{"gene":"VAC14","run_date":"2026-04-28T23:00:23","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 by directly contacting FIG4, FAB1/PIKfyve, VAC7, and ATG18; it mediates three distinct mechanisms for rapid interconversion of PI3P and PI(3,5)P2, and the ingls missense mutation in Vac14 prevents association with Fab1, generating a partial complex","method":"Structural prediction, yeast genetics, direct binding assays, mouse mutant analysis, epistasis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (structural prediction, binding assays, genetic epistasis, mouse mutant phenotyping) in a highly-cited study","pmids":["19037259"],"is_preprint":false},{"year":2003,"finding":"In yeast, Vac14 both positively regulates Fab1 kinase activity and directs the vacuolar localization and activation of the Fig4 PI(3,5)P2 phosphatase; Fig4 physically associates with Vac14 in a common membrane-associated complex, and in the absence of Vac14, Fig4 no longer localizes to the vacuole","method":"GFP localization, co-immunoprecipitation, in vitro phosphatase assay, genetic deletion analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro phosphatase assay, co-IP, and direct localization experiments with functional consequence, replicated across multiple genetic backgrounds","pmids":["14528018"],"is_preprint":false},{"year":2007,"finding":"Loss of Vac14 in mice results in massive neurodegeneration, vacuolation of neurons and fibroblasts, and defective endosome-to-TGN retrograde trafficking, establishing VAC14 as essential for PI(3,5)P2-dependent membrane trafficking in neural cells","method":"Vac14 knockout mouse, cellular vacuolation assay, membrane trafficking assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with defined cellular and trafficking phenotypes, highly cited","pmids":["17956977"],"is_preprint":false},{"year":2007,"finding":"Human VAC14 (ArPIKfyve/hVac14) forms a stable ternary complex with PIKfyve and Sac3 (the mammalian FIG4 ortholog) on endosomal membranes; Sac3 preferentially hydrolyzes PI(3,5)P2 in vitro, and siRNA knockdown of Sac3 elevates PI(3,5)P2 levels, while depletion of ArPIKfyve or PIKfyve reduces PI(3,5)P2","method":"Co-immunoprecipitation, co-fractionation, co-localization, in vitro phosphatase assay, siRNA knockdown with lipid measurements, in vitro carrier vesicle reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — reconstituted complex, in vitro enzymatic assay, siRNA functional validation, replicated with multiple orthogonal methods","pmids":["17556371"],"is_preprint":false},{"year":2004,"finding":"Human VAC14 (hVac14/ArPIKfyve) physically associates with PIKfyve, co-fractionates with it on intracellular membranes, and positively regulates PIKfyve lipid kinase activity; siRNA depletion of hVac14 reduces in vitro PIKfyve kinase activity and intracellular PI(3,5)P2, while ectopic expression increases PIKfyve activity and PI(3,5)P2 production","method":"Co-immunoprecipitation, siRNA knockdown, in vitro kinase assay, intracellular lipid labeling","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus co-IP plus siRNA functional validation with lipid measurements","pmids":["15542851"],"is_preprint":false},{"year":2002,"finding":"In yeast, Vac14 is an upstream activator of Fab1-catalyzed PI(3,5)P2 synthesis required for regulated PI(3,5)P2 production in response to hyperosmotic shock; vac14Δ cells make very little PI(3,5)P2 and fail to sort proteins to the multivesicular body; FAB1 overexpression suppresses vac14Δ phenotypes, placing Vac14 upstream of Fab1","method":"Genetic deletion, lipid measurements, protein trafficking assays, FAB1 overexpression epistasis, visual screening","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — epistasis experiment places Vac14 upstream of Fab1, supported by lipid measurements and trafficking assays","pmids":["12062051"],"is_preprint":false},{"year":2008,"finding":"ArPIKfyve (human VAC14) scaffolds the PIKfyve-ArPIKfyve-Sac3 (PAS) ternary complex