{"gene":"RAB31","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2020,"finding":"Active RAB31, phosphorylated by EGFR, engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form intraluminal vesicles (ILVs) independently of the ESCRT machinery. RAB31 interacts with the SPFH domain of flotillin and drives ILV formation via the flotillin domain. Simultaneously, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing MVE-lysosome fusion and enabling ILV secretion as exosomes.","method":"Co-immunoprecipitation, functional rescue, dominant-active/inactive mutants, knockdown/overexpression with phenotypic readouts (ILV formation, exosome secretion, lysosome fusion assays)","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, mutant analysis, knockdown, vesicle tracking), mechanistically rigorous, published in high-impact journal","pmids":["32958903"],"is_preprint":false},{"year":2007,"finding":"Gapex-5/RME-6 contains a VPS9 domain that acts as a guanine nucleotide exchange factor (GEF) for RAB31. In adipocytes, overexpression of RAB31 blocks insulin-stimulated Glut4 translocation, while knockdown potentiates it. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, reducing RAB31 activity and permitting Glut4 vesicles to translocate to the cell surface.","method":"Yeast two-hybrid, overexpression/knockdown in adipocytes, glucose uptake assays, subcellular localization","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, GEF activity assay, KD/OE with specific phenotypic readout), replicated across assays","pmids":["17189207"],"is_preprint":false},{"year":2011,"finding":"RIN3 specifically acts as a guanine nucleotide exchange factor (GEF) for RAB31 (but not RAB21), stimulating GTP-bound RAB31 formation in cell-free and in-cell GEF activity assays. Serine-to-alanine substitutions in the sequence between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on RAB31 but not RAB5. RIN3 colocalizes with RAB31 in enlarged vesicles and tubular structures in HeLa cells. RIN3 partially translocates CD-MPR from the TGN to peripheral vesicles in a RAB31-GEF-dependent manner.","method":"Cell-free and in-cell GEF activity assays, site-directed mutagenesis, colocalization by fluorescence microscopy, CD-MPR trafficking assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro GEF reconstitution, mutagenesis, and multiple cellular assays in a single rigorous study","pmids":["21586568"],"is_preprint":false},{"year":2002,"finding":"Recombinant RAB31 expressed as a GST-fusion protein binds [35S]GTPγS in a Mg2+-dependent manner (optimal at 5 µM free Mg2+, inhibited at higher concentrations). RAB31 displays low steady-state GTPase activity. The Q64L GTPase-dead mutation does not abolish GTPase activity of RAB31 (unlike most small GTPases).","method":"GST-fusion protein expression, radiolabeled nucleotide binding assay, GTPase activity assay, site-directed mutagenesis","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay with mutagenesis, single lab, characterization paper","pmids":["11784320"],"is_preprint":false},{"year":2009,"finding":"RAB31 is required for transport of mannose-6-phosphate receptors (specifically cation-dependent CD-MPR, but not CD63 or VSVG) from the TGN to endosomes. CD-MPR and RAB31 colocalize in TGN carriers containing clathrin and GGA1 coats. Constitutively active RAB31 redistributes CD-MPR from TGN to endosomes; dominant-negative RAB31 causes the reverse. siRNA depletion of RAB31 causes collapse of the Golgi apparatus.","method":"Dominant-active/inactive RAB31 mutants, siRNA knockdown, immunofluorescence colocalization, cargo trafficking assays","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (CA/DN mutants, siRNA, cargo tracking), specific cargo selectivity demonstrated","pmids":["19345684"],"is_preprint":false},{"year":2010,"finding":"RAB31 interacts with OCRL-1 (a PI(4,5)P2 5-phosphatase) as shown by yeast two-hybrid, GST-RAB31 pulldown, and co-immunoprecipitation from oligodendrocyte lysates. RAB31 and OCRL-1 colocalize in TGN, post-TGN carriers, and endosomes. siRNA depletion of RAB31 causes TGN collapse and markedly decreases OCRL-1 levels in the TGN and endosomes, indicating RAB31 recruits OCRL-1 to TGN domains where carriers form.","method":"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, siRNA knockdown, immunofluorescence colocalization","journal":"Journal of neuroscience research","confidence":"High","confidence_rationale":"Tier 2 / Strong — three independent binding assays plus functional knockdown with colocalization, multiple orthogonal methods","pmids":["19795375"],"is_preprint":false},{"year":2007,"finding":"Endogenous RAB22B/RAB31 is largely localized to the TGN in HeLa cells. Expression of dominant-negative GDP-bound RAB22B (but not wild-type) specifically disrupts TGN46 localization and inhibits anterograde exit of VSVG from the TGN, implicating RAB31 in anterograde TGN membrane dynamics.","method":"Specific antibody, overexpression of dominant-negative mutant, immunofluorescence, VSVG transport assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative approach with specific phenotypic readout, multiple markers tested, single lab","pmids":["17678623"],"is_preprint":false},{"year":2009,"finding":"RAB22B/RAB31 is expressed specifically in nestin/RC2-positive radial glia of the embryonic mouse brain and in GFAP-positive astrocytes of the adult brain. Silencing RAB22B in A431 cells causes abnormal trafficking of EGFR, Texas-red-EGF, and cation-independent M6PR. RAB22B associates with EGFR in a GTP-dependent manner as shown by affinity pulldown and co-immunoprecipitation.","method":"Specific antibody, immunofluorescence, siRNA knockdown, affinity pulldown, co-immunoprecipitation, receptor trafficking assays","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays plus KD with trafficking phenotype, single lab, two orthogonal binding methods","pmids":["19725050"],"is_preprint":false},{"year":2014,"finding":"RAB31 regulates trafficking of ligand-bound EGFR from early to late endosomes. Loss of RAB31 inhibits, and overexpression enhances, EGFR trafficking to late endosomes. RAB31 interacts with EGFR (by co-IP and affinity pulldown) and is recruited into a high-molecular-weight complex with EEA1 after EGF stimulation. Loss of EEA1 reduces RAB31-EGFR interaction and abolishes the effect of RAB31 on EGFR trafficking. Loss of GAPex5 also reduces RAB31-EGFR interaction.","method":"Co-immunoprecipitation, affinity pulldown, glycerol gradient sedimentation, siRNA knockdown, overexpression, pulse-chase EGFR trafficking assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, pulldown, sedimentation, KD/OE), specific rescue and epistasis assays, single lab","pmids":["24644286"],"is_preprint":false},{"year":2015,"finding":"RAB31 is recruited to early-stage phagocytic cups in macrophages at the PI(4,5)P2-to-PI(3,4,5)P3 phosphoinositide transition and persists on PI(3)P-enriched phagosomes. RAB31-GTP recruits the signaling adaptor APPL2 at phagocytic cups. siRNA depletion of either RAB31 or APPL2 reduces FcγR-mediated