished leading-edge formation and PDGF-stimulated migration. siRNA knockdown of Rabip4/4' inhibited PDGF-stimulated migration and impaired PDGF-induced translocation of αv integrins to the leading edge.","method":"GFP overexpression, siRNA knockdown, Rab4-binding-deficient mutant, scratch wound / Boyden chamber migration assay, F-actin staining, integrin localization, in NIH 3T3 fibroblasts","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function (siRNA), gain-of-function, and binding-mutant approaches with defined migration and integrin-trafficking phenotypes in a single lab","pmids":["17001082"],"is_preprint":false},{"year":2009,"finding":"The FYVE domain of RUFY1 inserts into PI3P-enriched membranes in a pH-dependent manner. Lowering pH substantially increases membrane binding (demonstrated in vitro with POPC/POPE/PI3P vesicles and in vivo). Two conserved histidine residues in the RHHCRXCG signature motif are required for pH-sensitivity; mutation of either His abolishes it. Protonation of these His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form.","method":"Lipid vesicle binding assay (in vitro, varying pH), site-directed mutagenesis of His residues, cell-based assays","journal":"Proteins","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, but RUFY1 FYVE domain is one of several tested; not independently replicated specifically for RUFY1","pmids":["19296456"],"is_preprint":false},{"year":2012,"finding":"RUFY1 (both Rabip4 and Rabip4' isoforms) regulates lysosome positioning. Silencing RUFY1 promoted outgrowth of plasma membrane protrusions and polarized clustering of lysosomes at their tips within the cortical actin network. Rabip4' physically binds the AP-3 adaptor complex via a direct interaction between the hinge region of AP-3 β3 subunit and the FYVE domain of Rabip4'. Rabip4' co-localizes with AP-3 on a tubular subdomain of early endosomes; this co-localization increased upon dominant-negative Rab4 expression. AP-3 knockdown caused even more dramatic lysosome accumulation in membrane protrusions.","method":"siRNA knockdown, direct binding assay (AP-3 β3 hinge/FYVE domain), co-localization, dominant-negative Rab4, confocal and electron microscopy","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay plus siRNA functional phenotype with co-localization, single lab, two orthogonal methods","pmids":["23144738"],"is_preprint":false},{"year":2018,"finding":"RUFY1 is specifically recruited to early endosomes in an EGF-dependent manner. Proteomic isolation of early endosomes from EGF-stimulated vs. unstimulated HeLa cells identified RUFY1 as one of five proteins enriched in a ligand-dependent manner. RNAi knockdown of RUFY1 impaired EGFR trafficking.","method":"Early endosome isolation by Percoll gradient + EEA1 immunoisolation, LC-MS/MS proteomics, RNAi knockdown with EGFR trafficking readout","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endosome isolation with mass spectrometry validation plus functional RNAi knockdown; single lab","pmids":["29523688"],"is_preprint":false},{"year":2018,"finding":"RUFY1 forms a complex with PODXL (Podocalyxin-like protein) in gastric cancer cells, as identified by mass spectrometry and confirmed by co-immunoprecipitation. Silencing RUFY1 attenuated PODXL-induced cell proliferation, migration, invasion, and activation of PI3K/AKT, NF-κB, and MAPK/ERK signaling pathways.","method":"Mass spectrometry, co-immunoprecipitation, siRNA knockdown, proliferation/migration/invasion assays, western blot for signaling pathways","journal":"Cancer science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP confirmation of complex and RNAi phenotype without defined molecular mechanism for the interaction; single lab","pmids":["30407695"],"is_preprint":false},{"year":2018,"finding":"Rab4A on sorting endosomes in melanocytes forms a complex that includes the RUFY1-encoded proteins Rabip4/4' (via rabenosyn-5 association). Rabenosyn-5 was found to associate with Rabip4/4' and differentially regulate cargo sorting from sorting endosomes to melanosomes. Knockdown of Rab4A caused mislocalization of melanosomal proteins to lysosomes, cell surface, and exosomes.","method":"RNAi screening in melanocytes, co-immunoprecipitation, co-localization, cargo trafficking assays","journal":"Journal of cell science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — RUFY1/Rabip4 involvement inferred from rabenosyn-5 association data; RUFY1 not directly knocked down in this study for the melanosome phenotype","pmids":["30154210"],"is_preprint":false},{"year":2016,"finding":"During Porphyromonas gingivalis infection of gingival epithelial cells, Rab4A mediates recruitment of RUFY1/Rabip4 (a Rab4A effector) to early endosomes containing the pathogen, following dissociation of the EXOC complex. Depletion of Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells, supporting RUFY1's role in the Rab4A-dependent fast recycling pathway exploited by the pathogen.","method":"Immunofluorescence co-localization, siRNA depletion of Rab4A, bacterial exit/accumulation assay in gingival epithelial cells","journal":"Cellular microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — RUFY1 involvement established by localization data only; functional depletion performed for Rab4A not RUFY1 itself","pmids":["26617273"],"is_preprint":false},{"year":2022,"finding":"RUFY1 binds to the Arf-like GTPase Arl8b; Arl8b regulates RUFY1 endosomal localization by controlling its interaction with Rab14. RUFY1 depletion delays CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. RUFY1 