through homomeric interactions mediated via its conserved C-terminal domain; ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve; disruption of ArPIKfyve-ArPIKfyve contact sites with a C-terminal peptide disassembles the PAS complex, reduces PIKfyve lipid kinase activity in vitro, and inhibits insulin-stimulated GLUT4 translocation","method":"Co-immunoprecipitation in transfected mammalian cells with truncation/point mutants, in vitro lipid kinase assay, GLUT4 translocation assay in 3T3-L1 adipocytes","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis of interaction domains, in vitro kinase assay, functional readout in adipocytes","pmids":["18950639"],"is_preprint":false},{"year":2009,"finding":"Within the PAS (PIKfyve-ArPIKfyve-Sac3) complex, the Cpn60_TCP1 domain of PIKfyve is the major determinant for associating the ArPIKfyve-Sac3 subcomplex; Sac3 assembled in the PAS complex retains active PI(3,5)P2 phosphatase activity; neither Sac3 nor PIKfyve enzymatic activities affect PAS complex formation or stability","method":"Co-immunoprecipitation with domain truncation/point mutants in triple-transfected COS cells, vacuolation assay as functional readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — systematic domain mapping with mutagenesis and functional phenotypic readout","pmids":["19840946"],"is_preprint":false},{"year":2010,"finding":"ArPIKfyve stabilizes Sac3 protein by attenuating proteasome-dependent degradation; ArPIKfyve elevates Sac3 steady-state levels and extends its half-life through direct association, without altering Sac3 mRNA levels; the CMT4J pathogenic Sac3(I41T) mutation prevents ArPIKfyve from extending Sac3 half-life, identifying failure of this stabilization mechanism as a molecular defect in CMT4J","method":"Cycloheximide chase, proteasome inhibitor treatment, co-expression experiments, half-life measurements, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including cycloheximide chase, proteasome inhibition, and mutant analysis establishing a novel regulatory mechanism","pmids":["20630877"],"is_preprint":false},{"year":2013,"finding":"Vac14 forms a dimer/multimer via conserved C-terminal motifs; multimerization is a prerequisite for Fab1 complex assembly, as monomeric Vac14 mutants cannot interact with Fab1 or Fig4; cells expressing monomeric Vac14 mutants have enlarged vacuoles that fail to fragment after hyperosmotic shock, indicating severely reduced PI(3,5)P2 levels","method":"Yeast two-hybrid, co-immunoprecipitation, vacuole morphology assay, hyperosmotic shock assay, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal binding assays with mutagenesis, functional phenotypic validation in yeast","pmids":["23389034"],"is_preprint":false},{"year":2012,"finding":"Endogenous 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 AMPA receptor (GluA2) levels due to diminished regulated AMPA receptor endocytosis; re-introduction of VAC14 into postsynaptic Vac14-/- cells reverses these effects","method":"Immunofluorescence localization, electrophysiology (mEPSC recording), surface receptor labeling, rescue by VAC14 re-expression in Vac14-/- neurons","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — direct localization with functional consequence, electrophysiology, and rescue experiment establishing postsynaptic role","pmids":["22842785"],"is_preprint":false},{"year":2006,"finding":"VAC14 interacts with the PDZ domain of neuronal nitric oxide synthase (nNOS) via a novel internal PDZ-recognition motif that is beta-finger independent; mutagenesis defined essential residues within this motif","method":"Binding assays with Vac14 deletion constructs and nNOS PDZ domain, mutational analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab pulldown/binding assay with mutagenesis, single method","pmids":["17161399"],"is_preprint":false},{"year":2014,"finding":"VAC14 interacts with Rab9 and the Rab7 GAP TBC1D15 as part of its protein interaction network, linking the VAC14 