{"gene":"VAC14","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2008,"finding":"VAC14/Vac14 is composed entirely of HEAT repeats and functions as a scaffold protein for the PI(3,5)P2 regulatory complex, making direct contact with Fig4, Fab1, Vac7, and Atg18. A missense mutation (ingls mouse) in Vac14 prevents association with Fab1, generating a partial complex. Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2.","method":"Yeast and mouse genetic analysis, co-immunoprecipitation, structural prediction, analysis of ingls mouse mutant with biochemical characterization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction mapping with multiple binding partners, multiple orthogonal methods (genetic epistasis, co-IP, in vivo mouse model), replicated across yeast and mammalian systems","pmids":["19037259"],"is_preprint":false},{"year":2003,"finding":"In yeast, Vac14 is required for the vacuolar localization of the PI(3,5)P2 phosphatase Fig4; in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. Fig4 physically associates with Vac14 in a common membrane-associated complex. Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of Fig4.","method":"GFP localization microscopy, co-immunoprecipitation, in vitro phosphatase assay, yeast genetics (deletion mutants)","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiment with functional consequence, co-IP, in vitro assay, multiple orthogonal methods in single study","pmids":["14528018"],"is_preprint":false},{"year":2007,"finding":"Loss of Vac14 in mice results in near-complete loss of PI(3,5)P2 and defects in endosome-to-TGN retrograde trafficking, establishing Vac14 as an essential regulator of PI(3,5)P2 synthesis required for membrane trafficking in vivo.","method":"Vac14 knockout mouse, lipid analysis, membrane trafficking assays, vacuole morphology","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout mouse with defined biochemical and cellular phenotype, multiple readouts","pmids":["17956977"],"is_preprint":false},{"year":2002,"finding":"Vac14 is an upstream activator of Fab1-catalysed PI(3,5)P2 synthesis; vac14Δ cells make very little PI(3,5)P2 and fail to respond to hyperosmotic shock. FAB1 overexpression corrects vac14Δ defects. Vac14 is essential for regulated PI(3,5)P2 synthesis and for sorting of proteins to the vacuole lumen via multivesicular bodies.","method":"Yeast genetics (deletion mutants, overexpression), phosphoinositide lipid analysis, GFP trafficking assays, hyperosmotic shock","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (FAB1 overexpression rescues vac14Δ), lipid measurements, trafficking assays, multiple orthogonal methods","pmids":["12062051"],"is_preprint":false},{"year":2004,"finding":"Human VAC14 (ArPIKfyve) physically associates with PIKfyve, co-fractionates and co-localizes with it on intracellular membranes, and positively regulates PIKfyve lipid kinase activity. siRNA knockdown of hVac14 decreases PIKfyve kinase activity and PI(3,5)P2 levels; ectopic expression increases PIKfyve kinase activity and PI(3,5)P2.","method":"Co-immunoprecipitation, co-fractionation, siRNA knockdown, in vitro lipid kinase assay, intracellular lipid labeling","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, in vitro kinase assay, siRNA loss-of-function and overexpression gain-of-function with biochemical readout","pmids":["15542851"],"is_preprint":false},{"year":2007,"finding":"Mammalian VAC14 (ArPIKfyve) is part of a stable ternary complex with PIKfyve and Sac3 (the mammalian Fig4 ortholog). Sac3 co-fractionates and co-localizes with ArPIKfyve and PIKfyve. The ternary complex couples PI(3,5)P2 synthesis (PIKfyve) with turnover (Sac3) and regulates early endosome dynamics.","method":"Co-immunoprecipitation of endogenous proteins, subcellular fractionation, co-localization, in vitro phosphatase assay, siRNA knockdown with lipid measurements, in vitro endosome reconstitution assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — endogenous co-IP, in vitro phosphatase assay, in vitro reconstitution of carrier vesicle formation, multiple orthogonal methods","pmids":["17556371"],"is_preprint":false},{"year":2008,"finding":"ArPIKfyve (VAC14) organizes the PIKfyve-ArPIKfyve-Sac3 (PAS) complex through both homomeric (self-interaction via conserved C-terminal domain) and heteromeric interactions. ArPIKfyve is the principal organizer, interacting with both Sac3 and PIKfyve; Sac3 is permissive for maximal PIKfyve-ArPIKfyve association. Introduction of C-terminal ArPIKfyve peptide disassembles the PAS complex and reduces PIKfyve lipid kinase activity in vitro.","method":"Co-immunoprecipitation in transfected cells with single/double/triple combinations, in vitro PIKfyve lipid kinase assay, dominant-negative peptide competition, GLUT4 translocation assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — systematic co-IP with truncation/domain mutants, in vitro kinase assay, functional readout (GLUT4 translocation), multiple orthogonal approaches","pmids":["18950639"],"is_preprint":false},{"year":2009,"finding":"Within the PAS complex, the Cpn60_TCP1 domain of PIKfyve is a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Sac3 assembled in the PAS complex remains an active PI(3,5)P2 phosphatase; phosphatase-deficient Sac3(D488A) eliminates the vacuolar phenotype caused by kinase-deficient PIKfyve, demonstrating that Sac3 activity within the complex drives PI(3,5)P2 turnover.","method":"Biochemical domain mapping with truncation/point mutants, vacuole morphology assay as functional readout, co-expression of catalytic mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — systematic mutagenesis of all three complex components, multiple functional readouts, internal controls with catalytic dead mutants","pmids":["19840946"],"is_preprint":false},{"year":2010,"finding":"ArPIKfyve (VAC14) stabilizes Sac3 protein by protecting it from rapid proteasome-dependent degradation. Knockdown of ArPIKfyve reduces Sac3 steady-state levels; overexpression of ArPIKfyve extends Sac3 half-life (from ~18.8 min). The pathogenic CMT4J mutation Sac3(I41T) cannot be stabilized by ArPIKfyve, suggesting failure of this stability mechanism underlies CMT4J pathogenesis.","method":"Cycloheximide chase assay, proteasome inhibitor experiments, siRNA knockdown, overexpression, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — cycloheximide chase with proteasome inhibitor, gain- and loss-of-function, disease mutant validation, multiple orthogonal methods","pmids":["20630877"],"is_preprint":false},{"year":2007,"finding":"ArPIKfyve (VAC14) physically associates with PIKfyve in 3T3-L1 adipocytes in an insulin-independent manner. Loss of either ArPIKfyve or PIKfyve by siRNA depletes PI(3,5)P2 and reduces insulin-stimulated glucose uptake, GLUT4 surface accumulation, and Akt phosphorylation, establishing the ArPIKfyve-PIKfyve-PI(3,5)P2 axis as required for insulin-regulated GLUT4 translocation.","method":"siRNA knockdown, co-immunoprecipitation, in vitro lipid kinase assay on isolated membranes, glucose uptake assay, GLUT4 surface quantification","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP in physiologically relevant cell type, siRNA knockdown of both components with multiple functional readouts, in vitro kinase assay","pmids":["17475247"],"is_preprint":false},{"year":2012,"finding":"VAC14 