interacts with the dynein-dynactin complex via its coiled-coil region, enabling dynein-dependent organelle clustering. RUFY1 thus acts on recycling endosomes to regulate endosome-to-TGN retrograde transport of CI-M6PR.","method":"Co-immunoprecipitation (Arl8b–RUFY1, RUFY1–Rab14, RUFY1–dynein-dynactin), siRNA depletion with CI-M6PR trafficking assay, lysosomal hydrolase delivery assay, organelle clustering assay, domain (coiled-coil) deletion mutants","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple reciprocal co-IPs, domain-deletion mutants, and two independent functional readouts (CI-M6PR retrieval and lysosomal hydrolase delivery) with mechanistic pathway placement","pmids":["36282215"],"is_preprint":false},{"year":2024,"finding":"In mouse oocytes, RUFY1 forms the protein matrix of non-membrane-bound compartments called endolysosomal vesicular assemblies (ELVAs), which are composed of endolysosomes, autophagosomes, and proteasomes. ELVAs sequester aggregated proteins (including TDP-43) in immature oocytes and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to accumulation of protein aggregates in embryos and is detrimental for embryo survival.","method":"Live-cell imaging, electron microscopy, proteomics, functional inhibition assays in mouse oocytes","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (live imaging, EM, proteomics, functional assays) establishing RUFY1 as structural matrix of ELVAs with defined cellular consequence","pmids":["38382525"],"is_preprint":false},{"year":2025,"finding":"Covalent modification of RUFY1 at glutamate E502 by an N-aryl aziridine compound disrupted RUFY1's interactions within the endosomal trafficking network and impaired receptor recycling, demonstrating that E502 is functionally important for RUFY1's role in endosomal trafficking.","method":"Chemoproteomics (activity-based protein profiling), covalent small-molecule targeting (N-aryl aziridine), receptor recycling functional assay","journal":"Journal of the American Chemical Society","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemoproteomic site identification with functional receptor recycling readout; single study, single lab","pmids":["40343844"],"is_preprint":false}],"current_model":"RUFY1 (Rabip4/Rabip4') is a Rab4 and Rab5 dual effector that localizes to early/sorting endosomes via its N-terminal RUN domain (membrane anchoring) and FYVE domain (PI3P binding, pH-sensitive), where it coordinates endosomal membrane dynamics including cargo sorting, GLUT4 and EGFR recycling, CI-M6PR retrograde retrieval to the TGN (via Arl8b, Rab14, and the dynein-dynactin complex), lysosome positioning (via AP-3 interaction), and cell migration via integrin trafficking; in mouse oocytes RUFY1 additionally serves as the structural matrix of degradative super-organelles (ELVAs) that sequester and degrade protein aggregates during oocyte maturation."},"narrative":{"mechanistic_narrative":"RUFY1 (Rabip4/Rabip4') is a dual Rab4/Rab5 effector that operates on early/sorting endosomes to coordinate cargo sorting and membrane dynamics [PMID:11172003, PMID:14617813]. It binds specifically the GTP-loaded forms of Rab4 and Rab5 simultaneously, and its endosomal targeting is bipartite: an N-terminal RUN domain anchors it to detergent-insoluble endosomal microdomains while a C-terminal FYVE domain binds PI3P, an interaction that is necessary for stable membrane association and is enhanced under acidic pH through protonation of conserved histidines in the FYVE RHHCRXCG motif [PMID:11509568, PMID:14617813, PMID:19296456]. From this endosomal platform RUFY1 governs recycling and retrograde traffic of multiple cargoes: it controls a backward transport step that retains the recycling cargo GLUT1 and modulates insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes [PMID:11172003, PMID:16522682], and it is recruited to endosomes in an EGF-dependent manner to regulate EGFR trafficking [PMID:29523688]. RUFY1 drives endosome-to-TGN retrograde retrieval of CI-M6PR — and consequent delivery of newly synthesized hydrolases to lysosomes — through interactions with Arl8b, Rab14, and the dynein-dynactin complex, the latter engaged via its coiled-coil region to enable dynein-dependent organelle clustering [PMID:36282215]. It also regulates lysosome positioning by directly binding the AP-3 adaptor (β3 hinge) through its FYVE domain [PMID:23144738] and promotes PDGF-stimulated cell migration by directing αv integrin trafficking to the leading edge [PMID:17001082]. In mouse oocytes RUFY1 takes on a distinct structural role as the protein matrix of endolysosomal vesicular assemblies (ELVAs), non-membrane-bound degradative super-organelles that sequester and clear protein aggregates during oocyte maturation [PMID:38382525].","teleology":[{"year":2001,"claim":"Established RUFY1 as a Rab4-specific effector on early endosomes, defining its place in endosomal recycling traffic rather than late or recycling endosome compartments.","evidence":"Yeast two-hybrid and mammalian binding to GTP-Rab4, co-localization with EEA1, and GLUT1 retention assays in CHO cells","pmids":["11172003"],"confidence":"High","gaps":["Direction of the transport step it controls inferred from GLUT1 retention, not directly traced","Structural basis of Rab4 recognition not resolved"]},{"year":2001,"claim":"Resolved how RUFY1 attaches to membranes, showing a division of labor between the RUN domain for microdomain anchoring and the FYVE domain for PI3P binding.","evidence":"Domain-deletion constructs, Triton X-100 solubility, wortmannin