complex to regulation of vesicular transport; overexpression of wild-type Vac14 or PIKfyve-binding deficient Vac14 L156R causes late endosomal/lysosomal vacuolation with late endosomal marker proteins on the vacuole membranes","method":"Protein affinity purification combined with MudPIT mass spectrometry, co-immunoprecipitation validation, immunofluorescence","journal":"Molecular & cellular proteomics : MCP","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP/pulldown validation of interactors identified by proteomics","pmids":["24578385"],"is_preprint":false},{"year":2004,"finding":"ArPIKfyve (VAC14) and PIKfyve physically associate in 3T3-L1 adipocytes in an insulin-independent manner; the ArPIKfyve-PIKfyve-PI(3,5)P2 pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport, with siRNA depletion of ArPIKfyve or PIKfyve reducing PI(3,5)P2, inhibiting insulin-stimulated Akt phosphorylation, and reducing GLUT4 surface accumulation","method":"Co-immunoprecipitation in 3T3-L1 adipocytes, siRNA knockdown, in vitro lipid labeling of membranes, glucose uptake assay, GLUT4 surface labeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus siRNA knockdown with multiple functional readouts (lipid measurements, glucose uptake, GLUT4 surface levels)","pmids":["15546865"],"is_preprint":false},{"year":2017,"finding":"Decreased VAC14 expression increases plasma membrane cholesterol, facilitating Salmonella docking and invasion; VAC14 therefore regulates susceptibility to Salmonella infection through modulation of cholesterol levels at the plasma membrane","method":"siRNA knockdown of VAC14, cholesterol measurement, Salmonella invasion assay, zebrafish infection model with ezetimibe treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with mechanistic validation (cholesterol measurement, invasion assay) and in vivo zebrafish model","pmids":["28827342"],"is_preprint":false},{"year":2015,"finding":"The ArPIKfyve-Sac3 heterodimer interacts with synphilin-1 (Sph1) in brain tissue; the ArPIKfyve-Sac3 complex prevents Sph1 aggregation through mechanisms involving increased cytosolic partitioning and basal autophagy-dependent removal of Sph1 aggregates; this effect requires active Sac3 phosphatase activity","method":"Mass spectrometry of brain-derived interactors, co-immunoprecipitation, modulation of ArPIKfyve/Sac3 levels by RNA silencing or overexpression, GFP-tagged Sph1 aggregation assay in multiple cell lines including primary neurons","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP/mass spectrometry identification plus functional validation in multiple cell types, but single laboratory","pmids":["26405034"],"is_preprint":false},{"year":2021,"finding":"BioID proximity labeling of Vac14 and Fig4 identified COPI subunit COPB1 and the GTPase Arf1 (required for COPI assembly) as proximity interactors of Vac14, validated by proximity ligation assays, suggesting a novel link between the VAC14-PIKfyve-FIG4 complex and COPI-dependent endosomal dynamics","method":"BioID proximity labeling, mass spectrometry, proximity ligation assay validation","journal":"Journal of proteome research","confidence":"Low","confidence_rationale":"Tier 3 — proximity labeling with limited validation (PLA only), single lab","pmids":["34554760"],"is_preprint":false},{"year":2025,"finding":"VAC14 forms a star-shaped pentamer scaffold (medium-resolution structure); two legs of VAC14 bind FIG4, with one leg also occupied by PIKfyve; VAC14 oligomerization is essential for Fab1/PIKfyve function, PI(3,5)P2 generation, VAC14 endosomal localization, and PIKfyve-VAC14-FIG4 complex formation; patient mutations in VAC14-VAC14 interfaces disrupt oligomerization, complex assembly, and colocalization with VPS35-containing endosomes","method":"CryoEM structure, AlphaFold2 prediction, yeast genetics with interface mutations, PI(3,5)P2 measurement, pull-down assays, fluorescence-detection size-exclusion chromatography (FSEC) of cell lysates, human VAC14 KO cell