Affinage

RUFY1

RUN and FYVE domain-containing protein 1 · UniProt Q96T51

Length
708 aa
Mass
79.8 kDa
Annotated
2026-04-28
28 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RUFY1 is an early endosomal scaffold protein that coordinates cargo sorting, receptor trafficking, and organelle positioning by integrating signals from multiple small GTPases and lipid determinants. It functions as a direct effector of GTP-bound Rab4 and Rab5, binding PI3P via its FYVE domain (with pH-sensitive membrane insertion) and associating with membranes and actin-linked networks through its RUN domain, to regulate trafficking of GLUT4, EGFR, integrins, and CI-M6PR through early and recycling endosomes (PMID:11172003, PMID:14617813, PMID:16522682, PMID:29523688). Its coiled-coil region recruits dynein–dynactin to endosomes, and interaction with Arl8b and Rab14 governs RUFY1 endosomal positioning required for retrograde CI-M6PR retrieval to the TGN and lysosomal hydrolase delivery (PMID:36282215). Beyond canonical endosomal trafficking, RUFY1 forms the structural protein matrix of endolysosomal vesicular assemblies (ELVAs) in oocytes, non-membrane-bound compartments that sequester and degrade protein aggregates during oocyte maturation, with loss of ELVA function impairing embryo survival (PMID:38382525). RUFY1 is phosphorylated by Etk/BMX kinase, which regulates its endosomal localization, and it interacts with the AP-3 adaptor complex to control lysosome positioning (PMID:11877430, PMID:23144738).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2001 High

    Identification of RUFY1 as a Rab4 effector on early endosomes established that a novel multidomain protein mediates Rab4-dependent sorting at the early-to-recycling endosome boundary, explaining how active Rab4 controls retrograde membrane traffic.

    Evidence Yeast two-hybrid, co-immunoprecipitation, confocal co-localization, and overexpression phenotyping in CHO cells

    PMID:11172003

    Open questions at the time
    • Endogenous stoichiometry of RUFY1–Rab4 complex unknown
    • No knockout or loss-of-function data at this stage
  2. 2001 High

    Dissecting the domain architecture revealed that the RUN domain drives primary membrane association through detergent-insoluble microdomains while the FYVE domain binds PI3P as a co-requirement, resolving how RUFY1 is dually anchored to endosomal membranes.

    Evidence Domain deletion constructs, membrane fractionation, wortmannin treatment, and PI3P-binding assays in CHO cells

    PMID:11509568

    Open questions at the time
    • Structural basis of RUN domain membrane interaction unresolved
    • No crystal or cryo-EM structure of any RUFY1 domain
  3. 2002 High

    Discovery that Etk/BMX tyrosine kinase phosphorylates RUFY1 and that phosphorylation is required for endosomal localization introduced a signaling-dependent regulatory layer controlling RUFY1 membrane targeting.

    Evidence Yeast two-hybrid, co-IP, phosphorylation assays, and localization of phospho-mutants in COS-1 and B82L cells

    PMID:11877430

    Open questions at the time
    • Specific phosphorylated tyrosine residues not identified at this stage
    • Physiological stimulus triggering Etk-mediated phosphorylation not defined
  4. 2003 High

    Demonstration that the Rabip4' splice variant simultaneously binds GTP-Rab4 and GTP-Rab5 established RUFY1 as a coincidence detector linking two major early endosomal GTPases, and dominant-negative experiments showed functional relevance for transferrin recycling.

    Evidence Co-immunoprecipitation with GTP/GDP-loaded Rab4 and Rab5, wortmannin treatment, dominant-negative expression, and transferrin recycling assay

    PMID:14617813

    Open questions at the time
    • Relative contribution of each GTPase interaction to distinct trafficking routes not separated
    • No structural model of ternary complex
  5. 2006 High

    Functional studies in adipocytes and fibroblasts demonstrated that RUFY1–Rab4 interaction is essential for correct GLUT4 endosomal sorting (insulin-stimulated glucose uptake) and for PDGF-stimulated αv integrin delivery to the leading edge during cell migration, broadening RUFY1's cargo repertoire beyond transferrin.

