| 2001 |
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. |
Yeast two-hybrid, co-localization in CHO cells, dominant-active/inactive Rab4 co-expression, subcellular fractionation |
Proceedings of the National Academy of Sciences of the United States of America |
High |
11172003
|
| 2001 |
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. |
Domain deletion constructs, subcellular fractionation, Triton X-100 solubility assay, wortmannin treatment, confocal microscopy in CHO cells |
The Journal of biological chemistry |
High |
11509568
|
| 2002 |
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. |
Yeast two-hybrid, co-immunoprecipitation, in-cell tyrosine phosphorylation assay, site-directed mutagenesis, confocal microscopy in COS-1 and B82L cells |
The Journal of biological chemistry |
Medium |
11877430
|
| 2003 |
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. |
Co-localization, wortmannin treatment, GST pulldown with Rab4-GTP and Rab5-GTP, dominant-negative overexpression, transferrin recycling assay |
Molecular biology of the cell |
High |
14617813
|
| 2006 |
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. |
Overexpression and Rab4-binding mutant in 3T3-L1 adipocytes, glucose uptake assay, confocal co-localization, insulin stimulation |
Journal of cell science |
Medium |
16522682
|
| 2006 |
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. |
GFP overexpression, siRNA knockdown, Rab4-binding-deficient mutant, scratch wound / Boyden chamber migration assay, F-actin staining, integrin localization, in NIH 3T3 fibroblasts |
The Journal of biological chemistry |
Medium |
17001082
|
| 2009 |
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. |
Lipid vesicle binding assay (in vitro, varying pH), site-directed mutagenesis of His residues, cell-based assays |
Proteins |
Medium |
19296456
|
| 2012 |
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. |
siRNA knockdown, direct binding assay (AP-3 β3 hinge/FYVE domain), co-localization, dominant-negative Rab4, confocal and electron microscopy |
PloS one |
Medium |
23144738
|
| 2018 |
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. |
Early endosome isolation by Percoll gradient + EEA1 immunoisolation, LC-MS/MS proteomics, RNAi knockdown with EGFR trafficking readout |
The Journal of biological chemistry |
Medium |
29523688
|
| 2018 |
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. |
Mass spectrometry, co-immunoprecipitation, siRNA knockdown, proliferation/migration/invasion assays, western blot for signaling pathways |
Cancer science |
Low |
30407695
|
| 2018 |
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. |
RNAi screening in melanocytes, co-immunoprecipitation, co-localization, cargo trafficking assays |
Journal of cell science |
Low |
30154210
|
| 2016 |
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. |
Immunofluorescence co-localization, siRNA depletion of Rab4A, bacterial exit/accumulation assay in gingival epithelial cells |
Cellular microbiology |
Low |
26617273
|
| 2022 |
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. |
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 |
The Journal of cell biology |
High |
36282215
|
| 2024 |
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. |
Live-cell imaging, electron microscopy, proteomics, functional inhibition assays in mouse oocytes |
Cell |
High |
38382525
|
| 2025 |
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. |
Chemoproteomics (activity-based protein profiling), covalent small-molecule targeting (N-aryl aziridine), receptor recycling functional assay |
Journal of the American Chemical Society |
Medium |
40343844
|