complementation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure combined with mutagenesis, in vivo functional assays, and human KO cell validation","pmids":["40305106"],"is_preprint":false},{"year":2016,"finding":"siRNA knockdown of VAC14 in stem cell-derived peripheral neuronal cells increases docetaxel sensitivity, measured by decreased neurite processes and branches; VAC14 heterozygous mice have greater nociceptive sensitivity prior to docetaxel treatment, demonstrating a neuroprotective role of VAC14 in peripheral neurons","method":"siRNA knockdown in neuronal cells, morphometric analysis of neurites, nociceptive behavioral testing in VAC14 heterozygous mice","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA KD with defined cellular phenotype plus in vivo mouse model, but mechanistic pathway not fully elucidated","pmids":["27143689"],"is_preprint":false},{"year":2016,"finding":"Biallelic VAC14 mutations (splice-site and missense variants in the dimerization domain) cause PI(3,5)P2 deficiency in patient fibroblasts manifesting as vacuole accumulation, which is rescued by transfection of wild-type VAC14 cDNA, demonstrating that VAC14 dimerization domain integrity is essential for its function","method":"Exome sequencing, fibroblast vacuolation assay, wild-type VAC14 rescue transfection","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — rescue experiment in patient cells confirms functional significance of identified variants","pmids":["27292112"],"is_preprint":false},{"year":2023,"finding":"PI(3,5)P2, produced by the VAC14-PIKfyve-FIG4 complex, inhibits the lysosomal chloride transporter ClC-7 (CLCN7); loss of FIG4 or VAC14 reduces PI(3,5)P2 and results in enlarged lysosomes that are rescued by CLCN7 knockout, and CLCN7 reduction improves neurological function in Fig4 null mice, placing CLCN7 downstream of VAC14/FIG4 signaling","method":"CLCN7 knockout in FIG4 null cells, lysosome morphology assay, lysosomal pH measurement, dominant-negative CLCN7 in Fig4 null mouse with neurological and lifespan readouts","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in both cell culture and mouse model with multiple functional readouts","pmids":["37363915"],"is_preprint":false}],"current_model":"VAC14 (ArPIKfyve) is a HEAT-repeat scaffold protein that forms a star-shaped pentameric complex with the lipid kinase PIKfyve/FAB1 and the PI(3,5)P2 phosphatase FIG4/Sac3, nucleating this complex through homomeric oligomerization and direct contacts with all partners; within this complex VAC14 activates PIKfyve kinase activity, directs FIG4 to endolysosomal membranes, stabilizes FIG4/Sac3 protein by protecting it from proteasomal degradation, and thereby controls the dynamic interconversion of PI3P and PI(3,5)P2 that is essential for endolysosomal trafficking, AMPA receptor endocytosis at synapses, GLUT4 translocation, and neuronal survival."},"narrative":{"teleology":[{"year":2002,"claim":"Establishing VAC14 as an upstream activator of PI(3,5)P₂ synthesis resolved how Fab1 kinase activity is regulated: Vac14 deletion nearly abolished PI(3,5)P₂ production in yeast, and FAB1 overexpression suppressed the phenotype, placing Vac14 genetically upstream.","evidence":"Yeast genetic deletion, lipid measurements, epistasis via FAB1 overexpression, and MVB sorting assays","pmids":["12062051"],"confidence":"High","gaps":["Mechanism of Vac14-mediated Fab1 activation unknown at this stage","Whether the relationship is via direct physical interaction was not demonstrated"]},{"year":2003,"claim":"Demonstrating that Vac14 physically associates with Fig4 and recruits it to the vacuolar membrane revealed that the same scaffold protein controls both PI(3,5)P₂ synthesis and turnover, establishing the concept of a coordinated regulatory complex.","evidence":"Co-immunoprecipitation, GFP localization, in vitro phosphatase assay, and genetic deletion analysis in yeast","pmids":["14528018"],"confidence":"High","gaps":["Stoichiometry of the