treatment, and fractionation in CHO cells","pmids":["11509568"],"confidence":"High","gaps":["What the RUN domain binds at the membrane is not defined","FYVE alone is insufficient, but the integrated targeting code is not fully reconstituted"]},{"year":2002,"claim":"Linked RUFY1 to tyrosine-kinase signaling and EGFR endocytosis, showing it can be recruited to the plasma membrane and phosphorylated by Etk/BMX.","evidence":"Yeast two-hybrid, co-IP, in-cell phosphorylation, and EGFR endocytosis assays in COS-1/B82L cells","pmids":["11877430"],"confidence":"Medium","gaps":["Not independently replicated","Functional consequence of RUFY1 tyrosine phosphorylation on cargo sorting unresolved"]},{"year":2003,"claim":"Demonstrated RUFY1 (Rabip4') binds GTP-Rab4 and GTP-Rab5 simultaneously and is functionally required for transferrin internalization and recycling, establishing it as a dual-Rab coordinator.","evidence":"GST pulldowns, wortmannin, dominant-negative overexpression, and transferrin recycling assays","pmids":["14617813"],"confidence":"High","gaps":["Whether Rab4 and Rab5 are bound at the same site or distinct sites not resolved","Stoichiometry of the dual-Rab complex unknown"]},{"year":2006,"claim":"Extended RUFY1 function to insulin-responsive GLUT4 traffic and to PDGF-driven cell migration via integrin delivery, showing its endosomal role shapes physiological cargo programs.","evidence":"Overexpression, Rab4-binding mutants, siRNA, glucose uptake/migration assays and integrin localization in adipocytes and fibroblasts","pmids":["16522682","17001082"],"confidence":"Medium","gaps":["Single-lab phenotypes for each cargo","Direct molecular link between RUFY1 and the integrin/GLUT4 sorting machinery not defined"]},{"year":2009,"claim":"Showed the RUFY1 FYVE domain membrane insertion is pH-sensitive, providing a mechanism for compartment-specific or maturation-dependent membrane binding.","evidence":"In vitro PI3P-vesicle binding at varying pH with His-residue mutagenesis","pmids":["19296456"],"confidence":"Medium","gaps":["RUFY1 FYVE was one of several domains tested; not RUFY1-specific replication","Physiological pH range over which this operates on real endosomes not established"]},{"year":2012,"claim":"Connected RUFY1 to lysosome positioning through a direct AP-3 interaction, broadening its role from cargo sorting to organelle distribution.","evidence":"Direct AP-3 β3 hinge/FYVE binding assay, siRNA, dominant-negative Rab4, confocal and EM","pmids":["23144738"],"confidence":"Medium","gaps":["Single lab","How AP-3 binding mechanistically restrains lysosome clustering not fully resolved"]},{"year":2018,"claim":"Confirmed ligand-triggered recruitment of RUFY1 to early endosomes and its requirement for EGFR trafficking through unbiased proteomics.","evidence":"Early endosome immunoisolation, LC-MS/MS, and RNAi with EGFR trafficking readout in HeLa","pmids":["29523688"],"confidence":"Medium","gaps":["Recruitment mechanism downstream of EGF not defined","Single lab"]},{"year":2022,"claim":"Defined RUFY1 as a retrograde-transport factor that links Arl8b, Rab14, and dynein-dynactin to drive CI-M6PR retrieval to the TGN and proper lysosomal hydrolase delivery.","evidence":"Reciprocal co-IPs, coiled-coil deletion mutants, and CI-M6PR/hydrolase trafficking and organelle-clustering assays","pmids":["36282215"],"confidence":"High","gaps":["Order of assembly of the Arl8b/Rab14/RUFY1 complex not fully resolved","How retrograde and recycling roles are partitioned on the same protein unclear"]},{"year":2024,"claim":"Revealed a non-canonical structural role for RUFY1 as the matrix of ELVAs, degradative super-organelles that clear protein aggregates in oocytes, distinct from its endosomal effector function.","evidence":"Live imaging, EM, proteomics and functional degradation-inhibition assays in mouse oocytes","pmids":["38382525"],"confidence":"High","gaps":["Whether RUFY1 forms ELVA-like structures in somatic cells unknown","How RUFY1 self-assembles into a non-membrane matrix not mechanistically defined"]},{"year":2025,"claim":"Identified RUFY1 glutamate E502 as a functionally important site whose covalent modification disrupts its trafficking-network interactions, providing a chemical handle on RUFY1 function.","evidence":"Activity-based chemoproteomics with covalent N-aryl aziridine targeting and receptor recycling assay","pmids":["40343844"],"confidence":"Medium","gaps":["Which specific interactions E502 modification disrupts not enumerated","Single study"]},{"year":null,"claim":"How RUFY1 switches between its endosomal effector role and its ELVA matrix/self-assembly role, and how its multiple cargo programs are coordinated on a single protein, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length RUFY1 or its multi-partner complexes","Mechanism switching effector vs. matrix function undefined","Regulation distributing RUFY1 among GLUT4, EGFR, integrin, and CI-M6PR pathways unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,3,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,12]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,3,7,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4,5]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[7,12]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,3,12]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,8,12]}],"complexes":["ELVA (endolysosomal vesicular assembly) matrix","AP-3 adaptor complex (interactor)","dynein-dynactin complex (interactor)"],"partners":["RAB4A","RAB5","RAB14","ARL8B","AP3B1","BMX","PODXL"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96T51","full_name":"RUN