phagocytosis, delays transition to PI(3,4,5)P3 and phagocytic cup closure, reduces PI3K/Akt signaling, and enhances p38 signaling.","method":"Live-cell imaging, siRNA knockdown, phosphoinositide reporters, FcγR-mediated phagocytosis assay, signaling pathway analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with functional knockdown, multiple orthogonal readouts (phosphoinositides, phagocytosis efficiency, signaling), single lab","pmids":["25568335"],"is_preprint":false},{"year":2012,"finding":"MUC1-C forms a complex with ERα on the RAB31 promoter and activates RAB31 gene transcription in an estrogen-dependent manner in ER+ breast cancer cells. In turn, RAB31 attenuates lysosomal degradation of MUC1-C, creating an autoinductive loop. Expression of inactive RAB31(S20N) mutant in nonmalignant breast epithelial cells confirmed RAB31 regulates MUC1-C expression.","method":"Chromatin immunoprecipitation, promoter-reporter assay, dominant-negative mutant, lysosomal degradation assay, mammosphere formation assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional mutant plus degradation assay, single lab, multiple orthogonal methods","pmids":["22792175"],"is_preprint":false},{"year":2022,"finding":"RAB31 is regulated by the transcription factor RUNX1, which binds the RAB31 promoter in megakaryocytic cells. Downregulation of RUNX1 or RAB31 (by siRNA or CRISPR/Cas9) causes striking enlargement of early endosomes (EEs), partially reversed by RAB31 reconstitution. RAB31 deficiency impairs trafficking of VWF (to α-granules), EGFR, and mannose-6-phosphate receptor at the level of EEs in megakaryocytic cells.","method":"Promoter-reporter assay, siRNA, CRISPR/Cas9 knockout, RAB31 reconstitution, immunofluorescence for EE markers, iPSC-derived megakaryocyte model","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with rescue, multiple cargo tracking, patient-derived iPSC model, multiple orthogonal methods","pmids":["35839075"],"is_preprint":false},{"year":2022,"finding":"RAB31 knockdown inhibits TGF-β receptor II complex endocytosis in hepatic stellate cells, impairing TGF-β/Smad signaling and preventing hepatic stellate cell activation. RAB31 is required as a prerequisite for TGF-βRII endocytosis-driven TGF-β signaling.","method":"Lentiviral knockdown, TGF-βRII endocytosis assay, Smad signaling analysis, mouse CCl4 fibrosis model","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with specific receptor endocytosis readout and signaling cascade, in vivo validation, single lab","pmids":["35091093"],"is_preprint":false},{"year":2021,"finding":"The Shigella effector IpaH4.5 harbors TBC-like dual-finger motifs and exhibits potent RabGAP activity specifically toward RAB31, inactivating it. This disrupts CD-MPR transport from the Golgi to endosomes and attenuates lysosomal cathepsin B activity, allowing Shigella to escape lysosomal degradation. Intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity.","method":"GAP activity assay (in vitro), co-immunoprecipitation, confocal microscopy (MPR trafficking), Magic Red-RR cathepsin B activity assay, intracellular persistence assay","journal":"Journal of medical microbiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro GAP activity assay plus multiple functional cellular assays, mechanistically rigorous, single lab","pmids":["34296983"],"is_preprint":false},{"year":2022,"finding":"The C-terminal hypervariable domain (HVD) of RAB31, together with the interswitch loop and N-terminal domain, constitutes a membrane targeting domain (MTD) that dictates Golgi (rather than early endosomal) localization of RAB31. Replacing the RAB31 HVD with the RAB22 HVD shifts RAB31 to early endosomes. RAB31 Golgi localization is further influenced by differential interaction with the early endosomal effector Rabenosyn-5, which stabilizes RAB22 at endosomes but does not stabilize RAB31 there.","method":"Domain-swap chimera mutants, live fluorescence microscopy, Rabenosyn-5 knockout cells, co-localization analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimera mutagenesis with localization readout, KO cells, single lab, multiple constructs tested","pmids":["35863437"],"is_preprint":false},{"year":2022,"finding":"RAB31 interacts with RAGE (receptor for advanced glycation end products) intracellular domain, identified by GST pulldown combined with mass spectrometry and confirmed by co-immunoprecipitation and immunostaining. This interaction is enhanced by glycation-serum stimulation and is associated with membrane redistribution of RAB31. RAB31 promotes RAGE endocytosis and inhibits AGE-induced β-cell apoptosis through the pAKT/BCL2 pathway.","method":"GST pulldown combined with mass spectrometry, co-immunoprecipitation, immunostaining, RAGE endocytosis assay, apoptosis assay","journal":"Endocrine journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GST pulldown with MS confirmation plus reciprocal Co-IP plus functional assay, single lab","pmids":["35314532"],"is_preprint":false},{"year":2018,"finding":"NCSTN mutations reduce miR-30a-3p levels, which negatively regulates RAB31 expression. Enhanced RAB31 levels accelerate degradation of activated EGFR, leading to abnormal keratinocyte differentiation. The miR-30a-3p/RAB31/EGFR signaling axis was demonstrated in familial acne inversa patient samples and NcstnΔKC mice.","method":"miRNA microarray, Ncstn keratinocyte-specific knockout mice, luciferase reporter assay (miRNA target validation), EGFR degradation assay, keratinocyte differentiation assay","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — validated miRNA-target interaction plus functional EGFR degradation readout, mouse model, single lab","pmids":["30120935"],"is_preprint":false},{"year":2014,"finding":"RAB31 interacts with GLI1 as confirmed by co-immunoprecipitation and immunofluorescence in gastric cancer cells. RAB31 silencing suppresses cell viability, promotes cell cycle arrest, enhances apoptosis, and affects cell cycle/apoptotic proteins, effects mediated through GLI1.","method":"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, cell viability/apoptosis assays, luciferase reporter assay (miRNA target validation)","journal":"Frontiers in oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for interaction, functional assays without reconstitution; note: paper subsequently retracted (PMID 41114346)","pmids":["30534536"],"is_preprint":false},{"year":2014,"finding":"RAB31 is expressed in neural progenitor cells (NPCs). Silencing RAB31 hinders, while overexpression enhances, differentiation of NPCs to astrocytes, establishing a role for RAB31 in NPC fate determination.","method":"Primary NPC culture, siRNA knockdown, overexpression, differentiation assay with GFAP/nestin markers","journal":"FEBS letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — KD/OE with phenotypic differentiation readout, no mechanistic pathway placement, single lab","pmids":["24999186"],"is_preprint":false},{"year":2022,"finding":"RAB31 interacts with MAPK6, and RAB31 knockdown reduces MAPK6 protein levels by