    Evidence Overexpression and Rab4-binding mutants in 3T3-L1 adipocytes (glucose uptake) and NIH 3T3 fibroblasts (wound-healing, siRNA knockdown, integrin localization)

    PMID:16522682 PMID:17001082

    Open questions at the time
    • Whether RUFY1 directly contacts cargo or acts purely as a scaffold unclear
    • Contribution of Rab5 binding versus Rab4 binding to each cargo pathway not dissected
  6. 2009 High

    Biochemical reconstitution showed that FYVE domain membrane insertion is pH-dependent via two conserved histidine residues, providing a molecular mechanism for how RUFY1 preferentially associates with the acidic lumen-facing leaflet of early endosomes.

    Evidence In vitro lipid vesicle binding at varying pH, histidine mutagenesis, in vivo endosomal localization

    PMID:19296456

    Open questions at the time
    • Whether pH sensing contributes to cargo-specific sorting decisions unknown
    • No full-length structural data
  7. 2012 High

    Identification of a direct interaction between RUFY1's FYVE domain and the AP-3 β3-subunit hinge on tubular early endosome subdomains expanded RUFY1's function to include cooperation with coat machinery for lysosome positioning.

    Evidence In vitro pull-down, siRNA knockdown, co-localization in HeLa cells

    PMID:23144738

    Open questions at the time
    • Whether RUFY1–AP-3 interaction is regulated by phosphorylation or GTPase state unknown
    • How RUFY1 coordinates AP-3 and Rab4 binding simultaneously not resolved
  8. 2018 Medium

    Proteomic and knockdown studies placed RUFY1 in EGF-dependent early endosome complexes and showed it is required for EGFR trafficking, while parallel work linked it to Rab4A-rabenosyn-5 sorting complexes in melanocytes, reinforcing its general role as an endosomal sorting hub for diverse cargoes.

    Evidence Endosome proteomics and siRNA in HeLa (EGFR); RNAi screening and co-IP in melanocytes (melanosome maturation)

    PMID:29523688 PMID:30154210

    Open questions at the time
    • Direct knockdown of RUFY1 in melanocytes not performed
    • Whether RUFY1 sorts EGFR toward degradation or recycling not clarified
  9. 2022 High

    Discovery that RUFY1 binds Arl8b and acts as a dynein–dynactin adaptor for retrograde CI-M6PR retrieval from endosomes to the TGN established a new trafficking axis (Arl8b–RUFY1–Rab14–dynein) and demonstrated functional consequences for lysosomal hydrolase delivery.

    Evidence Co-IP, siRNA, pulse-chase trafficking, domain mapping in HeLa cells

    PMID:36282215

    Open questions at the time
    • Structural basis of coiled-coil–dynein interaction not determined
    • Whether Arl8b–RUFY1 axis operates in all cell types or is tissue-specific unknown
  10. 2024 High

    Identification of RUFY1 as the structural matrix protein of ELVAs in mouse oocytes revealed a non-canonical function: building a non-membrane-bound compartment that sequesters and degrades protein aggregates, with disruption causing embryonic lethality from aggregate accumulation.

    Evidence Live-cell imaging, electron microscopy, proteomics, functional inhibition in mouse oocytes

    PMID:38382525

    Open questions at the time
    • Whether ELVA-like structures exist in somatic cells unknown
    • Mechanism by which RUFY1 polymerizes into a non-membrane matrix not defined
    • Which RUFY1 domains are necessary for ELVA formation not dissected
  11. 2025 Medium

    Chemoproteomic identification of glutamate E502 as a functionally critical residue showed that covalent modification at this site disrupts RUFY1's endosomal trafficking network and receptor recycling, providing the first chemical tool to acutely perturb RUFY1 function.

    Evidence Chemoproteomics with N-aryl aziridine fragments, receptor recycling assay, LC-MS/MS

    PMID:40343844

    Open questions at the time
    • Selectivity of the covalent modifier for RUFY1 over other targets not fully established
    • Which specific interaction E502 modification disrupts (GTPase, dynein, AP-3) not determined

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the structural basis of RUFY1's multivalent GTPase/adaptor interactions, how RUFY1 polymerizes into the ELVA matrix in oocytes, whether ELVA-like functions exist in somatic cells, and which post-translational modifications dynamically regulate its switching between trafficking and scaffolding roles.
  • No high-resolution structure of RUFY1 or any of its complexes
  • Mechanism of ELVA matrix assembly unknown
  • Tissue-specific regulation of RUFY1 isoforms poorly characterized