and FYVE domain-containing protein 1","aliases":["FYVE-finger protein EIP1","La-binding protein 1","Rab4-interacting protein","Zinc finger FYVE domain-containing protein 12"],"length_aa":708,"mass_kda":79.8,"function":"Activating adapter involved in cargo sorting from early/recycling endosomes. Regulates retrieval of proteins from endosomes to the trans-Golgi network through interaction with the dynein-dynactin complex (PubMed:36282215). Dual effector of RAB4B and RAB14, mediates a cooperative interaction allowing endosomal tethering and fusion (PubMed:20534812). Binds phospholipid vesicles containing phosphatidylinositol 3-phosphate and participates in early endosomal trafficking (PubMed:14617813). In oocytes, self-assembles to form a protein matrix which hold together endolysosomes, autophagosomes and proteasomes and generate non-membrane-bound compartments called endo-lysosomal vesicular assemblies (ELVAs). In immature oocytes, ELVAs sequester ubiquitinated protein aggregates and degrade them upon oocyte maturation (By similarity)","subcellular_location":"Early endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q96T51/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RUFY1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RUFY1","total_profiled":1310},"omim":[{"mim_id":"620083","title":"NEURODEVELOPMENTAL DISORDER WITH CRANIOFACIAL DYSMORPHISM AND SKELETAL DEFECTS; NEDCDS","url":"https://www.omim.org/entry/620083"},{"mim_id":"610328","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 2; RUFY2","url":"https://www.omim.org/entry/610328"},{"mim_id":"610327","title":"RUN AND FYVE DOMAINS-CONTAINING PROTEIN 1; RUFY1","url":"https://www.omim.org/entry/610327"},{"mim_id":"601035","title":"HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN H1; HNRNPH1","url":"https://www.omim.org/entry/601035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RUFY1"},"hgnc":{"alias_symbol":["FLJ22251","ZFYVE12","RABIP4"],"prev_symbol":[]},"alphafold":{"accession":"Q96T51","domains":[{"cath_id":"1.20.58.900","chopping":"106-286","consensus_level":"high","plddt":85.4308,"start":106,"end":286},{"cath_id":"-","chopping":"310-503","consensus_level":"medium","plddt":90.7499,"start":310,"end":503},{"cath_id":"3.30.40.10","chopping":"643-703","consensus_level":"high","plddt":83.3469,"start":643,"end":703}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96T51","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96T51-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96T51-F1-predicted_aligned_error_v6.png","plddt_mean":75.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RUFY1","jax_strain_url":"https://www.jax.org/strain/search?query=RUFY1"},"sequence":{"accession":"Q96T51","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96T51.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96T51/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96T51"}},"corpus_meta":[{"pmid":"11172003","id":"PMC_11172003","title":"A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic.","date":"2001","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/11172003","citation_count":87,"is_preprint":false},{"pmid":"14617813","id":"PMC_14617813","title":"Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes.","date":"2003","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/14617813","citation_count":58,"is_preprint":false},{"pmid":"11877430","id":"PMC_11877430","title":"Interaction between tyrosine kinase Etk and a RUN domain- and FYVE domain-containing protein RUFY1. A possible role of ETK in regulation of vesicle trafficking.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11877430","citation_count":53,"is_preprint":false},{"pmid":"28738127","id":"PMC_28738127","title":"Early-Onset Alzheimer Disease and Candidate Risk Genes Involved in Endolysosomal Transport.","date":"2017","source":"JAMA neurology","url":"https://pubmed.ncbi.nlm.nih.gov/28738127","citation_count":50,"is_preprint":false},{"pmid":"19296456","id":"PMC_19296456","title":"Membrane insertion of the FYVE domain is modulated by pH.","date":"2009","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/19296456","citation_count":50,"is_preprint":false},{"pmid":"11509568","id":"PMC_11509568","title":"Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4.","date":"2001","source":"The Journal of biological 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Rabip4 plays a role in the endocytotic trafficking of Glut 4 in 3T3-L1 adipocytes.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16522682","citation_count":34,"is_preprint":false},{"pmid":"30154210","id":"PMC_30154210","title":"Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles.","date":"2018","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/30154210","citation_count":32,"is_preprint":false},{"pmid":"35871080","id":"PMC_35871080","title":"Identification of fusions with potential clinical significance in melanoma.","date":"2022","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/35871080","citation_count":30,"is_preprint":false},{"pmid":"17001082","id":"PMC_17001082","title":"The Rab4A effector protein Rabip4 is involved in migration of NIH 3T3 fibroblasts.","date":"2006","source":"The 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It localizes to early endosomes (co-localizing with EEA1) but not recycling (Rab11) or late (Rab7) endosomes. Co-expression with active Rab4 enlarges early endosomes and promotes colocalization of Rab5 and Rab11 markers with Rab4. RUFY1 expression causes intracellular retention of the recycling molecule GLUT1, suggesting it controls a backward transport step from recycling to sorting endosomes.