promoting its degradation. MAPK6 overexpression restores the decreased migration potential caused by RAB31 knockdown, placing RAB31 upstream of MAPK6 in cervical cancer cell migration.","method":"Co-immunoprecipitation, MAPK6 degradation assay, overexpression rescue, migration assay, in vivo xenograft","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus degradation assay plus genetic rescue establishing epistasis, single lab","pmids":["34975321"],"is_preprint":false},{"year":2023,"finding":"RAB31 overexpression in gastric cancer cells increases exosome secretion (number), while RAB31 depletion reduces both the number and size of secreted exosomes. Injection of exosomes derived from RAB31-overexpressing cells promotes pulmonary metastasis in vivo. PSMA1 was identified as an exosomal protein overexpressed in concert with RAB31.","method":"Exosome nanoparticle tracking analysis, electron microscopy, in vivo exosome injection/metastasis model, protein mass spectrometry","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — exosome quantification with multiple methods plus in vivo functional test, single lab","pmids":["37222416"],"is_preprint":false},{"year":2023,"finding":"RAB31 in glioma-derived endothelial cells drives enrichment of MYO1C into extracellular vesicles (EVs). RAB31 knockdown reduces MYO1C enrichment in secretory EVs and attenuates promotion of glioma cell invasion by GhEC-EVs. This EV export mechanism is also dependent on RAB27B and FAS.","method":"siRNA knockdown, EV isolation, Western blot for MYO1C in EVs, invasion assay","journal":"FEBS open bio","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single KD approach with EV cargo tracking, no reconstitution, single lab","pmids":["37953466"],"is_preprint":false}],"current_model":"RAB31 is a Rab5-subfamily small GTPase that cycles between GDP- and GTP-bound states under control of multiple GEFs (Gapex-5/RME-6, RIN3) and is regulated by EGFR-mediated phosphorylation; in its active (GTP-bound) form it localizes predominantly to the trans-Golgi network and early endosomes, where it mediates selective TGN-to-endosome transport of mannose-6-phosphate receptors (via recruitment of OCRL-1 and interaction with EEA1/GAPex5), drives ESCRT-independent intraluminal vesicle formation by engaging flotillin proteins in lipid raft microdomains, suppresses MVE-lysosome fusion by recruiting the RAB7-GAP TBC1D2B, promotes FcγR-mediated phagocytosis through APPL2 recruitment, facilitates TGF-βRII endocytosis to sustain TGF-β/Smad signaling, and regulates EGFR trafficking from early to late endosomes; its localization is determined by a membrane targeting domain comprising the C-terminal hypervariable domain and interswitch loop, and it is transcriptionally regulated by RUNX1 and ERα/MUC1-C complexes."},"narrative":{"mechanistic_narrative":"RAB31 is a Rab5-subfamily small GTPase that governs selective membrane trafficking between the trans-Golgi network (TGN), early endosomes, and multivesicular endosomes [PMID:19345684, PMID:35863437]. Its nucleotide cycle is driven by VPS9-domain GEFs: Gapex-5/RME-6, which couples RAB31 activity to insulin-regulated Glut4 vesicle translocation in adipocytes [PMID:17189207], and RIN3, which specifically loads GTP onto RAB31 to mobilize CD-MPR out of the TGN [PMID:21586568]. In its active form RAB31 directs TGN-to-endosome transport of mannose-6-phosphate receptors, recruiting the PI(4,5)P2 5-phosphatase OCRL-1 to TGN carriers and acting at clathrin/GGA1-coated domains; its depletion collapses the Golgi [PMID:19345684, PMID:19795375]. RAB31 distinguishes itself from the related RAB22 through a membrane targeting domain built from its C-terminal hypervariable domain, interswitch loop, and N-terminal region, which enforces Golgi over endosomal localization [PMID:35863437]. On the endosomal/MVE axis, EGFR-phosphorylated RAB31 engages flotillins in lipid raft microdomains to drive ESCRT-independent intraluminal vesicle formation while recruiting the RAB7-GAP TBC1D2B to block MVE-lysosome fusion, thereby routing cargo toward exosome secretion [PMID:32958903]; consistently, RAB31 controls EGFR transit from early to late endosomes via an EEA1- and Gapex-5-dependent complex [PMID:24644286]. Beyond constitutive trafficking, RAB31 supports FcγR-mediated phagocytosis by recruiting APPL2 at phagocytic cups during the phosphoinositide transition [PMID:25568335], sustains TGF-βRII endocytosis required for TGF-β/Smad signaling [PMID:35091093], and its expression is transcriptionally driven by RUNX1 in megakaryocytes, where it is needed for early-endosomal trafficking of VWF, EGFR, and M6PR [PMID:35839075]. RAB31 transcription is also activated by an ERα/MUC1-C complex in ER+ breast cancer, forming an autoinductive loop in which RAB31 stabilizes MUC1-C against lysosomal degradation [PMID:22792175]. The Shigella effector IpaH4.5 acts as a RAB31-specific RabGAP, inactivating it to subvert CD-MPR-dependent cathepsin trafficking and evade lysosomal killing [PMID:34296983].","teleology":[{"year":2002,"claim":"Established the basic biochemistry of RAB31 as a guanine nucleotide-binding protein, defining it as a functional small GTPase with atypical catalytic properties.","evidence":"GST-fusion recombinant protein with radiolabeled GTPγS binding and GTPase assays plus Q64L mutagenesis","pmids":["11784320"],"confidence":"Medium","gaps":["No effectors or cellular pathway assigned","Atypical retention of activity by Q64L mutant left unexplained","Single-lab in vitro characterization"]},{"year":2007,"claim":"Identified the first physiological GEF (Gapex-5/RME-6) and a cellular role, linking RAB31 activity to insulin-regulated Glut4 vesicle trafficking and revealing its TGN localization.","evidence":"Yeast two-hybrid, GEF activity, and KD/OE with glucose uptake readouts in adipocytes; specific antibody localization to TGN with DN-mutant VSVG transport assay","pmids":["17189207","17678623"],"confidence":"High","gaps":["Direct cargo of RAB31 in Glut4 pathway not defined","Connection between TGN dynamics and Glut4 retention unresolved"]},{"year":2009,"claim":"Defined RAB31's core trafficking function: selective TGN-to-endosome transport of mannose-6-phosphate receptors and broader receptor sorting, with Golgi integrity dependent on RAB31.","evidence":"CA/DN mutants, siRNA, cargo trafficking and colocalization assays for CD-MPR; reciprocal binding and KD trafficking assays for EGFR/CI-M6PR in A431 cells","pmids":["19345684","19725050"],"confidence":"High","gaps":["Cargo selectivity mechanism (why CD-MPR but not CD63/VSVG) unexplained","Effectors mediating carrier formation not yet identified"]},{"year":2010,"claim":"Identified OCRL-1 as a direct RAB31 effector recruited to TGN carrier-forming domains, linking RAB31 to phosphoinositide control at the TGN.","evidence":"Yeast two-hybrid, GST pulldown, co-IP, and siRNA depletion with colocalization in oligodendrocytes","pmids":["19795375"],"confidence":"High","gaps":["Functional consequence of OCRL-1 phosphatase activity for carrier budding not directly