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0008092 cytoskeletal protein binding 2 GO:0008289 lipid binding 2
Localization
GO:0005768 endosome 6 GO:0005856 cytoskeleton 2 GO:0005829 cytosol 1
Pathway
R-HSA-5653656 Vesicle-mediated transport 5 R-HSA-9609507 Protein localization 3 R-HSA-162582 Signal Transduction 2
Partners
AP3B1ARL8BBMXRAB14RAB4ARAB5RBSN

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2001 RUFY1 (Rabip4) was identified as a direct effector of Rab4 GTPase, binding specifically to the GTP-bound (active) form of Rab4 in yeast two-hybrid and mammalian cell assays. RUFY1 localizes to early endosomes (co-localizing with EEA1) but not recycling (Rab11-positive) or late (Rab7-positive) endosomes. Co-expression of RUFY1 with active Rab4 causes enlargement of early endosomes and intracellular retention of GLUT1, suggesting RUFY1 controls early endosomal traffic via a backward transport step from recycling to sorting endosomes. Yeast two-hybrid, co-immunoprecipitation in mammalian cells, confocal co-localization, overexpression phenotypic assays in CHO cells Proceedings of the National Academy of Sciences of the United States of America High 11172003
2001 The RUN domain of RUFY1 (Rabip4) drives primary membrane/endosomal association through Triton X-100-insoluble microdomains, while the FYVE domain binds phosphatidylinositol 3-phosphate (PI3P) and is necessary but not sufficient for membrane targeting. Both the FYVE domain and the Rab4-binding site are required for full function. RUFY1 associates with an actin-linked network but is not directly bound to actin. Domain deletion/truncation constructs expressed in CHO cells, membrane fractionation, wortmannin treatment, confocal microscopy, PI3P-binding assay The Journal of biological chemistry High 11509568
2002 Etk/BMX tyrosine kinase interacts with RUFY1 through its SH3 and SH2 domains and phosphorylates RUFY1 on tyrosine residues. RUFY1 mutants lacking phosphorylation sites fail to localize to endosomes. The FYVE domain of RUFY1 is recruited to the plasma membrane via its proline-rich motif binding to the SH3 domain of Etk, independently of its lipid-binding activity. Yeast two-hybrid, co-immunoprecipitation, phosphorylation assays, subcellular localization of phosphorylation-site mutants, overexpression in COS-1 and B82L cells The Journal of biological chemistry High 11877430
2003 The RUFY1 splice variant Rabip4' (with an 108 aa N-terminal extension) is a peripheral membrane protein on early endosomes whose membrane association requires the FYVE domain and PI3P (sensitive to wortmannin). Rabip4'/RUFY1 binds simultaneously and specifically to GTP-bound Rab4 and Rab5, and a dominant-negative Rabip4' mutant reduces transferrin internalization and recycling from early endosomes. Co-immunoprecipitation with GTP/GDP-loaded Rab4 and Rab5, wortmannin treatment, dominant-negative expression, transferrin recycling assay, co-localization with EEA1 Molecular biology of the cell High 14617813
2006 RUFY1 (Rabip4) defines an endocytic sorting platform for GLUT4 in adipocytes. Expression of RUFY1 increases insulin-stimulated GLUT4 translocation and glucose uptake. A Rab4-binding-deficient mutant of RUFY1 increases plasma membrane GLUT4 and impairs GLUT4 trafficking from endosomes to its sequestration compartments, indicating RUFY1-Rab4 interaction is required for correct GLUT4 endosomal sorting. Overexpression and dominant-negative mutant expression in 3T3-L1 adipocytes, glucose uptake assay, GLUT4 localization by immunofluorescence, subcellular fractionation Journal of cell science High 16522682
2006 RUFY1 (Rabip4/Rabip4') mediates PDGF-stimulated cell migration in NIH 3T3 fibroblasts by regulating αv integrin trafficking to the leading edge. PDGF redistributes RUFY1 toward the cell periphery where it co-localizes with F-actin. Rab4-binding-deficient RUFY1 mutants impair leading edge formation and block PDGF-stimulated migration; siRNA knockdown of RUFY1 inhibits PDGF-induced αv integrin translocation and cell migration. GFP-tagged constructs and Rab4-binding mutants in NIH 3T3 cells, siRNA knockdown, wound-healing migration assay, immunofluorescence co-localization with F-actin and αv integrins The Journal of biological chemistry High 17001082