localizes to endocytic organelles in fibroblasts and neurons, and shows pronounced synaptic localization in hippocampal neurons. Loss of VAC14 enhances miniature excitatory postsynaptic current amplitude and increases surface levels of AMPA receptor subunit GluA2, due to diminished regulated endocytosis of AMPA receptors. Re-introduction of VAC14 in postsynaptic cells reverses these effects.","method":"Immunofluorescence localization, electrophysiology (mEPSC recording) in Vac14-/- neurons, surface biotinylation of AMPA receptors, rescue by postsynaptic VAC14 re-expression","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous localization, knockout electrophysiology, surface receptor quantification, and specific postsynaptic rescue experiment with multiple orthogonal methods","pmids":["22842785"],"is_preprint":false},{"year":2013,"finding":"Vac14 forms a homodimer/multimer, and Vac14 multimerization is prerequisite for Fab1 complex assembly. Monomeric Vac14 mutants (with mutations in conserved C-terminal motifs) fail to interact with Fab1 or Fig4, have enlarged vacuoles, and cannot generate PI(3,5)P2 in response to hyperosmotic shock, demonstrating that Vac14 self-interaction is the first molecular event in Fab1 complex assembly.","method":"Co-immunoprecipitation with Vac14 mutants, yeast vacuole morphology, hyperosmotic shock PI(3,5)P2 synthesis assay, two-hybrid analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic C-terminal mutagenesis, multiple orthogonal methods (co-IP, lipid analysis, morphology), clear epistatic ordering","pmids":["23389034"],"is_preprint":false},{"year":2006,"finding":"VAC14 interacts with the PDZ domain of neuronal nitric oxide synthase (nNOS) through a novel internal PDZ-recognition motif that is beta-finger independent. Deletion mapping and mutational analysis of Vac14 defined the essential residues of this internal motif.","method":"In vitro binding assays with Vac14 deletion constructs, mutational analysis of PDZ interaction motif","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding assay with multiple deletion/point mutants characterizing the interaction, single lab, no reciprocal validation in cellular context","pmids":["17161399"],"is_preprint":false},{"year":2004,"finding":"In 3T3-L1 adipocytes, hyperosmotic stress induces a marked PI(3,5)P2 increase that is fully dependent on the ArPIKfyve-PIKfyve axis; siRNA depletion of either ArPIKfyve or PIKfyve abolishes the hyperosmotically activated PI(3,5)P2 rise.","method":"siRNA knockdown of ArPIKfyve and PIKfyve, 32P lipid labeling and measurement in 3T3-L1 adipocytes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown of both components with biochemical lipid readout, single lab, single method per component","pmids":["15546865"],"is_preprint":false},{"year":2017,"finding":"Decreased VAC14 expression increases plasma membrane cholesterol, which facilitates Salmonella docking and invasion. The mechanism linking VAC14 to cholesterol homeostasis at the plasma membrane was established through siRNA knockdown and cholesterol measurement experiments.","method":"siRNA knockdown of VAC14, plasma membrane cholesterol quantification, Salmonella invasion assay, zebrafish infection model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined biochemical mechanism (cholesterol), multiple model systems (cells and zebrafish), single lab","pmids":["28827342"],"is_preprint":false},{"year":2014,"finding":"VAC14 interacts with Rab9 and the Rab7 GAP TBC1D15, linking the VAC14 complex to endolysosomal vesicular transport regulation. These interactions were validated by reciprocal co-immunoprecipitation and proximity ligation assay.","method":"Protein affinity purification combined with MudPIT mass spectrometry, reciprocal co-immunoprecipitation, proximity ligation assay","journal":"Molecular & cellular proteomics : MCP","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS interactome with orthogonal validation by reciprocal co-IP and PLA, single lab","pmids":["24578385"],"is_preprint":false},{"year":2015,"finding":"The ArPIKfyve-Sac3 heterodimer (VAC14-Sac3 binary complex) interacts with Synphilin-1 (Sph1) in brain, as identified by mass spectrometry. Modulation of ArPIKfyve/Sac3 levels alters aggregation properties of Sph1-GFP through mechanisms involving increased cytosolic partitioning and basal autophagy-mediated clearance.","method":"Mass spectrometry of brain-derived ArPIKfyve-Sac3 interactors, RNA silencing and overexpression in mammalian cell lines including primary neurons, GFP aggregation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS identification with functional validation by modulation experiments, single lab, multiple cell types","pmids":["26405034"],"is_preprint":false},{"year":2021,"finding":"BioID proximity labeling of Vac14 identified COPI subunit COPB1 and the GTPase Arf1 (required for COPI assembly) as proximal interactors of Vac14, validated by proximity ligation assay, suggesting a functional link between Vac14 and Golgi-associated COPI complex in endosomal dynamics.","method":"BioID proximity-dependent biotin labeling, mass spectrometry, proximity ligation assay","journal":"Journal of proteome research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — proximity labeling (not direct interaction), PLA validation, single lab, functional consequence not directly tested","pmids":["34554760"],"is_preprint":false},{"year":2025,"finding":"VAC14 forms a star-shaped pentameric scaffold (medium-resolution structure). Atomic-resolution AlphaFold2 prediction combined with cryo-EM maps revealed that disease-linked mutations reside at VAC14-VAC14 interfaces. Mutations disrupting VAC14 oligomerization cause defects in PI(3,5)P2 generation, VAC14 localization, and Fab1/PIKfyve activity. In human VAC14 KO cells, patient mutations are defective in PIKfyve-VAC14-FIG4 complex formation (pull-down), VAC14 oligomerization (fluorescence-detection size-exclusion chromatography), and colocalization with VPS35-positive endosomes.","method":"CryoEM structure, AlphaFold2 modeling, yeast genetics with oligomerization interface mutants, lipid analysis, pull-down assay in human KO cells, fluorescence-detection size-exclusion chromatography, colocalization microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural data combined with mutagenesis in both yeast and human cells, multiple orthogonal methods (structure, lipid assay, pull-down, FSEC, localization)","pmids":["40305106"],"is_preprint":false},{"year":2016,"finding":"VAC14 variants in the dimerization domain (p.Ala582Ser/p.Ser583Leu) cause pediatric neurological disease with PI(3,5)P2 deficiency. Vacuolization of patient fibroblasts was rescued by transfection of wild-type VAC14 cDNA, establishing that the patient variants are loss-of-function.","method":"Exome sequencing, patient fibroblast vacuole assay, rescue by wild-type VAC14 transfection","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional rescue experiment in patient fibroblasts confirming loss-of-function, single cellular assay, two unrelated families","pmids":["27292112"],"is_preprint":false},{"year":2023,"finding":"PI(3,5)P2 synthesized by the FIG4-VAC14 pathway inhibits the lysosomal chloride transporter ClC-7. Knockout of CLCN7 corrects lysosomal swelling and