tested","Whether OCRL-1 recruitment is GTP-dependent not resolved"]},{"year":2011,"claim":"Demonstrated GEF specificity by reconstitution, establishing RIN3 as a RAB31-selective GEF that mobilizes CD-MPR from the TGN.","evidence":"Cell-free and in-cell GEF assays, site-directed mutagenesis, colocalization, and CD-MPR trafficking assay","pmids":["21586568"],"confidence":"High","gaps":["Physiological signal activating RIN3 toward RAB31 unknown","Relationship between RIN3 and Gapex-5 GEF inputs not reconciled"]},{"year":2012,"claim":"Placed RAB31 in a transcription-trafficking feedback loop in breast cancer, where ERα/MUC1-C activate RAB31 and RAB31 stabilizes MUC1-C against lysosomal degradation.","evidence":"ChIP, promoter-reporter, DN mutant (S20N), and lysosomal degradation assays in ER+ breast cancer cells","pmids":["22792175"],"confidence":"Medium","gaps":["Mechanism by which RAB31 diverts MUC1-C from lysosomes not detailed","Generality beyond ER+ context untested"]},{"year":2014,"claim":"Extended RAB31 function to endosomal maturation, showing it routes ligand-bound EGFR from early to late endosomes via an EEA1/Gapex-5-containing complex.","evidence":"Co-IP, affinity pulldown, glycerol gradient sedimentation, KD/OE, and pulse-chase EGFR trafficking assays","pmids":["24644286"],"confidence":"High","gaps":["Stoichiometry and assembly order of the RAB31-EEA1-Gapex5 complex unresolved","How RAB31 reconciles pro-degradation versus exosome-routing fates of EGFR not addressed"]},{"year":2015,"claim":"Revealed RAB31 as a coordinator of phagosome maturation, recruiting APPL2 at the phosphoinositide transition to support FcγR-mediated phagocytosis and PI3K/Akt signaling.","evidence":"Live-cell imaging with phosphoinositide reporters, siRNA, phagocytosis efficiency, and signaling pathway analysis in macrophages","pmids":["25568335"],"confidence":"High","gaps":["Whether APPL2 recruitment is direct GTP-dependent binding not biochemically confirmed","GEF driving RAB31 activation at the phagocytic cup unidentified"]},{"year":2020,"claim":"Resolved a mechanism for ESCRT-independent exosome biogenesis, showing EGFR-phosphorylated RAB31 uses flotillins to form ILVs while recruiting TBC1D2B to inactivate RAB7 and block lysosomal fusion.","evidence":"Co-IP, domain mapping, DA/DN mutants, KD/OE with ILV formation, exosome secretion, and lysosome fusion assays","pmids":["32958903"],"confidence":"High","gaps":["The EGFR phosphosite(s) on RAB31 and their regulatory effect not fully mapped","Balance between flotillin ILV pathway and canonical ESCRT routing unclear"]},{"year":2021,"claim":"Showed RAB31 is a target of bacterial subversion, with the Shigella RabGAP effector IpaH4.5 inactivating it to disrupt M6PR-dependent cathepsin delivery and evade lysosomal killing.","evidence":"In vitro GAP activity assay, co-IP, MPR trafficking microscopy, cathepsin B activity, and intracellular persistence assays","pmids":["34296983"],"confidence":"High","gaps":["Endogenous host RabGAP for RAB31 still unknown","Whether IpaH4.5 also affects RAB31's exosomal/endosomal roles untested"]},{"year":2022,"claim":"Identified the structural determinant of RAB31 organelle targeting and a transcriptional driver, while expanding its physiological roles to megakaryocyte granule biogenesis, TGF-β signaling, and RAGE endocytosis.","evidence":"Domain-swap chimeras and Rabenosyn-5 KO localization; RUNX1 promoter/CRISPR KO with cargo tracking in iPSC-megakaryocytes; KD TGF-βRII endocytosis with CCl4 fibrosis model; GST-MS/co-IP with RAGE endocytosis and β-cell apoptosis assays","pmids":["35863437","35839075","35091093","35314532"],"confidence":"High","gaps":["How the HVD/interswitch MTD physically discriminates Golgi from endosomal membranes mechanistically unresolved","Whether RAGE and TGF-βRII handling share the EGFR endosomal machinery untested"]},{"year":2023,"claim":"Linked RAB31-driven exosome biogenesis to tumor progression, showing RAB31 controls exosome number/size and selective EV cargo loading that promotes metastasis and invasion.","evidence":"Nanoparticle tracking, electron microscopy, mass spectrometry, and in vivo exosome injection/metastasis models in gastric cancer and glioma EC systems","pmids":["37222416","37953466"],"confidence":"Medium","gaps":["Mechanism of selective cargo (PSMA1, MYO1C) sorting into RAB31-dependent EVs undefined","Glioma EV study rests on single-KD evidence without reconstitution"]},{"year":null,"claim":"How RAB31 mechanistically partitions a single cargo (e.g. EGFR) between degradative late-endosome routing, exosomal secretion, and recycling, and which GEF/effector combinations select each fate, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model reconciling pro-degradation versus exosome-secretory RAB31 functions","Spatiotemporal regulation of competing effectors (OCRL-1, flotillin, TBC1D2B, APPL2, EEA1) not integrated","Structural basis of effector selection beyond the membrane targeting domain unknown"]}],"mechanism_profile":{"molecular_activity":[],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[4,5,6,14]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[4,8,11,14]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,1]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[4,5,8,11]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,12,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,13]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,20]}],"complexes":[],"partners":["FLOT1","TBC1D2B","OCRL","EEA1","EGFR","APPL2","RIN3","GAPEX5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13636","full_name":"Ras-related protein Rab-31","aliases":["Ras-related protein Rab-22B"],"length_aa":195,"mass_kda":21.7,"function":"The small GTPases Rab are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes (PubMed:11784320). Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different set of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion. Required for the integrity and for normal function of the Golgi apparatus and the trans-Golgi network. Plays a role in insulin-stimulated translocation of GLUT4 to the cell membrane. Plays a role in M6PR transport from the trans-Golgi network to endosomes. Plays a role in the internalization of EGFR from the cell membrane into endosomes. Plays a role in the maturation of phagosomes that engulf pathogens, such as S.aureus and M.tuberculosis","subcellular_location":"Golgi apparatus, trans-Golgi network; Golgi apparatus, trans-Golgi network membrane; Early endosome; Cytoplasmic vesicle, phagosome; Cytoplasmic vesicle, phagosome membrane","url":"https://www.uniprot.org/uniprotkb/Q13636/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAB31","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/RAB31","total_profiled":1310},"omim":[{"mim_id":"619152","title":"TBC1 DOMAIN FAMILY, MEMBER 2B; TBC1D2B","url":"https://www.omim.org/entry/619152"},{"mim_id":"612966","title":"RAS-ASSOCIATED PROTEIN RAB22A; RAB22A","url":"https://www.omim.org/entry/612966"},{"mim_id":"612906","title":"RAS-ASSOCIATED PROTEIN RAB32; RAB32","url":"https://www.omim.org/entry/612906"},{"mim_id":"610223","title":"RAS AND RAB INTERACTOR 3; RIN3","url":"https://www.omim.org/entry/610223"},{"mim_id":"605694","title":"RAS-ASSOCIATED PROTEIN RAB31; RAB31","url":"https://www.omim.org/entry/605694"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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RAB31 interacts with the SPFH domain of flotillin and drives ILV formation via the flotillin domain. Simultaneously, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing MVE-lysosome fusion and enabling ILV secretion as exosomes.