2009 The FYVE domain of RUFY1 inserts into PI3P-enriched membranes, and this membrane insertion is substantially enhanced at acidic pH. Two adjacent histidine residues in the conserved R(R/K)HHCRXCG motif are required for pH-sensitivity; mutation of either histidine abolishes pH-dependent modulation of membrane binding. In vitro lipid vesicle binding assays at varying pH, mutagenesis of histidine residues, in vivo endosomal localization assays Proteins High 19296456
2012 RUFY1 (rabip4') interacts directly with the AP-3 adaptor complex via binding of the β3 subunit hinge region to the FYVE domain of RUFY1. RUFY1 co-localizes with AP-3 on a tubular subdomain of early endosomes. Silencing RUFY1 promotes plasma membrane protrusions and polarized lysosome clustering at their tips; AP-3 knockdown causes even more dramatic lysosome accumulation in protrusions, revealing a cooperative role for RUFY1 and AP-3 in lysosome positioning. Direct binding assay (in vitro pull-down of AP-3 β3 hinge and RUFY1 FYVE domain), siRNA knockdown, co-localization by immunofluorescence, dominant-negative Rab4 experiments PloS one High 23144738
2016 During intracellular P. gingivalis infection, Rab4A mediates dissociation of the EXOC complex from early endosomes and recruits RUFY1/Rabip4 (Rab4A effector) along with Rab14, enabling bacteria to exploit the fast recycling pathway for exiting infected cells. Depletion of Rab4A leads to accumulation of bacteria in early endosomes and disturbs bacterial exit. siRNA knockdown of Rab4A and VAMP2, co-localization imaging, bacterial exit assay in gingival epithelial cells Cellular microbiology Medium 26617273
2018 Rab4A forms an endosomal complex that includes rabenosyn-5, and rabenosyn-5 associates differentially with rabaptin-5 or Rabip4/4' (isoforms encoded by RUFY1) to regulate cargo sorting from sorting endosomes. RNAi knockdown of Rab4A causes defective melanosome maturation and mislocalization of melanosomal proteins, phenocopied by knockdown of rabenosyn-5. RNAi screening in melanocytes, co-immunoprecipitation, subcellular localization, cargo-sorting assay Journal of cell science Medium 30154210
2018 RUFY1 was identified as an EGF-dependent early endosome-associated protein by proteomics, and siRNA knockdown of RUFY1 impairs EGFR trafficking from early endosomes. Early endosome isolation and LC-MS/MS proteomics, siRNA knockdown, EGFR trafficking assay in HeLa cells The Journal of biological chemistry Medium 29523688
2018 RUFY1 interacts with PODXL (podocalyxin-like protein) as demonstrated by co-immunoprecipitation and mass spectrometry in gastric cancer cells. Silencing RUFY1 attenuates PODXL-induced cell proliferation, migration, invasion, and activation of PI3K/AKT, NF-κB, and MAPK/ERK signaling pathways. Mass spectrometry, co-immunoprecipitation, western blot, siRNA knockdown of RUFY1, cell proliferation/migration/invasion assays Cancer science Medium 30407695
2022 RUFY1 binds directly to Arl8b (an Arf-like GTP-binding protein) and acts as a dynein adaptor on recycling endosomes to mediate CI-M6PR retrieval from endosomes to the TGN. Arl8b regulates RUFY1 endosomal localization by controlling its interaction with Rab14. RUFY1 depletion delays CI-M6PR retrieval and impairs delivery of newly synthesized hydrolases to lysosomes. The coiled-coil region of RUFY1 binds dynein-dynactin and is required for dynein-dependent organelle clustering. Co-immunoprecipitation, siRNA knockdown, co-localization by immunofluorescence, pulse-chase trafficking assay for hydrolases and CI-M6PR, domain mapping experiments The Journal of cell biology High 36282215
2024 In mouse oocytes, RUFY1 forms the protein matrix of endolysosomal vesicular assemblies (ELVAs), non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes. ELVAs sequester aggregated proteins (including TDP-43) in immature oocytes and degrade them upon oocyte maturation. Inhibiting ELVA degradative activity leads to protein aggregate accumulation in embryos and impaired embryo survival. Live-cell imaging, electron microscopy, proteomics, RUFY1 localization by fluorescence, functional inhibition assays, protein aggregate detection Cell High 38382525
2025 Covalent modification of RUFY1 at glutamate E502 by an N-aryl aziridine compound disrupts RUFY1's interactions within the endosomal trafficking network and impairs receptor recycling, demonstrating that E502 is functionally important for RUFY1's role in endosomal trafficking. Chemoproteomics, covalent labeling with N-aryl aziridine fragments, receptor recycling assay, target deconvolution by LC-MS/MS Journal of the American Chemical Society Medium 40343844