\",\n      \"method\": \"Yeast two-hybrid, co-localization in CHO cells, dominant-active/inactive Rab4 co-expression, subcellular fractionation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal yeast two-hybrid and mammalian cell binding assays, multiple orthogonal approaches (localization, functional assay with active/inactive mutants, GLUT1 trafficking), replicated in subsequent papers\",\n      \"pmids\": [\"11172003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The subcellular localization of RUFY1 (Rabip4) is primarily driven by its N-terminal RUN domain to Triton X-100-insoluble endosomal microdomains; the FYVE domain binds phosphatidylinositol 3-phosphate (PI3P) specifically and is necessary but not sufficient for membrane association. A construct containing the RUN domain (residues 1–212) was membrane-associated and localized to a filamentous/honeycomb network, while the FYVE domain alone was cytosolic. The PI3P-binding activity of the FYVE domain further stabilizes RUFY1 in PI3P-enriched lipid microdomains.\",\n      \"method\": \"Domain deletion constructs, subcellular fractionation, Triton X-100 solubility assay, wortmannin treatment, confocal microscopy in CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple deletion constructs tested with orthogonal biochemical (fractionation) and imaging methods, internally consistent results\",\n      \"pmids\": [\"11509568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RUFY1 interacts with the tyrosine kinase Etk/BMX through Etk's SH3 and SH2 domains, and RUFY1 is tyrosine-phosphorylated by Etk in cells. A RUFY1 mutant lacking the phosphorylation sites failed to localize to endosomes. The FYVE domain of RUFY1, via its proline-rich motif, is targeted to the plasma membrane through interaction with the SH3 domain of Etk (lipid-binding activity of the FYVE domain is not required for this). Overexpression of Etk increased EGFR plasma membrane localization and delayed EGF-induced EGFR endocytosis; these effects were blocked by the FYVE domain of RUFY1.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in-cell tyrosine phosphorylation assay, site-directed mutagenesis, confocal microscopy in COS-1 and B82L cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, mutagenesis, and functional EGFR endocytosis assay in single study; not independently replicated\",\n      \"pmids\": [\"11877430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RUFY1 isoform Rabip4' is a peripheral membrane protein that localizes to early endosomes (co-localizing with EEA1 and internalized transferrin). Membrane association requires the FYVE domain and is disrupted by the PI3K inhibitor wortmannin. Rabip4' binds specifically and simultaneously to the GTP-bound forms of both Rab4 and Rab5. Expression of a dominant-negative Rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, indicating functional coordination of Rab4 and Rab5 activities.\",\n      \"method\": \"Co-localization, wortmannin treatment, GST pulldown with Rab4-GTP and Rab5-GTP, dominant-negative overexpression, transferrin recycling assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct GTP-form-specific binding assays, dominant-negative functional readout, multiple orthogonal methods in single study, independently consistent with prior Rab4 data\",\n      \"pmids\": [\"14617813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RUFY1 (Rabip4) controls GLUT4 trafficking in adipocytes. Expression of Rabip4 increased glucose uptake and GLUT4 translocation in response to insulin by approximately 30%. Insulin redistributes Rabip4 to the plasma membrane. Rabip4 defines a subdomain of early endosomes distinct from EEA1, Rab11, transferrin receptor, and GLUT4 sequestration compartments but reachable by internalized transferrin and GLUT4. A Rabip4 mutant unable to bind Rab4 caused accumulation of non-functional GLUT4 at the plasma membrane and perturbed trafficking from endosomes to the GLUT4 sequestration compartment.\",\n      \"method\": \"Overexpression and Rab4-binding mutant in 3T3-L1 adipocytes, glucose uptake assay, confocal co-localization, insulin stimulation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional glucose uptake assay plus Rab4-binding mutant with co-localization, single lab\",\n      \"pmids\": [\"16522682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RUFY1 (Rabip4/Rabip4') regulates cell migration. PDGF treatment redistributes endogenous Rabip4/4' toward the cell periphery where they co-localize with F-actin in membrane ruffles. GFP-Rabip4 overexpression induces constitutive localization at leading edges with increased cortical F-actin and increased migration. A Rab4-binding-deficient mutant of Rabip4 abolished leading-edge formation and PDGF-stimulated migration. siRNA knockdown of Rabip4/4' inhibited PDGF-stimulated migration and impaired PDGF-induced translocation of αv integrins to the leading edge.\",\n      \"method\": \"GFP overexpression, siRNA knockdown, Rab4-binding-deficient mutant, scratch wound / Boyden chamber migration assay, F-actin staining, integrin localization, in NIH 3T3 fibroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function (siRNA), gain-of-function, and binding-mutant approaches with defined migration and integrin-trafficking phenotypes in a single lab\",\n      \"pmids\": [\"17001082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The FYVE domain of RUFY1 inserts into PI3P-enriched membranes in a pH-dependent manner. Lowering pH substantially increases membrane binding (demonstrated in vitro with POPC/POPE/PI3P vesicles and in vivo). Two conserved histidine residues in the RHHCRXCG signature motif are required for pH-sensitivity; mutation of either His abolishes it. Protonation of these His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form.