\",\n      \"method\": \"Co-immunoprecipitation, functional rescue, dominant-active/inactive mutants, knockdown/overexpression with phenotypic readouts (ILV formation, exosome secretion, lysosome fusion assays)\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, mutant analysis, knockdown, vesicle tracking), mechanistically rigorous, published in high-impact journal\",\n      \"pmids\": [\"32958903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Gapex-5/RME-6 contains a VPS9 domain that acts as a guanine nucleotide exchange factor (GEF) for RAB31. In adipocytes, overexpression of RAB31 blocks insulin-stimulated Glut4 translocation, while knockdown potentiates it. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, reducing RAB31 activity and permitting Glut4 vesicles to translocate to the cell surface.\",\n      \"method\": \"Yeast two-hybrid, overexpression/knockdown in adipocytes, glucose uptake assays, subcellular localization\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, GEF activity assay, KD/OE with specific phenotypic readout), replicated across assays\",\n      \"pmids\": [\"17189207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RIN3 specifically acts as a guanine nucleotide exchange factor (GEF) for RAB31 (but not RAB21), stimulating GTP-bound RAB31 formation in cell-free and in-cell GEF activity assays. Serine-to-alanine substitutions in the sequence between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on RAB31 but not RAB5. RIN3 colocalizes with RAB31 in enlarged vesicles and tubular structures in HeLa cells. RIN3 partially translocates CD-MPR from the TGN to peripheral vesicles in a RAB31-GEF-dependent manner.\",\n      \"method\": \"Cell-free and in-cell GEF activity assays, site-directed mutagenesis, colocalization by fluorescence microscopy, CD-MPR trafficking assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro GEF reconstitution, mutagenesis, and multiple cellular assays in a single rigorous study\",\n      \"pmids\": [\"21586568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Recombinant RAB31 expressed as a GST-fusion protein binds [35S]GTPγS in a Mg2+-dependent manner (optimal at 5 µM free Mg2+, inhibited at higher concentrations). RAB31 displays low steady-state GTPase activity. The Q64L GTPase-dead mutation does not abolish GTPase activity of RAB31 (unlike most small GTPases).\",\n      \"method\": \"GST-fusion protein expression, radiolabeled nucleotide binding assay, GTPase activity assay, site-directed mutagenesis\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay with mutagenesis, single lab, characterization paper\",\n      \"pmids\": [\"11784320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RAB31 is required for transport of mannose-6-phosphate receptors (specifically cation-dependent CD-MPR, but not CD63 or VSVG) from the TGN to endosomes. CD-MPR and RAB31 colocalize in TGN carriers containing clathrin and GGA1 coats. Constitutively active RAB31 redistributes CD-MPR from TGN to endosomes; dominant-negative RAB31 causes the reverse. siRNA depletion of RAB31 causes collapse of the Golgi apparatus.\",\n      \"method\": \"Dominant-active/inactive RAB31 mutants, siRNA knockdown, immunofluorescence colocalization, cargo trafficking assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (CA/DN mutants, siRNA, cargo tracking), specific cargo selectivity demonstrated\",\n      \"pmids\": [\"19345684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RAB31 interacts with OCRL-1 (a PI(4,5)P2 5-phosphatase) as shown by yeast two-hybrid, GST-RAB31 pulldown, and co-immunoprecipitation from oligodendrocyte lysates. RAB31 and OCRL-1 colocalize in TGN, post-TGN carriers, and endosomes. siRNA depletion of RAB31 causes TGN collapse and markedly decreases OCRL-1 levels in the TGN and endosomes, indicating RAB31 recruits OCRL-1 to TGN domains where carriers form.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, co-immunoprecipitation, siRNA knockdown, immunofluorescence colocalization\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — three independent binding assays plus functional knockdown with colocalization, multiple orthogonal methods\",\n      \"pmids\": [\"19795375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Endogenous RAB22B/RAB31 is largely localized to the TGN in HeLa cells. Expression of dominant-negative GDP-bound RAB22B (but not wild-type) specifically disrupts TGN46 localization and inhibits anterograde exit of VSVG from the TGN, implicating RAB31 in anterograde TGN membrane dynamics.\",\n      \"method\": \"Specific antibody, overexpression of dominant-negative mutant, immunofluorescence, VSVG transport assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative approach with specific phenotypic readout, multiple markers tested, single lab\",\n      \"pmids\": [\"17678623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RAB22B/RAB31 is expressed specifically in nestin/RC2-positive radial glia of the embryonic mouse brain and in GFAP-positive astrocytes of the adult brain. Silencing RAB22B in A431 cells causes abnormal trafficking of EGFR, Texas-red-EGF, and cation-independent M6PR. RAB22B associates with EGFR in a GTP-dependent manner as shown by affinity pulldown and co-immunoprecipitation.\",\n      \"method\": \"Specific antibody, immunofluorescence, siRNA knockdown, affinity pulldown, co-immunoprecipitation, receptor trafficking assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays plus KD with trafficking phenotype, single lab, two orthogonal binding methods\",\n      \"pmids\": [\"19725050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAB31 regulates trafficking of ligand-bound EGFR from early to late endosomes. Loss of RAB31 inhibits, and overexpression enhances, EGFR trafficking to late endosomes. RAB31 interacts with EGFR (by co-IP and affinity pulldown) and is recruited into a high-molecular-weight complex with EEA1 after EGF stimulation. Loss of EEA1 reduces RAB31-EGFR interaction and abolishes the effect of RAB31 on EGFR trafficking. Loss of GAPex5 also reduces RAB31-EGFR interaction.