partially corrects lysosomal hyperacidification in FIG4 null cells, and dominant-negative CLCN7 in Fig4 null mice improved growth, neurological function, and lifespan by 20%, placing VAC14/FIG4-dependent PI(3,5)P2 upstream of ClC-7 in lysosomal regulation.","method":"CLCN7/CLCN6 knockout in FIG4 null cells, dominant-negative CLCN7 in Fig4 null mouse, lysosomal morphology and pH assays, in vivo phenotyping","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with knockout and dominant-negative in both cell and mouse models, multiple phenotypic readouts, functional pathway ordering","pmids":["37363915"],"is_preprint":false},{"year":2020,"finding":"VAC14 homodimerization can be altered by missense mutations at the C-terminal dimerization domain; p.Leu648Phe and p.Ala562Val mutations enhance VAC14 homodimer formation compared to wildtype, while p.Arg623His does not, as measured in stably transfected SH-SY5Y cells.","method":"Stable overexpression in SH-SY5Y cells, co-immunoprecipitation to assess homodimer formation, patient fibroblast analysis","journal":"Parkinsonism & related disorders","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP based homodimerization assay, single lab, no functional consequence directly tested for homodimer enhancement","pmids":["32949958"],"is_preprint":false},{"year":2016,"finding":"siRNA knockdown of VAC14 in stem cell-derived peripheral neuronal cells increased docetaxel sensitivity, measured by decreased neurite processes and branches, indicating VAC14 is required for peripheral neuronal resilience to taxane-induced damage.","method":"siRNA knockdown in stem cell-derived peripheral neurons, neurite morphology quantification after docetaxel treatment","journal":"Clinical cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single siRNA knockdown assay with morphological readout, single lab, no direct molecular mechanism identified","pmids":["27143689"],"is_preprint":false},{"year":2024,"finding":"Under ethanol stress in yeast, Vac14 dissociates from Atg18, its interaction partner in the Fab1 complex; this dissociation is linked to reduced PI(3,5)P2 levels caused by Fab1 downregulation, resulting in Atg18 membrane detachment and vacuole fusion.","method":"Fluorescence microscopy with PI(3,5)P2 sensor, genetic analysis with hyperactive Fab1 mutant, co-localization of Vac14 and Atg18","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, primarily indirect evidence from sensor redistribution and fluorescence imaging, Vac14-Atg18 interaction inferred from colocalization loss","pmids":[],"is_preprint":true}],"current_model":"VAC14 (ArPIKfyve) functions as a HEAT-repeat scaffold protein that forms a star-shaped pentameric complex, organizing the PIKfyve lipid kinase and the FIG4/Sac3 PI(3,5)P2 phosphatase into a ternary PAS complex on endosomal membranes; VAC14 oligomerization is the prerequisite step for complex assembly, VAC14 directly activates PIKfyve kinase activity, stabilizes FIG4/Sac3 from proteasomal degradation, and directs FIG4 localization to the vacuole/endosome, thereby coordinating the synthesis and turnover of PI(3,5)P2 to regulate endolysosomal trafficking, AMPA receptor endocytosis at synapses, GLUT4 translocation in adipocytes, and lysosomal homeostasis via downstream inhibition of the ClC-7 chloride transporter."},"narrative":{"mechanistic_narrative":"VAC14 is a HEAT-repeat scaffold protein that organizes the synthesis and turnover of the signaling lipid PI(3,5)P2 on endosomal/vacuolar membranes by assembling the PIKfyve(Fab1) lipid kinase and the FIG4/Sac3 phosphatase into a single ternary complex, thereby governing endolysosomal membrane trafficking [PMID:19037259, PMID:17556371]. VAC14 self-associates into a star-shaped pentameric scaffold, and this oligomerization is the prerequisite first molecular event for assembly of the kinase–phosphatase complex; monomeric or oligomerization-defective mutants fail to bind Fab1/FIG4, mislocalize, and cannot generate PI(3,5)P2 [PMID:23389034, PMID:40305106]. Within the assembled complex VAC14 positively regulates PIKfyve/Fab1 kinase activity and PI(3,5)P2 production [PMID:12062051, PMID:15542851, PMID:18950639], directs FIG4/Sac3 to the vacuole/endosome, and stabilizes Sac3 against proteasomal degradation [PMID:14528018, PMID:20630877]. Loss of VAC14 collapses cellular PI(3,5)P2 and disrupts endosome-to-TGN retrograde trafficking and vacuolar homeostasis [PMID:17956977]. Through this lipid output VAC14 controls multiple physiological processes: regulated endocytosis of AMPA receptors at synapses [PMID:22842785], insulin-stimulated GLUT4 translocation in adipocytes [PMID:17475247], and lysosomal size and acidification via PI(3,5)P2-dependent inhibition of the chloride transporter ClC-7 [PMID:37363915]. Biallelic and dimerization-domain mutations in VAC14 cause a pediatric neurological disease characterized by PI(3,5)P2 deficiency and cellular vacuolization that is rescued by wild-type VAC14 [PMID:27292112].","teleology":[{"year":2002,"claim":"Established that VAC14 acts genetically upstream of the Fab1 kinase, answering whether VAC14 is a regulator rather than a catalytic component of PI(3,5)P2 synthesis.","evidence":"Yeast deletion/overexpression genetics with phosphoinositide measurement and hyperosmotic shock, where FAB1 overexpression rescues vac14Δ","pmids":["12062051"],"confidence":"High","gaps":["Molecular basis of activation not defined","Did not resolve whether VAC14 acts on the kinase directly or through other partners"]},{"year":2003,"claim":"Showed VAC14 dually controls PI(3,5)P2 metabolism by directing the phosphatase Fig4 to the vacuole while also activating the Fab1 kinase, revealing coordination of synthesis and turnover.","evidence":"GFP localization, co-IP, in vitro phosphatase assay and deletion genetics in yeast","pmids":["14528018"],"confidence":"High","gaps":["Stoichiometry of the membrane complex unknown","Mechanism of Fig4 recruitment not defined at residue level"]},{"year":2004,"claim":"Extended the model to mammals, showing human VAC14 physically binds and activates PIKfyve, establishing functional conservation of the scaffold-kinase relationship.","evidence":"Reciprocal co-IP, co-fractionation, siRNA knockdown and in vitro lipid kinase assay; plus adipocyte hyperosmotic PI(3,5)P2 measurements","pmids":["15542851","15546865"],"confidence":"High","gaps":["Did not yet define the phosphatase arm in mammals","Domains mediating binding not mapped"]},{"year":2007,"claim":"Defined the in vivo requirement for VAC14 and the full mammalian ternary (PAS) complex, demonstrating that VAC14 couples PI(3,5)P2 synthesis (PIKfyve) with turnover (Sac3) to drive endosomal dynamics.","evidence":"Vac14 knockout mouse with lipid and retrograde-trafficking phenotypes; endogenous co-IP, fractionation, in vitro phosphatase and endosome reconstitution assays","pmids":["17956977","17556371","17475247"],"confidence":"High","gaps":["Structural architecture of the complex unknown","How synthesis and turnover are temporally balanced not resolved"]},{"year":2008,"claim":"Identified VAC14 as the central organizer of the PAS complex via homomeric and heteromeric contacts, answering which subunit nucleates assembly and showing its C-terminus is required for kinase activation.","evidence":"HEAT-repeat scaffold characterization with