Source papers

Stage 0 corpus · 28 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2001 A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic. Proceedings of the National Academy of Sciences of the United States of America 86 11172003
2003 Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes. Molecular biology of the cell 58 14617813
2002 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. The Journal of biological chemistry 53 11877430
2009 Membrane insertion of the FYVE domain is modulated by pH. Proteins 50 19296456
2001 Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4. The Journal of biological chemistry 49 11509568
2017 Early-Onset Alzheimer Disease and Candidate Risk Genes Involved in Endolysosomal Transport. JAMA neurology 48 28738127
2015 Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity. PLoS genetics 45 26658939
2024 Mouse oocytes sequester aggregated proteins in degradative super-organelles. Cell 44 38382525
2006 The Rab4 effector Rabip4 plays a role in the endocytotic trafficking of Glut 4 in 3T3-L1 adipocytes. Journal of cell science 34 16522682
2023 Prediction of treatment response to antipsychotic drugs for precision medicine approach to schizophrenia: randomized trials and multiomics analysis. Military Medical Research 32 37269009
2018 Rab4A organizes endosomal domains for sorting cargo to lysosome-related organelles. Journal of cell science 32 30154210
2022 Identification of fusions with potential clinical significance in melanoma. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 28 35871080
2007 Evolutionarily conserved structural and functional roles of the FYVE domain. Biochemical Society symposium 27 17233583
2006 The Rab4A effector protein Rabip4 is involved in migration of NIH 3T3 fibroblasts. The Journal of biological chemistry 27 17001082
2018 Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor-dependent manner. The Journal of biological chemistry 23 29523688
2021 Transcriptomic Profiling of Skeletal Muscle Reveals Candidate Genes Influencing Muscle Growth and Associated Lipid Composition in Portuguese Local Pig Breeds. Animals : an open access journal from MDPI 22 34065673
2018 Podocalyxin-like protein promotes gastric cancer progression through interacting with RUN and FYVE domain containing 1 protein. Cancer science 20 30407695
2012 AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes. PloS one 20 23144738
2016 Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells. Cellular microbiology 17 26617273
2022 RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes. The Journal of cell biology 14 36282215
2020 5' UTR variants in the quantitative trait gene Hnrnph1 support reduced 5' UTR usage and hnRNP H protein as a molecular mechanism underlying reduced methamphetamine sensitivity. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 12 32401417
2004 Characterization of RUSC1 and RUSC2 genes in silico. Oncology reports 8 15375525
2025 Proteome-Wide Covalent Targeting of Acidic Residues with Tunable N-Aryl Aziridines. Journal of the American Chemical Society 7 40343844
2017 Identification of five genetic variants as novel determinants of type 2 diabetes mellitus in Japanese by exome-wide association studies. Oncotarget 5 29113320
2024 Blood DNA Methylation Analysis Reveals a Distinctive Epigenetic Signature of Vasospasm in Aneurysmal Subarachnoid Hemorrhage. Translational stroke research 4 38649590
2024 DNA Methylation Patterns Associated with Tinnitus in Young Adults-A Pilot Study. Journal of the Association for Research in Otolaryngology : JARO 2 39147981
2025 Acquired RUFY1-RET rearrangement as a mechanism of resistance to lorlatinib in a patient with CD74-ROS1 rearranged non-small cell lung cancer: A case report. Oncotarget 0 39907599
2025 Impact of Epigenome-Wide Methylation and Breast Cancer Recurrence in Women Tested Negative for BRCA Genes: The Breast Methylation Risk (BREMERI) Study. Cancers 0 41097660