\",\n      \"method\": \"Co-immunoprecipitation, affinity pulldown, glycerol gradient sedimentation, siRNA knockdown, overexpression, pulse-chase EGFR trafficking assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, pulldown, sedimentation, KD/OE), specific rescue and epistasis assays, single lab\",\n      \"pmids\": [\"24644286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RAB31 is recruited to early-stage phagocytic cups in macrophages at the PI(4,5)P2-to-PI(3,4,5)P3 phosphoinositide transition and persists on PI(3)P-enriched phagosomes. RAB31-GTP recruits the signaling adaptor APPL2 at phagocytic cups. siRNA depletion of either RAB31 or APPL2 reduces FcγR-mediated phagocytosis, delays transition to PI(3,4,5)P3 and phagocytic cup closure, reduces PI3K/Akt signaling, and enhances p38 signaling.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, phosphoinositide reporters, FcγR-mediated phagocytosis assay, signaling pathway analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with functional knockdown, multiple orthogonal readouts (phosphoinositides, phagocytosis efficiency, signaling), single lab\",\n      \"pmids\": [\"25568335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MUC1-C forms a complex with ERα on the RAB31 promoter and activates RAB31 gene transcription in an estrogen-dependent manner in ER+ breast cancer cells. In turn, RAB31 attenuates lysosomal degradation of MUC1-C, creating an autoinductive loop. Expression of inactive RAB31(S20N) mutant in nonmalignant breast epithelial cells confirmed RAB31 regulates MUC1-C expression.\",\n      \"method\": \"Chromatin immunoprecipitation, promoter-reporter assay, dominant-negative mutant, lysosomal degradation assay, mammosphere formation assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional mutant plus degradation assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"22792175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAB31 is regulated by the transcription factor RUNX1, which binds the RAB31 promoter in megakaryocytic cells. Downregulation of RUNX1 or RAB31 (by siRNA or CRISPR/Cas9) causes striking enlargement of early endosomes (EEs), partially reversed by RAB31 reconstitution. RAB31 deficiency impairs trafficking of VWF (to α-granules), EGFR, and mannose-6-phosphate receptor at the level of EEs in megakaryocytic cells.\",\n      \"method\": \"Promoter-reporter assay, siRNA, CRISPR/Cas9 knockout, RAB31 reconstitution, immunofluorescence for EE markers, iPSC-derived megakaryocyte model\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with rescue, multiple cargo tracking, patient-derived iPSC model, multiple orthogonal methods\",\n      \"pmids\": [\"35839075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAB31 knockdown inhibits TGF-β receptor II complex endocytosis in hepatic stellate cells, impairing TGF-β/Smad signaling and preventing hepatic stellate cell activation. RAB31 is required as a prerequisite for TGF-βRII endocytosis-driven TGF-β signaling.\",\n      \"method\": \"Lentiviral knockdown, TGF-βRII endocytosis assay, Smad signaling analysis, mouse CCl4 fibrosis model\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with specific receptor endocytosis readout and signaling cascade, in vivo validation, single lab\",\n      \"pmids\": [\"35091093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The Shigella effector IpaH4.5 harbors TBC-like dual-finger motifs and exhibits potent RabGAP activity specifically toward RAB31, inactivating it. This disrupts CD-MPR transport from the Golgi to endosomes and attenuates lysosomal cathepsin B activity, allowing Shigella to escape lysosomal degradation. Intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity.\",\n      \"method\": \"GAP activity assay (in vitro), co-immunoprecipitation, confocal microscopy (MPR trafficking), Magic Red-RR cathepsin B activity assay, intracellular persistence assay\",\n      \"journal\": \"Journal of medical microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro GAP activity assay plus multiple functional cellular assays, mechanistically rigorous, single lab\",\n      \"pmids\": [\"34296983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The C-terminal hypervariable domain (HVD) of RAB31, together with the interswitch loop and N-terminal domain, constitutes a membrane targeting domain (MTD) that dictates Golgi (rather than early endosomal) localization of RAB31. Replacing the RAB31 HVD with the RAB22 HVD shifts RAB31 to early endosomes. RAB31 Golgi localization is further influenced by differential interaction with the early endosomal effector Rabenosyn-5, which stabilizes RAB22 at endosomes but does not stabilize RAB31 there.\",\n      \"method\": \"Domain-swap chimera mutants, live fluorescence microscopy, Rabenosyn-5 knockout cells, co-localization analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimera mutagenesis with localization readout, KO cells, single lab, multiple constructs tested\",\n      \"pmids\": [\"35863437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAB31 interacts with RAGE (receptor for advanced glycation end products) intracellular domain, identified by GST pulldown combined with mass spectrometry and confirmed by co-immunoprecipitation and immunostaining. This interaction is enhanced by glycation-serum stimulation and is associated with membrane redistribution of RAB31. RAB31 promotes RAGE endocytosis and inhibits AGE-induced β-cell apoptosis through the pAKT/BCL2 pathway.\",\n      \"method\": \"GST pulldown combined with mass spectrometry, co-immunoprecipitation, immunostaining, RAGE endocytosis assay, apoptosis assay\",\n      \"journal\": \"Endocrine journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pulldown with MS confirmation plus reciprocal Co-IP plus functional assay, single lab\",\n      \"pmids\": [\"35314532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NCSTN mutations reduce miR-30a-3p levels, which negatively regulates RAB31 expression. Enhanced RAB31 levels accelerate degradation of activated EGFR, leading to abnormal keratinocyte differentiation. The miR-30a-3p/RAB31/EGFR signaling axis was demonstrated in familial acne inversa patient samples and NcstnΔKC mice.\",\n      \"method\": \"miRNA microarray, Ncstn keratinocyte-specific knockout mice, luciferase reporter assay (miRNA target validation), EGFR degradation assay, keratinocyte differentiation assay\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — validated miRNA-target interaction plus functional EGFR degradation readout, mouse model, single lab\",\n      \"pmids\": [\"30120935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAB31 interacts with GLI1 as confirmed by co-immunoprecipitation and immunofluorescence in gastric cancer cells. RAB31 silencing suppresses cell viability, promotes cell cycle arrest, enhances apoptosis, and affects cell cycle/apoptotic proteins, effects mediated through GLI1.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, siRNA knockdown, cell viability/apoptosis assays, luciferase reporter assay (miRNA target validation)\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for interaction, functional assays without reconstitution; note: paper subsequently retracted (PMID 41114346)\",\n      \"pmids\": [\"30534536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAB31 is expressed in neural progenitor cells (NPCs). Silencing RAB31 hinders, while overexpression enhances, differentiation of NPCs to astrocytes, establishing a role for RAB31 in NPC fate determination.