ingls mouse mutant; systematic co-IP with truncation mutants, in vitro kinase assay, dominant-negative peptide competition and GLUT4 readout","pmids":["19037259","18950639"],"confidence":"High","gaps":["Oligomeric state not quantified","Atomic structure of interfaces not available"]},{"year":2009,"claim":"Mapped the PIKfyve Cpn60_TCP1 domain as the docking site for the VAC14-Sac3 subcomplex and confirmed that Sac3 phosphatase activity within the complex drives PI(3,5)P2 turnover.","evidence":"Domain mapping with truncation/point mutants and vacuole morphology readout using catalytic-dead Sac3 and PIKfyve mutants","pmids":["19840946"],"confidence":"High","gaps":["Spatial relationship of kinase and phosphatase active sites unresolved","Regulatory switching between synthesis and turnover not addressed"]},{"year":2010,"claim":"Revealed a protein-stability function for VAC14, showing it protects Sac3 from proteasomal degradation, and linked failure of this mechanism to CMT4J disease via an unstabilizable Sac3 mutant.","evidence":"Cycloheximide chase with proteasome inhibitors, siRNA/overexpression and co-IP with the Sac3(I41T) disease mutant","pmids":["20630877"],"confidence":"High","gaps":["Degradation pathway/E3 ligase not identified","Whether stabilization requires full complex assembly not tested"]},{"year":2012,"claim":"Connected VAC14 to neuronal physiology, demonstrating it controls AMPA receptor surface levels through regulated endocytosis at synapses.","evidence":"Endogenous localization, mEPSC electrophysiology in Vac14-/- neurons, AMPA receptor surface biotinylation and postsynaptic rescue","pmids":["22842785"],"confidence":"High","gaps":["Direct lipid-to-receptor-trafficking link not mechanistically dissected","Identity of relevant endocytic machinery not defined"]},{"year":2013,"claim":"Ordered complex assembly by showing VAC14 self-multimerization is the obligatory first step that precedes recruitment of Fab1 and Fig4.","evidence":"Co-IP with C-terminal Vac14 mutants, two-hybrid, vacuole morphology and hyperosmotic PI(3,5)P2 assays in yeast","pmids":["23389034"],"confidence":"High","gaps":["Exact oligomer number not determined","Trigger initiating multimerization unknown"]},{"year":2016,"claim":"Provided human disease causation, showing dimerization-domain VAC14 variants produce PI(3,5)P2 deficiency and cellular vacuolization rescuable by wild-type VAC14.","evidence":"Exome sequencing in two families with patient fibroblast vacuole rescue","pmids":["27292112"],"confidence":"Medium","gaps":["Single cellular phenotype assayed","Tissue-specific basis of neurological presentation not established"]},{"year":2023,"claim":"Placed VAC14/FIG4-derived PI(3,5)P2 upstream of the lysosomal chloride transporter ClC-7, defining a downstream effector for lysosomal size and pH control.","evidence":"CLCN7 knockout in FIG4-null cells and dominant-negative CLCN7 in Fig4-null mice with lysosomal and in vivo phenotyping","pmids":["37363915"],"confidence":"High","gaps":["Whether PI(3,5)P2 inhibits ClC-7 directly or via intermediaries not resolved","Generalizability beyond FIG4-null context untested for VAC14 alone"]},{"year":2025,"claim":"Delivered structural understanding, showing VAC14 forms a star-shaped pentameric scaffold whose interface mutations disrupt oligomerization, complex formation, localization and PI(3,5)P2 generation, unifying the assembly and disease models.","evidence":"Cryo-EM, AlphaFold2 modeling, oligomerization-interface mutagenesis in yeast and human KO cells, lipid analysis, pull-down, FSEC and VPS35-endosome colocalization","pmids":["40305106"],"confidence":"High","gaps":["High-resolution structure of full kinase-phosphatase-loaded complex not solved","Dynamics of assembly/disassembly during signaling not captured"]},{"year":null,"claim":"How VAC14 complex assembly and PI(3,5)P2 output are dynamically regulated by upstream signals and connected to its peripheral partners (nNOS, Rab9/TBC1D15, Synphilin-1, COPI/Arf1, cholesterol homeostasis) remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Functional significance of non-core partners largely untested","No mechanism linking VAC14 to plasma-membrane cholesterol established at molecular level","Signal-dependent regulation of pentamer assembly unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,6,11,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,4,6,8]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,18]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,5,10,18]},{"term_id":"GO:0005773","term_label":"vacuole","supporting_discovery_ids":[1,3,7]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[20]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,5,10]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,4,9]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[2,1]}],"complexes":["PIKfyve-VAC14-FIG4/Sac3 (PAS) complex","Fab1 PI(3,5)P2 regulatory complex"],"partners":["PIKFYVE","FIG4","VAC7","ATG18","RAB9A","TBC1D15","NOS1","SNCAIP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q08AM6","full_name":"Protein VAC14 homolog","aliases":["Tax1-binding protein 2"],"length_aa":782,"mass_kda":88.0,"function":"Scaffold protein component of the PI(3,5)P2 regulatory complex which regulates both the synthesis and turnover of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2). Pentamerizes into a star-shaped structure and nucleates the assembly of the complex. The pentamer binds a single copy each of PIKFYVE and FIG4 and coordinates both PIKfyve kinase activity and FIG4 phosphatase activity, being required to maintain normal levels of phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 5-phosphate (PtdIns(5)P) (PubMed:33098764). Plays a role in the biogenesis of endosome carrier vesicles (ECV) / multivesicular bodies (MVB) transport intermediates from early endosomes","subcellular_location":"Endosome membrane; Microsome membrane","url":"https://www.uniprot.org/uniprotkb/Q08AM6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VAC14","classification":"Not Classified","n_dependent_lines":115,"n_total_lines":1208,"dependency_fraction":0.09519867549668874},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/VAC14","total_profiled":1310},"omim":[{"mim_id":"617054","title":"STRIATONIGRAL DEGENERATION, CHILDHOOD-ONSET; SNDC","url":"https://www.omim.org/entry/617054"},{"mim_id":"611228","title":"CHARCOT-MARIE-TOOTH DISEASE, DEMYELINATING, TYPE 4J; CMT4J","url":"https://www.omim.org/entry/611228"},{"mim_id":"609414","title":"PHOSPHOINOSITIDE KINASE, FYVE FINGER-CONTAINING; PIKFYVE","url":"https://www.omim.org/entry/609414"},{"mim_id":"609390","title":"FIG4 PHOSPHOINOSITIDE 5-PHOSPHATASE; FIG4","url":"https://www.omim.org/entry/609390"},{"mim_id":"604632","title":"VAC14 COMPONENT OF PIKFYVE COMPLEX; VAC14","url":"https://www.omim.org/entry/604632"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/VAC14"},"hgnc":{"alias_symbol":["FLJ10305","ArPIKfyve"],"prev_symbol":["TAX1BP2"]},"alphafold":{"accession":"Q08AM6","domains":[{"cath_id":"1.25.10.10","chopping":"2-170","consensus_level":"medium","plddt":93.2782,"start":2,"end":170},{"cath_id":"1.25.10","chopping":"398-475","consensus_level":"medium","plddt":94.1632,"start":398,"end":475},{"cath_id":"-","chopping":"629-724_745-782","consensus_level":"medium","plddt":87.2781,"start":629,"end":782}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08AM6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q08AM6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q08AM6-F1-predicted_aligned_error_v6.png","plddt_mean":83.