\",\n      \"method\": \"Lipid vesicle binding assay (in vitro, varying pH), site-directed mutagenesis of His residues, cell-based assays\",\n      \"journal\": \"Proteins\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, but RUFY1 FYVE domain is one of several tested; not independently replicated specifically for RUFY1\",\n      \"pmids\": [\"19296456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RUFY1 (both Rabip4 and Rabip4' isoforms) regulates lysosome positioning. Silencing RUFY1 promoted outgrowth of plasma membrane protrusions and polarized clustering of lysosomes at their tips within the cortical actin network. Rabip4' physically binds the AP-3 adaptor complex via a direct interaction between the hinge region of AP-3 β3 subunit and the FYVE domain of Rabip4'. Rabip4' co-localizes with AP-3 on a tubular subdomain of early endosomes; this co-localization increased upon dominant-negative Rab4 expression. AP-3 knockdown caused even more dramatic lysosome accumulation in membrane protrusions.\",\n      \"method\": \"siRNA knockdown, direct binding assay (AP-3 β3 hinge/FYVE domain), co-localization, dominant-negative Rab4, confocal and electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay plus siRNA functional phenotype with co-localization, single lab, two orthogonal methods\",\n      \"pmids\": [\"23144738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RUFY1 is specifically recruited to early endosomes in an EGF-dependent manner. Proteomic isolation of early endosomes from EGF-stimulated vs. unstimulated HeLa cells identified RUFY1 as one of five proteins enriched in a ligand-dependent manner. RNAi knockdown of RUFY1 impaired EGFR trafficking.\",\n      \"method\": \"Early endosome isolation by Percoll gradient + EEA1 immunoisolation, LC-MS/MS proteomics, RNAi knockdown with EGFR trafficking readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endosome isolation with mass spectrometry validation plus functional RNAi knockdown; single lab\",\n      \"pmids\": [\"29523688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RUFY1 forms a complex with PODXL (Podocalyxin-like protein) in gastric cancer cells, as identified by mass spectrometry and confirmed by co-immunoprecipitation. Silencing RUFY1 attenuated PODXL-induced cell proliferation, migration, invasion, and activation of PI3K/AKT, NF-κB, and MAPK/ERK signaling pathways.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, siRNA knockdown, proliferation/migration/invasion assays, western blot for signaling pathways\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP confirmation of complex and RNAi phenotype without defined molecular mechanism for the interaction; single lab\",\n      \"pmids\": [\"30407695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rab4A on sorting endosomes in melanocytes forms a complex that includes the RUFY1-encoded proteins Rabip4/4' (via rabenosyn-5 association). Rabenosyn-5 was found to associate with Rabip4/4' and differentially regulate cargo sorting from sorting endosomes to melanosomes. Knockdown of Rab4A caused mislocalization of melanosomal proteins to lysosomes, cell surface, and exosomes.\",\n      \"method\": \"RNAi screening in melanocytes, co-immunoprecipitation, co-localization, cargo trafficking assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — RUFY1/Rabip4 involvement inferred from rabenosyn-5 association data; RUFY1 not directly knocked down in this study for the melanosome phenotype\",\n      \"pmids\": [\"30154210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"During Porphyromonas gingivalis infection of gingival epithelial cells, Rab4A mediates recruitment of RUFY1/Rabip4 (a Rab4A effector) to early endosomes containing the pathogen, following dissociation of the EXOC complex. Depletion of Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells, supporting RUFY1's role in the Rab4A-dependent fast recycling pathway exploited by the pathogen.\",\n      \"method\": \"Immunofluorescence co-localization, siRNA depletion of Rab4A, bacterial exit/accumulation assay in gingival epithelial cells\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — RUFY1 involvement established by localization data only; functional depletion performed for Rab4A not RUFY1 itself\",\n      \"pmids\": [\"26617273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RUFY1 binds to the Arf-like GTPase Arl8b; Arl8b regulates RUFY1 endosomal localization by controlling its interaction with Rab14. RUFY1 depletion delays CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. RUFY1 interacts with the dynein-dynactin complex via its coiled-coil region, enabling dynein-dependent organelle clustering. RUFY1 thus acts on recycling endosomes to regulate endosome-to-TGN retrograde transport of CI-M6PR.