\",\n      \"method\": \"Primary NPC culture, siRNA knockdown, overexpression, differentiation assay with GFAP/nestin markers\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — KD/OE with phenotypic differentiation readout, no mechanistic pathway placement, single lab\",\n      \"pmids\": [\"24999186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAB31 interacts with MAPK6, and RAB31 knockdown reduces MAPK6 protein levels by promoting its degradation. MAPK6 overexpression restores the decreased migration potential caused by RAB31 knockdown, placing RAB31 upstream of MAPK6 in cervical cancer cell migration.\",\n      \"method\": \"Co-immunoprecipitation, MAPK6 degradation assay, overexpression rescue, migration assay, in vivo xenograft\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus degradation assay plus genetic rescue establishing epistasis, single lab\",\n      \"pmids\": [\"34975321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RAB31 overexpression in gastric cancer cells increases exosome secretion (number), while RAB31 depletion reduces both the number and size of secreted exosomes. Injection of exosomes derived from RAB31-overexpressing cells promotes pulmonary metastasis in vivo. PSMA1 was identified as an exosomal protein overexpressed in concert with RAB31.\",\n      \"method\": \"Exosome nanoparticle tracking analysis, electron microscopy, in vivo exosome injection/metastasis model, protein mass spectrometry\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — exosome quantification with multiple methods plus in vivo functional test, single lab\",\n      \"pmids\": [\"37222416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RAB31 in glioma-derived endothelial cells drives enrichment of MYO1C into extracellular vesicles (EVs). RAB31 knockdown reduces MYO1C enrichment in secretory EVs and attenuates promotion of glioma cell invasion by GhEC-EVs. This EV export mechanism is also dependent on RAB27B and FAS.\",\n      \"method\": \"siRNA knockdown, EV isolation, Western blot for MYO1C in EVs, invasion assay\",\n      \"journal\": \"FEBS open bio\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single KD approach with EV cargo tracking, no reconstitution, single lab\",\n      \"pmids\": [\"37953466\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RAB31 is a Rab5-subfamily small GTPase that cycles between GDP- and GTP-bound states under control of multiple GEFs (Gapex-5/RME-6, RIN3) and is regulated by EGFR-mediated phosphorylation; in its active (GTP-bound) form it localizes predominantly to the trans-Golgi network and early endosomes, where it mediates selective TGN-to-endosome transport of mannose-6-phosphate receptors (via recruitment of OCRL-1 and interaction with EEA1/GAPex5), drives ESCRT-independent intraluminal vesicle formation by engaging flotillin proteins in lipid raft microdomains, suppresses MVE-lysosome fusion by recruiting the RAB7-GAP TBC1D2B, promotes FcγR-mediated phagocytosis through APPL2 recruitment, facilitates TGF-βRII endocytosis to sustain TGF-β/Smad signaling, and regulates EGFR trafficking from early to late endosomes; its localization is determined by a membrane targeting domain comprising the C-terminal hypervariable domain and interswitch loop, and it is transcriptionally regulated by RUNX1 and ERα/MUC1-C complexes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAB31 is a Rab5-subfamily small GTPase that governs selective membrane trafficking between the trans-Golgi network (TGN), early endosomes, and multivesicular endosomes [#4, #14]. Its nucleotide cycle is driven by VPS9-domain GEFs: Gapex-5/RME-6, which couples RAB31 activity to insulin-regulated Glut4 vesicle translocation in adipocytes [#1], and RIN3, which specifically loads GTP onto RAB31 to mobilize CD-MPR out of the TGN [#2]. In its active form RAB31 directs TGN-to-endosome transport of mannose-6-phosphate receptors, recruiting the PI(4,5)P2 5-phosphatase OCRL-1 to TGN carriers and acting at clathrin/GGA1-coated domains; its depletion collapses the Golgi [#4, #5]. RAB31 distinguishes itself from the related RAB22 through a membrane targeting domain built from its C-terminal hypervariable domain, interswitch loop, and N-terminal region, which enforces Golgi over endosomal localization [#14]. On the endosomal/MVE axis, EGFR-phosphorylated RAB31 engages flotillins in lipid raft microdomains to drive ESCRT-independent intraluminal vesicle formation while recruiting the RAB7-GAP TBC1D2B to block MVE-lysosome fusion, thereby routing cargo toward exosome secretion [#0]; consistently, RAB31 controls EGFR transit from early to late endosomes via an EEA1- and Gapex-5-dependent complex [#8]. Beyond constitutive trafficking, RAB31 supports Fc\\u03b3R-mediated phagocytosis by recruiting APPL2 at phagocytic cups during the phosphoinositide transition [#9], sustains TGF-\\u03b2RII endocytosis required for TGF-\\u03b2/Smad signaling [#12], and its expression is transcriptionally driven by RUNX1 in megakaryocytes, where it is needed for early-endosomal trafficking of VWF, EGFR, and M6PR [#11]. RAB31 transcription is also activated by an ER\\u03b1/MUC1-C complex in ER+ breast cancer, forming an autoinductive loop in which RAB31 stabilizes MUC1-C against lysosomal degradation [#10]. The Shigella effector IpaH4.5 acts as a RAB31-specific RabGAP, inactivating it to subvert CD-MPR-dependent cathepsin trafficking and evade lysosomal killing [#13].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established the basic biochemistry of RAB31 as a guanine nucleotide-binding protein, defining it as a functional small GTPase with atypical catalytic properties.\",\n      \"evidence\": \"GST-fusion recombinant protein with radiolabeled GTP\\u03b3S binding and GTPase assays plus Q64L mutagenesis\",\n      \"pmids\": [\"11784320\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No effectors or cellular pathway assigned\", \"Atypical retention of activity by Q64L mutant left unexplained\", \"Single-lab in vitro characterization\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified the first physiological GEF (Gapex-5/RME-6) and a cellular role, linking RAB31 activity to insulin-regulated Glut4 vesicle trafficking and revealing its TGN localization.\",\n      \"evidence\": \"Yeast two-hybrid, GEF activity, and KD/OE with glucose uptake readouts in adipocytes; specific antibody localization to TGN with DN-mutant VSVG transport assay\",\n      \"pmids\": [\"17189207\", \"17678623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct cargo of RAB31 in Glut4 pathway not defined\", \"Connection between TGN dynamics and Glut4 retention unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined RAB31's core trafficking function: selective TGN-to-endosome transport of mannose-6-phosphate receptors and broader receptor sorting, with Golgi integrity dependent on RAB31.