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VAC14","jax_strain_url":"https://www.jax.org/strain/search?query=VAC14"},"sequence":{"accession":"Q08AM6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q08AM6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q08AM6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q08AM6"}},"corpus_meta":[{"pmid":"19037259","id":"PMC_19037259","title":"VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P(2) in yeast and mouse.","date":"2008","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/19037259","citation_count":198,"is_preprint":false},{"pmid":"17956977","id":"PMC_17956977","title":"Loss of Vac14, a regulator of the signaling lipid phosphatidylinositol 3,5-bisphosphate, results in neurodegeneration in mice.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17956977","citation_count":194,"is_preprint":false},{"pmid":"14528018","id":"PMC_14528018","title":"Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase.","date":"2003","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/14528018","citation_count":163,"is_preprint":false},{"pmid":"17556371","id":"PMC_17556371","title":"Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. 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A missense mutation (ingls mouse) in Vac14 prevents association with Fab1, generating a partial complex. Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2.\",\n      \"method\": \"Yeast and mouse genetic analysis, co-immunoprecipitation, structural prediction, analysis of ingls mouse mutant with biochemical characterization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction mapping with multiple binding partners, multiple orthogonal methods (genetic epistasis, co-IP, in vivo mouse model), replicated across yeast and mammalian systems\",\n      \"pmids\": [\"19037259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In yeast, Vac14 is required for the vacuolar localization of the PI(3,5)P2 phosphatase Fig4; in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. Fig4 physically associates with Vac14 in a common membrane-associated complex. Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of Fig4.\",\n      \"method\": \"GFP localization microscopy, co-immunoprecipitation, in vitro phosphatase assay, yeast genetics (deletion mutants)\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiment with functional consequence, co-IP, in vitro assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"14528018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Loss of Vac14 in mice results in near-complete loss of PI(3,5)P2 and defects in endosome-to-TGN retrograde trafficking, establishing Vac14 as an essential regulator of PI(3,5)P2 synthesis required for membrane trafficking in vivo.\",\n      \"method\": \"Vac14 knockout mouse, lipid analysis, membrane trafficking assays, vacuole morphology\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout mouse with defined biochemical and cellular phenotype, multiple readouts\",\n      \"pmids\": [\"17956977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Vac14 is an upstream activator of Fab1-catalysed PI(3,5)P2 synthesis; vac14Δ cells make very little PI(3,5)P2 and fail to respond to hyperosmotic shock. FAB1 overexpression corrects vac14Δ defects. Vac14 is essential for regulated PI(3,5)P2 synthesis and for sorting of proteins to the vacuole lumen via multivesicular bodies.\",\n      \"method\": \"Yeast genetics (deletion mutants, overexpression), phosphoinositide lipid analysis, GFP trafficking assays, hyperosmotic shock\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (FAB1 overexpression rescues vac14Δ), lipid measurements, trafficking assays, multiple orthogonal methods\",\n      \"pmids\": [\"12062051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human VAC14 (ArPIKfyve) physically associates with PIKfyve, co-fractionates and co-localizes with it on intracellular membranes, and positively regulates PIKfyve lipid kinase activity. siRNA knockdown of hVac14 decreases PIKfyve kinase activity and PI(3,5)P2 levels; ectopic expression increases PIKfyve kinase activity and PI(3,5)P2.\",\n      \"method\": \"Co-immunoprecipitation, co-fractionation, siRNA knockdown, in vitro lipid kinase assay, intracellular lipid labeling\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, in vitro kinase assay, siRNA loss-of-function and overexpression gain-of-function with biochemical readout\",\n      \"pmids\": [\"15542851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mammalian VAC14 (ArPIKfyve) is part of a stable ternary complex with PIKfyve and Sac3 (the mammalian Fig4 ortholog). Sac3 co-fractionates and co-localizes with ArPIKfyve and PIKfyve. The ternary complex couples PI(3,5)P2 synthesis (PIKfyve) with turnover (Sac3) and regulates early endosome dynamics.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, subcellular fractionation, co-localization, in vitro phosphatase assay, siRNA knockdown with lipid measurements, in vitro endosome reconstitution assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — endogenous co-IP, in vitro phosphatase assay, in vitro reconstitution of carrier vesicle formation, multiple orthogonal methods\",\n      \"pmids\": [\"17556371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ArPIKfyve (VAC14) organizes the PIKfyve-ArPIKfyve-Sac3 (PAS) complex through both homomeric (self-interaction via conserved C-terminal domain) and heteromeric interactions. ArPIKfyve is the principal organizer, interacting with both Sac3 and PIKfyve; Sac3 is permissive for maximal PIKfyve-ArPIKfyve association. Introduction of C-terminal ArPIKfyve peptide disassembles the PAS complex and reduces PIKfyve lipid kinase activity in vitro.\",\n      \"method\": \"Co-immunoprecipitation in transfected cells with single/double/triple combinations, in vitro PIKfyve lipid kinase assay, dominant-negative peptide competition, GLUT4 translocation assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — systematic co-IP with truncation/domain mutants, in vitro kinase assay, functional readout (GLUT4 translocation), multiple orthogonal approaches\",\n      \"pmids\": [\"18950639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Within the PAS complex, the Cpn60_TCP1 domain of PIKfyve is a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Sac3 assembled in the PAS complex remains an active PI(3,5)P2 phosphatase; phosphatase-deficient Sac3(D488A) eliminates the vacuolar phenotype caused by kinase-deficient PIKfyve, demonstrating that Sac3 activity within the complex drives PI(3,5)P2 turnover.\",\n      \"method\": \"Biochemical domain mapping with truncation/point mutants, vacuole morphology assay as functional readout, co-expression of catalytic mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — systematic mutagenesis of all three complex components, multiple functional readouts, internal controls with catalytic dead mutants\",\n      \"pmids\": [\"19840946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ArPIKfyve (VAC14) stabilizes Sac3 protein by protecting it from rapid proteasome-dependent degradation. Knockdown of ArPIKfyve reduces Sac3 steady-state levels; overexpression of ArPIKfyve extends Sac3 half-life (from ~18.8 min). The pathogenic CMT4J mutation Sac3(I41T) cannot be stabilized by ArPIKfyve, suggesting failure of this stability mechanism underlies CMT4J pathogenesis.