\",\n      \"method\": \"Co-immunoprecipitation (Arl8b–RUFY1, RUFY1–Rab14, RUFY1–dynein-dynactin), siRNA depletion with CI-M6PR trafficking assay, lysosomal hydrolase delivery assay, organelle clustering assay, domain (coiled-coil) deletion mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple reciprocal co-IPs, domain-deletion mutants, and two independent functional readouts (CI-M6PR retrieval and lysosomal hydrolase delivery) with mechanistic pathway placement\",\n      \"pmids\": [\"36282215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In mouse oocytes, RUFY1 forms the protein matrix of non-membrane-bound compartments called endolysosomal vesicular assemblies (ELVAs), which are composed of endolysosomes, autophagosomes, and proteasomes. ELVAs sequester aggregated proteins (including TDP-43) in immature oocytes and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to accumulation of protein aggregates in embryos and is detrimental for embryo survival.\",\n      \"method\": \"Live-cell imaging, electron microscopy, proteomics, functional inhibition assays in mouse oocytes\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (live imaging, EM, proteomics, functional assays) establishing RUFY1 as structural matrix of ELVAs with defined cellular consequence\",\n      \"pmids\": [\"38382525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Covalent modification of RUFY1 at glutamate E502 by an N-aryl aziridine compound disrupted RUFY1's interactions within the endosomal trafficking network and impaired receptor recycling, demonstrating that E502 is functionally important for RUFY1's role in endosomal trafficking.\",\n      \"method\": \"Chemoproteomics (activity-based protein profiling), covalent small-molecule targeting (N-aryl aziridine), receptor recycling functional assay\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemoproteomic site identification with functional receptor recycling readout; single study, single lab\",\n      \"pmids\": [\"40343844\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RUFY1 (Rabip4/Rabip4') is a Rab4 and Rab5 dual effector that localizes to early/sorting endosomes via its N-terminal RUN domain (membrane anchoring) and FYVE domain (PI3P binding, pH-sensitive), where it coordinates endosomal membrane dynamics including cargo sorting, GLUT4 and EGFR recycling, CI-M6PR retrograde retrieval to the TGN (via Arl8b, Rab14, and the dynein-dynactin complex), lysosome positioning (via AP-3 interaction), and cell migration via integrin trafficking; in mouse oocytes RUFY1 additionally serves as the structural matrix of degradative super-organelles (ELVAs) that sequester and degrade protein aggregates during oocyte maturation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RUFY1 (Rabip4/Rabip4') is a dual Rab4/Rab5 effector that operates on early/sorting endosomes to coordinate cargo sorting and membrane dynamics [#0, #3]. It binds specifically the GTP-loaded forms of Rab4 and Rab5 simultaneously, and its endosomal targeting is bipartite: an N-terminal RUN domain anchors it to detergent-insoluble endosomal microdomains while a C-terminal FYVE domain binds PI3P, an interaction that is necessary for stable membrane association and is enhanced under acidic pH through protonation of conserved histidines in the FYVE RHHCRXCG motif [#1, #3, #6]. From this endosomal platform RUFY1 governs recycling and retrograde traffic of multiple cargoes: it controls a backward transport step that retains the recycling cargo GLUT1 and modulates insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes [#0, #4], and it is recruited to endosomes in an EGF-dependent manner to regulate EGFR trafficking [#8]. RUFY1 drives endosome-to-TGN retrograde retrieval of CI-M6PR — and consequent delivery of newly synthesized hydrolases to lysosomes — through interactions with Arl8b, Rab14, and the dynein-dynactin complex, the latter engaged via its coiled-coil region to enable dynein-dependent organelle clustering [#12]. It also regulates lysosome positioning by directly binding the AP-3 adaptor (β3 hinge) through its FYVE domain [#7] and promotes PDGF-stimulated cell migration by directing αv integrin trafficking to the leading edge [#5]. In mouse oocytes RUFY1 takes on a distinct structural role as the protein matrix of endolysosomal vesicular assemblies (ELVAs), non-membrane-bound degradative super-organelles that sequester and clear protein aggregates during oocyte maturation [#13].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established RUFY1 as a Rab4-specific effector on early endosomes, defining its place in endosomal recycling traffic rather than late or recycling endosome compartments.\",\n      \"evidence\": \"Yeast two-hybrid and mammalian binding to GTP-Rab4, co-localization with EEA1, and GLUT1 retention assays in CHO cells\",\n      \"pmids\": [\"11172003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direction of the transport step it controls inferred from GLUT1 retention, not directly traced\", \"Structural basis of Rab4 recognition not resolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Resolved how RUFY1 attaches to membranes, showing a division of labor between the RUN domain for microdomain anchoring and the FYVE domain for PI3P binding.\",\n      \"evidence\": \"Domain-deletion constructs, Triton X-100 solubility, wortmannin treatment, and fractionation in CHO cells\",\n      \"pmids\": [\"11509568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What the RUN domain binds at the membrane is not defined\", \"FYVE alone is insufficient, but the integrated targeting code is not fully reconstituted\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked RUFY1 to tyrosine-kinase signaling and EGFR endocytosis, showing it can be recruited to the plasma membrane and phosphorylated by Etk/BMX.