\",\n      \"evidence\": \"CA/DN mutants, siRNA, cargo trafficking and colocalization assays for CD-MPR; reciprocal binding and KD trafficking assays for EGFR/CI-M6PR in A431 cells\",\n      \"pmids\": [\"19345684\", \"19725050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cargo selectivity mechanism (why CD-MPR but not CD63/VSVG) unexplained\", \"Effectors mediating carrier formation not yet identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified OCRL-1 as a direct RAB31 effector recruited to TGN carrier-forming domains, linking RAB31 to phosphoinositide control at the TGN.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, co-IP, and siRNA depletion with colocalization in oligodendrocytes\",\n      \"pmids\": [\"19795375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of OCRL-1 phosphatase activity for carrier budding not directly tested\", \"Whether OCRL-1 recruitment is GTP-dependent not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated GEF specificity by reconstitution, establishing RIN3 as a RAB31-selective GEF that mobilizes CD-MPR from the TGN.\",\n      \"evidence\": \"Cell-free and in-cell GEF assays, site-directed mutagenesis, colocalization, and CD-MPR trafficking assay\",\n      \"pmids\": [\"21586568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signal activating RIN3 toward RAB31 unknown\", \"Relationship between RIN3 and Gapex-5 GEF inputs not reconciled\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed RAB31 in a transcription-trafficking feedback loop in breast cancer, where ER\\u03b1/MUC1-C activate RAB31 and RAB31 stabilizes MUC1-C against lysosomal degradation.\",\n      \"evidence\": \"ChIP, promoter-reporter, DN mutant (S20N), and lysosomal degradation assays in ER+ breast cancer cells\",\n      \"pmids\": [\"22792175\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RAB31 diverts MUC1-C from lysosomes not detailed\", \"Generality beyond ER+ context untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended RAB31 function to endosomal maturation, showing it routes ligand-bound EGFR from early to late endosomes via an EEA1/Gapex-5-containing complex.\",\n      \"evidence\": \"Co-IP, affinity pulldown, glycerol gradient sedimentation, KD/OE, and pulse-chase EGFR trafficking assays\",\n      \"pmids\": [\"24644286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the RAB31-EEA1-Gapex5 complex unresolved\", \"How RAB31 reconciles pro-degradation versus exosome-routing fates of EGFR not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed RAB31 as a coordinator of phagosome maturation, recruiting APPL2 at the phosphoinositide transition to support Fc\\u03b3R-mediated phagocytosis and PI3K/Akt signaling.\",\n      \"evidence\": \"Live-cell imaging with phosphoinositide reporters, siRNA, phagocytosis efficiency, and signaling pathway analysis in macrophages\",\n      \"pmids\": [\"25568335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether APPL2 recruitment is direct GTP-dependent binding not biochemically confirmed\", \"GEF driving RAB31 activation at the phagocytic cup unidentified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved a mechanism for ESCRT-independent exosome biogenesis, showing EGFR-phosphorylated RAB31 uses flotillins to form ILVs while recruiting TBC1D2B to inactivate RAB7 and block lysosomal fusion.\",\n      \"evidence\": \"Co-IP, domain mapping, DA/DN mutants, KD/OE with ILV formation, exosome secretion, and lysosome fusion assays\",\n      \"pmids\": [\"32958903\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The EGFR phosphosite(s) on RAB31 and their regulatory effect not fully mapped\", \"Balance between flotillin ILV pathway and canonical ESCRT routing unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed RAB31 is a target of bacterial subversion, with the Shigella RabGAP effector IpaH4.5 inactivating it to disrupt M6PR-dependent cathepsin delivery and evade lysosomal killing.\",\n      \"evidence\": \"In vitro GAP activity assay, co-IP, MPR trafficking microscopy, cathepsin B activity, and intracellular persistence assays\",\n      \"pmids\": [\"34296983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous host RabGAP for RAB31 still unknown\", \"Whether IpaH4.5 also affects RAB31's exosomal/endosomal roles untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the structural determinant of RAB31 organelle targeting and a transcriptional driver, while expanding its physiological roles to megakaryocyte granule biogenesis, TGF-\\u03b2 signaling, and RAGE endocytosis.\",\n      \"evidence\": \"Domain-swap chimeras and Rabenosyn-5 KO localization; RUNX1 promoter/CRISPR KO with cargo tracking in iPSC-megakaryocytes; KD TGF-\\u03b2RII endocytosis with CCl4 fibrosis model; GST-MS/co-IP with RAGE endocytosis and \\u03b2-cell apoptosis assays\",\n      \"pmids\": [\"35863437\", \"35839075\", \"35091093\", \"35314532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the HVD/interswitch MTD physically discriminates Golgi from endosomal membranes mechanistically unresolved\", \"Whether RAGE and TGF-\\u03b2RII handling share the EGFR endosomal machinery untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked RAB31-driven exosome biogenesis to tumor progression, showing RAB31 controls exosome number/size and selective EV cargo loading that promotes metastasis and invasion.\",\n      \"evidence\": \"Nanoparticle tracking, electron microscopy, mass spectrometry, and in vivo exosome injection/metastasis models in gastric cancer and glioma EC systems\",\n      \"pmids\": [\"37222416\", \"37953466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of selective cargo (PSMA1, MYO1C) sorting into RAB31-dependent EVs undefined\", \"Glioma EV study rests on single-KD evidence without reconstitution\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAB31 mechanistically partitions a single cargo (e.g. EGFR) between degradative late-endosome routing, exosomal secretion, and recycling, and which GEF/effector combinations select each fate, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model reconciling pro-degradation versus exosome-secretory RAB31 functions\", \"Spatiotemporal regulation of competing effectors (OCRL-1, flotillin, TBC1D2B, APPL2, EEA1) not integrated\", \"Structural basis of effector selection beyond the membrane targeting domain unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GTPase activity\", \"supporting_discovery_ids\": [3, 0, 2]},\n      {\"term_id\": \"molecular function regulator activity\", \"supporting_discovery_ids\": [0, 9, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [4, 5, 6, 14]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [4, 8, 11, 14]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [4, 5, 8, 11]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 12, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 13]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 20]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"FLOT1\", \"TBC1D2B\", \"OCRL\", \"EEA1\", \"EGFR\", \"APPL2\", \"RIN3\", \"GAPEX5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}