\",\n      \"method\": \"Cycloheximide chase assay, proteasome inhibitor experiments, siRNA knockdown, overexpression, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cycloheximide chase with proteasome inhibitor, gain- and loss-of-function, disease mutant validation, multiple orthogonal methods\",\n      \"pmids\": [\"20630877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ArPIKfyve (VAC14) physically associates with PIKfyve in 3T3-L1 adipocytes in an insulin-independent manner. Loss of either ArPIKfyve or PIKfyve by siRNA depletes PI(3,5)P2 and reduces insulin-stimulated glucose uptake, GLUT4 surface accumulation, and Akt phosphorylation, establishing the ArPIKfyve-PIKfyve-PI(3,5)P2 axis as required for insulin-regulated GLUT4 translocation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, in vitro lipid kinase assay on isolated membranes, glucose uptake assay, GLUT4 surface quantification\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP in physiologically relevant cell type, siRNA knockdown of both components with multiple functional readouts, in vitro kinase assay\",\n      \"pmids\": [\"17475247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VAC14 localizes to endocytic organelles in fibroblasts and neurons, and shows pronounced synaptic localization in hippocampal neurons. Loss of VAC14 enhances miniature excitatory postsynaptic current amplitude and increases surface levels of AMPA receptor subunit GluA2, due to diminished regulated endocytosis of AMPA receptors. Re-introduction of VAC14 in postsynaptic cells reverses these effects.\",\n      \"method\": \"Immunofluorescence localization, electrophysiology (mEPSC recording) in Vac14-/- neurons, surface biotinylation of AMPA receptors, rescue by postsynaptic VAC14 re-expression\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous localization, knockout electrophysiology, surface receptor quantification, and specific postsynaptic rescue experiment with multiple orthogonal methods\",\n      \"pmids\": [\"22842785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Vac14 forms a homodimer/multimer, and Vac14 multimerization is prerequisite for Fab1 complex assembly. Monomeric Vac14 mutants (with mutations in conserved C-terminal motifs) fail to interact with Fab1 or Fig4, have enlarged vacuoles, and cannot generate PI(3,5)P2 in response to hyperosmotic shock, demonstrating that Vac14 self-interaction is the first molecular event in Fab1 complex assembly.\",\n      \"method\": \"Co-immunoprecipitation with Vac14 mutants, yeast vacuole morphology, hyperosmotic shock PI(3,5)P2 synthesis assay, two-hybrid analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic C-terminal mutagenesis, multiple orthogonal methods (co-IP, lipid analysis, morphology), clear epistatic ordering\",\n      \"pmids\": [\"23389034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"VAC14 interacts with the PDZ domain of neuronal nitric oxide synthase (nNOS) through a novel internal PDZ-recognition motif that is beta-finger independent. Deletion mapping and mutational analysis of Vac14 defined the essential residues of this internal motif.\",\n      \"method\": \"In vitro binding assays with Vac14 deletion constructs, mutational analysis of PDZ interaction motif\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding assay with multiple deletion/point mutants characterizing the interaction, single lab, no reciprocal validation in cellular context\",\n      \"pmids\": [\"17161399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In 3T3-L1 adipocytes, hyperosmotic stress induces a marked PI(3,5)P2 increase that is fully dependent on the ArPIKfyve-PIKfyve axis; siRNA depletion of either ArPIKfyve or PIKfyve abolishes the hyperosmotically activated PI(3,5)P2 rise.\",\n      \"method\": \"siRNA knockdown of ArPIKfyve and PIKfyve, 32P lipid labeling and measurement in 3T3-L1 adipocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown of both components with biochemical lipid readout, single lab, single method per component\",\n      \"pmids\": [\"15546865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Decreased VAC14 expression increases plasma membrane cholesterol, which facilitates Salmonella docking and invasion. The mechanism linking VAC14 to cholesterol homeostasis at the plasma membrane was established through siRNA knockdown and cholesterol measurement experiments.\",\n      \"method\": \"siRNA knockdown of VAC14, plasma membrane cholesterol quantification, Salmonella invasion assay, zebrafish infection model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined biochemical mechanism (cholesterol), multiple model systems (cells and zebrafish), single lab\",\n      \"pmids\": [\"28827342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VAC14 interacts with Rab9 and the Rab7 GAP TBC1D15, linking the VAC14 complex to endolysosomal vesicular transport regulation. These interactions were validated by reciprocal co-immunoprecipitation and proximity ligation assay.\",\n      \"method\": \"Protein affinity purification combined with MudPIT mass spectrometry, reciprocal co-immunoprecipitation, proximity ligation assay\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS interactome with orthogonal validation by reciprocal co-IP and PLA, single lab\",\n      \"pmids\": [\"24578385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ArPIKfyve-Sac3 heterodimer (VAC14-Sac3 binary complex) interacts with Synphilin-1 (Sph1) in brain, as identified by mass spectrometry. Modulation of ArPIKfyve/Sac3 levels alters aggregation properties of Sph1-GFP through mechanisms involving increased cytosolic partitioning and basal autophagy-mediated clearance.\",\n      \"method\": \"Mass spectrometry of brain-derived ArPIKfyve-Sac3 interactors, RNA silencing and overexpression in mammalian cell lines including primary neurons, GFP aggregation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS identification with functional validation by modulation experiments, single lab, multiple cell types\",\n      \"pmids\": [\"26405034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BioID proximity labeling of Vac14 identified COPI subunit COPB1 and the GTPase Arf1 (required for COPI assembly) as proximal interactors of Vac14, validated by proximity ligation assay, suggesting a functional link between Vac14 and Golgi-associated COPI complex in endosomal dynamics.\",\n      \"method\": \"BioID proximity-dependent biotin labeling, mass spectrometry, proximity ligation assay\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — proximity labeling (not direct interaction), PLA validation, single lab, functional consequence not directly tested\",\n      \"pmids\": [\"34554760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VAC14 forms a star-shaped pentameric scaffold (medium-resolution structure). Atomic-resolution AlphaFold2 prediction combined with cryo-EM maps revealed that disease-linked mutations reside at VAC14-VAC14 interfaces. Mutations disrupting VAC14 oligomerization cause defects in PI(3,5)P2 generation, VAC14 localization, and Fab1/PIKfyve activity. In human VAC14 KO cells, patient mutations are defective in PIKfyve-VAC14-FIG4 complex formation (pull-down), VAC14 oligomerization (fluorescence-detection size-exclusion chromatography), and colocalization with VPS35-positive endosomes.