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in-cell phosphorylation, and EGFR endocytosis assays in COS-1/B82L cells\",\n      \"pmids\": [\"11877430\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not independently replicated\", \"Functional consequence of RUFY1 tyrosine phosphorylation on cargo sorting unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated RUFY1 (Rabip4') binds GTP-Rab4 and GTP-Rab5 simultaneously and is functionally required for transferrin internalization and recycling, establishing it as a dual-Rab coordinator.\",\n      \"evidence\": \"GST pulldowns, wortmannin, dominant-negative overexpression, and transferrin recycling assays\",\n      \"pmids\": [\"14617813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rab4 and Rab5 are bound at the same site or distinct sites not resolved\", \"Stoichiometry of the dual-Rab complex unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended RUFY1 function to insulin-responsive GLUT4 traffic and to PDGF-driven cell migration via integrin delivery, showing its endosomal role shapes physiological cargo programs.\",\n      \"evidence\": \"Overexpression, Rab4-binding mutants, siRNA, glucose uptake/migration assays and integrin localization in adipocytes and fibroblasts\",\n      \"pmids\": [\"16522682\", \"17001082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab phenotypes for each cargo\", \"Direct molecular link between RUFY1 and the integrin/GLUT4 sorting machinery not defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed the RUFY1 FYVE domain membrane insertion is pH-sensitive, providing a mechanism for compartment-specific or maturation-dependent membrane binding.\",\n      \"evidence\": \"In vitro PI3P-vesicle binding at varying pH with His-residue mutagenesis\",\n      \"pmids\": [\"19296456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RUFY1 FYVE was one of several domains tested; not RUFY1-specific replication\", \"Physiological pH range over which this operates on real endosomes not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected RUFY1 to lysosome positioning through a direct AP-3 interaction, broadening its role from cargo sorting to organelle distribution.\",\n      \"evidence\": \"Direct AP-3 β3 hinge/FYVE binding assay, siRNA, dominant-negative Rab4, confocal and EM\",\n      \"pmids\": [\"23144738\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How AP-3 binding mechanistically restrains lysosome clustering not fully resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirmed ligand-triggered recruitment of RUFY1 to early endosomes and its requirement for EGFR trafficking through unbiased proteomics.\",\n      \"evidence\": \"Early endosome immunoisolation, LC-MS/MS, and RNAi with EGFR trafficking readout in HeLa\",\n      \"pmids\": [\"29523688\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recruitment mechanism downstream of EGF not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined RUFY1 as a retrograde-transport factor that links Arl8b, Rab14, and dynein-dynactin to drive CI-M6PR retrieval to the TGN and proper lysosomal hydrolase delivery.\",\n      \"evidence\": \"Reciprocal co-IPs, coiled-coil deletion mutants, and CI-M6PR/hydrolase trafficking and organelle-clustering assays\",\n      \"pmids\": [\"36282215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of assembly of the Arl8b/Rab14/RUFY1 complex not fully resolved\", \"How retrograde and recycling roles are partitioned on the same protein unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a non-canonical structural role for RUFY1 as the matrix of ELVAs, degradative super-organelles that clear protein aggregates in oocytes, distinct from its endosomal effector function.\",\n      \"evidence\": \"Live imaging, EM, proteomics and functional degradation-inhibition assays in mouse oocytes\",\n      \"pmids\": [\"38382525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RUFY1 forms ELVA-like structures in somatic cells unknown\", \"How RUFY1 self-assembles into a non-membrane matrix not mechanistically defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified RUFY1 glutamate E502 as a functionally important site whose covalent modification disrupts its trafficking-network interactions, providing a chemical handle on RUFY1 function.\",\n      \"evidence\": \"Activity-based chemoproteomics with covalent N-aryl aziridine targeting and receptor recycling assay\",\n      \"pmids\": [\"40343844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which specific interactions E502 modification disrupts not enumerated\", \"Single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RUFY1 switches between its endosomal effector role and its ELVA matrix/self-assembly role, and how its multiple cargo programs are coordinated on a single protein, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length RUFY1 or its multi-partner complexes\", \"Mechanism switching effector vs. matrix function undefined\", \"Regulation distributing RUFY1 among GLUT4, EGFR, integrin, and CI-M6PR pathways unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 3, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 3, 7, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4, 5]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [7, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 8, 12]}\n    ],\n    \"complexes\": [\"ELVA (endolysosomal vesicular assembly) matrix\", \"AP-3 adaptor complex (interactor)\", \"dynein-dynactin complex (interactor)\"],\n    \"partners\": [\"RAB4A\", \"RAB5\", \"RAB14\", \"ARL8B\", \"AP3B1\", \"BMX\", \"PODXL\", \"PODXL\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}