\",\n      \"method\": \"CryoEM structure, AlphaFold2 modeling, yeast genetics with oligomerization interface mutants, lipid analysis, pull-down assay in human KO cells, fluorescence-detection size-exclusion chromatography, colocalization microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural data combined with mutagenesis in both yeast and human cells, multiple orthogonal methods (structure, lipid assay, pull-down, FSEC, localization)\",\n      \"pmids\": [\"40305106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"VAC14 variants in the dimerization domain (p.Ala582Ser/p.Ser583Leu) cause pediatric neurological disease with PI(3,5)P2 deficiency. Vacuolization of patient fibroblasts was rescued by transfection of wild-type VAC14 cDNA, establishing that the patient variants are loss-of-function.\",\n      \"method\": \"Exome sequencing, patient fibroblast vacuole assay, rescue by wild-type VAC14 transfection\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional rescue experiment in patient fibroblasts confirming loss-of-function, single cellular assay, two unrelated families\",\n      \"pmids\": [\"27292112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PI(3,5)P2 synthesized by the FIG4-VAC14 pathway inhibits the lysosomal chloride transporter ClC-7. Knockout of CLCN7 corrects lysosomal swelling and partially corrects lysosomal hyperacidification in FIG4 null cells, and dominant-negative CLCN7 in Fig4 null mice improved growth, neurological function, and lifespan by 20%, placing VAC14/FIG4-dependent PI(3,5)P2 upstream of ClC-7 in lysosomal regulation.\",\n      \"method\": \"CLCN7/CLCN6 knockout in FIG4 null cells, dominant-negative CLCN7 in Fig4 null mouse, lysosomal morphology and pH assays, in vivo phenotyping\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with knockout and dominant-negative in both cell and mouse models, multiple phenotypic readouts, functional pathway ordering\",\n      \"pmids\": [\"37363915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"VAC14 homodimerization can be altered by missense mutations at the C-terminal dimerization domain; p.Leu648Phe and p.Ala562Val mutations enhance VAC14 homodimer formation compared to wildtype, while p.Arg623His does not, as measured in stably transfected SH-SY5Y cells.\",\n      \"method\": \"Stable overexpression in SH-SY5Y cells, co-immunoprecipitation to assess homodimer formation, patient fibroblast analysis\",\n      \"journal\": \"Parkinsonism & related disorders\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP based homodimerization assay, single lab, no functional consequence directly tested for homodimer enhancement\",\n      \"pmids\": [\"32949958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"siRNA knockdown of VAC14 in stem cell-derived peripheral neuronal cells increased docetaxel sensitivity, measured by decreased neurite processes and branches, indicating VAC14 is required for peripheral neuronal resilience to taxane-induced damage.\",\n      \"method\": \"siRNA knockdown in stem cell-derived peripheral neurons, neurite morphology quantification after docetaxel treatment\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single siRNA knockdown assay with morphological readout, single lab, no direct molecular mechanism identified\",\n      \"pmids\": [\"27143689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Under ethanol stress in yeast, Vac14 dissociates from Atg18, its interaction partner in the Fab1 complex; this dissociation is linked to reduced PI(3,5)P2 levels caused by Fab1 downregulation, resulting in Atg18 membrane detachment and vacuole fusion.\",\n      \"method\": \"Fluorescence microscopy with PI(3,5)P2 sensor, genetic analysis with hyperactive Fab1 mutant, co-localization of Vac14 and Atg18\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, primarily indirect evidence from sensor redistribution and fluorescence imaging, Vac14-Atg18 interaction inferred from colocalization loss\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"VAC14 (ArPIKfyve) functions as a HEAT-repeat scaffold protein that forms a star-shaped pentameric complex, organizing the PIKfyve lipid kinase and the FIG4/Sac3 PI(3,5)P2 phosphatase into a ternary PAS complex on endosomal membranes; VAC14 oligomerization is the prerequisite step for complex assembly, VAC14 directly activates PIKfyve kinase activity, stabilizes FIG4/Sac3 from proteasomal degradation, and directs FIG4 localization to the vacuole/endosome, thereby coordinating the synthesis and turnover of PI(3,5)P2 to regulate endolysosomal trafficking, AMPA receptor endocytosis at synapses, GLUT4 translocation in adipocytes, and lysosomal homeostasis via downstream inhibition of the ClC-7 chloride transporter.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VAC14 is a HEAT-repeat scaffold protein that organizes the synthesis and turnover of the signaling lipid PI(3,5)P2 on endosomal/vacuolar membranes by assembling the PIKfyve(Fab1) lipid kinase and the FIG4/Sac3 phosphatase into a single ternary complex, thereby governing endolysosomal membrane trafficking [#0, #5]. VAC14 self-associates into a star-shaped pentameric scaffold, and this oligomerization is the prerequisite first molecular event for assembly of the kinase–phosphatase complex; monomeric or oligomerization-defective mutants fail to bind Fab1/FIG4, mislocalize, and cannot generate PI(3,5)P2 [#11, #18]. Within the assembled complex VAC14 positively regulates PIKfyve/Fab1 kinase activity and PI(3,5)P2 production [#3, #4, #6], directs FIG4/Sac3 to the vacuole/endosome, and stabilizes Sac3 against proteasomal degradation [#1, #8]. Loss of VAC14 collapses cellular PI(3,5)P2 and disrupts endosome-to-TGN retrograde trafficking and vacuolar homeostasis [#2]. Through this lipid output VAC14 controls multiple physiological processes: regulated endocytosis of AMPA receptors at synapses [#10], insulin-stimulated GLUT4 translocation in adipocytes [#9], and lysosomal size and acidification via PI(3,5)P2-dependent inhibition of the chloride transporter ClC-7 [#20]. Biallelic and dimerization-domain mutations in VAC14 cause a pediatric neurological disease characterized by PI(3,5)P2 deficiency and cellular vacuolization that is rescued by wild-type VAC14 [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that VAC14 acts genetically upstream of the Fab1 kinase, answering whether VAC14 is a regulator rather than a catalytic component of PI(3,5)P2 synthesis.\",\n      \"evidence\": \"Yeast deletion/overexpression genetics with phosphoinositide measurement and hyperosmotic shock, where FAB1 overexpression rescues vac14\\u0394\",\n      \"pmids\": [\"12062051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of activation not defined\", \"Did not resolve whether VAC14 acts on the kinase directly or through other partners\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed VAC14 dually controls PI(3,5)P2 metabolism by directing the phosphatase Fig4 to the vacuole while also activating the Fab1 kinase, revealing coordination of synthesis and turnover.\",\n      \"evidence\": \"GFP localization, co-IP, in vitro phosphatase assay and deletion genetics in yeast\",\n      \"pmids\": [\"14528018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the membrane complex unknown\", \"Mechanism of Fig4 recruitment not defined at residue level\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Extended the model to mammals, showing human VAC14 physically binds and activates PIKfyve, establishing functional conservation of the scaffold-kinase relationship.\",\n      \"evidence\": \"Reciprocal co-IP, co-fractionation, siRNA knockdown and in vitro lipid kinase assay; plus adipocyte hyperosmotic PI(3,5)P2 measurements\",\n      \"pmids\": [\"15542851\", \"15546865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not yet define the phosphatase arm in mammals\", \"Domains mediating binding not mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the in vivo requirement for VAC14 and the full mammalian ternary (PAS) complex, demonstrating that VAC14 couples PI(3,5)P2 synthesis (PIKfyve) with turnover (Sac3) to drive endosomal dynamics.\",\n      \"evidence\": \"Vac14 knockout mouse with lipid and retrograde-trafficking phenotypes; endogenous co-IP, fractionation, in vitro phosphatase and endosome reconstitution assays\",\n      \"pmids\": [\"17956977\", \"17556371\", \"17475247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural architecture of the complex unknown\", \"How synthesis and turnover are temporally balanced not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified VAC14 as the central organizer of the PAS complex via homomeric and heteromeric contacts, answering which subunit nucleates assembly and showing its C-terminus is required for kinase activation.\",\n      \"evidence\": \"HEAT-repeat scaffold characterization with ingls mouse mutant; systematic co-IP with truncation mutants, in vitro kinase assay, dominant-negative peptide competition and GLUT4 readout\",\n      \"pmids\": [\"19037259\", \"18950639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Oligomeric state not quantified\", \"Atomic structure of interfaces not available\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mapped the PIKfyve Cpn60_TCP1 domain as the docking site for the VAC14-Sac3 subcomplex and confirmed that Sac3 phosphatase activity within the complex drives PI(3,5)P2 turnover.\",\n      \"evidence\": \"Domain mapping with truncation/point mutants and vacuole morphology readout using catalytic-dead Sac3 and PIKfyve mutants\",\n      \"pmids\": [\"19840946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial relationship of kinase and phosphatase active sites unresolved\", \"Regulatory switching between synthesis and turnover not addressed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed a protein-stability function for VAC14, showing it protects Sac3 from proteasomal degradation, and linked failure of this mechanism to CMT4J disease via an unstabilizable Sac3 mutant.\",\n      \"evidence\": \"Cycloheximide chase with proteasome inhibitors, siRNA/overexpression and co-IP with the Sac3(I41T) disease mutant\",\n      \"pmids\": [\"20630877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degradation pathway/E3 ligase not identified\", \"Whether stabilization requires full complex assembly not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected VAC14 to neuronal physiology, demonstrating it controls AMPA receptor surface levels through regulated endocytosis at synapses.\",\n      \"evidence\": \"Endogenous localization, mEPSC electrophysiology in Vac14-/- neurons, AMPA receptor surface biotinylation and postsynaptic rescue\",\n      \"pmids\": [\"22842785\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct lipid-to-receptor-trafficking link not mechanistically dissected\", \"Identity of relevant endocytic machinery not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Ordered complex assembly by showing VAC14 self-multimerization is the obligatory first step that precedes recruitment of Fab1 and Fig4.\",\n      \"evidence\": \"Co-IP with C-terminal Vac14 mutants, two-hybrid, vacuole morphology and hyperosmotic PI(3,5)P2 assays in yeast\",\n      \"pmids\": [\"23389034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact oligomer number not determined\", \"Trigger initiating multimerization unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided human disease causation, showing dimerization-domain VAC14 variants produce PI(3,5)P2 deficiency and cellular vacuolization rescuable by wild-type VAC14.\",\n      \"evidence\": \"Exome sequencing in two families with patient fibroblast vacuole rescue\",\n      \"pmids\": [\"27292112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cellular phenotype assayed\", \"Tissue-specific basis of neurological presentation not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed VAC14/FIG4-derived PI(3,5)P2 upstream of the lysosomal chloride transporter ClC-7, defining a downstream effector for lysosomal size and pH control.\",\n      \"evidence\": \"CLCN7 knockout in FIG4-null cells and dominant-negative CLCN7 in Fig4-null mice with lysosomal and in vivo phenotyping\",\n      \"pmids\": [\"37363915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PI(3,5)P2 inhibits ClC-7 directly or via intermediaries not resolved\", \"Generalizability beyond FIG4-null context untested for VAC14 alone\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Delivered structural understanding, showing VAC14 forms a star-shaped pentameric scaffold whose interface mutations disrupt oligomerization, complex formation, localization and PI(3,5)P2 generation, unifying the assembly and disease models.\",\n      \"evidence\": \"Cryo-EM, AlphaFold2 modeling, oligomerization-interface mutagenesis in yeast and human KO cells, lipid analysis, pull-down, FSEC and VPS35-endosome colocalization\",\n      \"pmids\": [\"40305106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of full kinase-phosphatase-loaded complex not solved\", \"Dynamics of assembly/disassembly during signaling not captured\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How VAC14 complex assembly and PI(3,5)P2 output are dynamically regulated by upstream signals and connected to its peripheral partners (nNOS, Rab9/TBC1D15, Synphilin-1, COPI/Arf1, cholesterol homeostasis) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Functional significance of non-core partners largely untested\", \"No mechanism linking VAC14 to plasma-membrane cholesterol established at molecular level\", \"Signal-dependent regulation of pentamer assembly unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 6, 11, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 4, 6, 8]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 5, 10, 18]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 5, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 4, 9]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [2, 1]}\n    ],\n    \"complexes\": [\"PIKfyve-VAC14-FIG4/Sac3 (PAS) complex\", \"Fab1 PI(3,5)P2 regulatory complex\"],\n    \"partners\": [\"PIKFYVE\", \"FIG4\", \"VAC7\", \"ATG18\", \"RAB9A\", \"TBC1D15\", \"NOS1\", \"SNCAIP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}