complex unknown","Whether the interaction is conserved in mammals was not yet tested"]},{"year":2004,"claim":"Identification of human VAC14 (ArPIKfyve) as a direct PIKfyve-binding partner that positively regulates its kinase activity extended the yeast findings to mammals and linked the pathway to insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes.","evidence":"Co-IP in mammalian cells and 3T3-L1 adipocytes, siRNA knockdown with in vitro kinase assay and lipid measurements, GLUT4 surface labeling","pmids":["15542851","15546865"],"confidence":"High","gaps":["How VAC14 stimulates PIKfyve kinase activity mechanistically was not resolved","Whether the mammalian complex also includes a phosphatase subunit was not yet shown"]},{"year":2007,"claim":"Generation of Vac14 knockout mice revealed that VAC14 is essential for neuronal survival: loss causes massive neurodegeneration and cellular vacuolation, and the ternary PIKfyve-ArPIKfyve-Sac3 (PAS) complex was biochemically reconstituted in mammalian cells, confirming that kinase and phosphatase coexist on the same scaffold.","evidence":"Vac14 KO mouse phenotyping, membrane trafficking assays, co-IP/co-fractionation/co-localization of endogenous mammalian complex, in vitro phosphatase assay","pmids":["17956977","17556371"],"confidence":"High","gaps":["Specific neuronal cell types and circuits affected were not defined","Whether Sac3/FIG4 stability depends on VAC14 was unknown"]},{"year":2008,"claim":"Mapping the HEAT-repeat architecture of VAC14 and demonstrating that C-terminal-mediated homomeric interaction is required for PAS complex assembly established VAC14 as the obligate scaffold organizing both enzymatic partners.","evidence":"Structural prediction, truncation/point mutant co-IP, in vitro kinase assay, GLUT4 translocation in adipocytes, yeast epistasis and mouse mutant analysis","pmids":["19037259","18950639"],"confidence":"High","gaps":["Atomic-resolution structure of the multimer was lacking","Precise oligomeric state (dimer vs. higher order) was debated"]},{"year":2009,"claim":"Identifying the PIKfyve Cpn60_TCP1 domain as the determinant for docking with the ArPIKfyve-Sac3 subcomplex, and showing that enzymatic activities of neither partner affect complex stability, clarified that scaffold assembly precedes and is independent of catalysis.","evidence":"Domain truncation/point mutant co-IP in triple-transfected COS cells, vacuolation assay","pmids":["19840946"],"confidence":"High","gaps":["Whether post-translational modifications regulate assembly was not tested","No structural data for the PIKfyve-VAC14 interface"]},{"year":2010,"claim":"Discovering that VAC14 stabilizes FIG4/Sac3 by attenuating its proteasomal degradation added a non-catalytic regulatory function to the scaffold and explained why the CMT4J-associated Sac3(I41T) mutation reduces Sac3 levels—it disrupts VAC14-mediated stabilization.","evidence":"Cycloheximide chase, proteasome inhibitor treatment, co-expression experiments, half-life measurements, siRNA knockdown","pmids":["20630877"],"confidence":"High","gaps":["Whether VAC14 shields a specific Sac3 degron was not determined","Ubiquitin ligase responsible for Sac3 turnover was not identified"]},{"year":2012,"claim":"Demonstrating that VAC14 localizes to synapses and that its loss elevates surface AMPA receptors by impairing regulated endocytosis established a postsynaptic function for PI(3,5)P₂ signaling in excitatory neurotransmission.","evidence":"Immunofluorescence, mEPSC electrophysiology, surface GluA2 labeling, rescue by VAC14 re-expression in Vac14⁻/⁻ neurons","pmids":["22842785"],"confidence":"High","gaps":["Whether PI(3,5)P₂ acts directly on endocytic machinery or via an effector at the synapse is unclear","Behavioral consequences of enhanced AMPA signaling not characterized"]},{"year":2013,"claim":"Precise mutagenesis in yeast confirmed that Vac14 multimerization through C-terminal motifs is an absolute prerequisite for Fab1 and Fig4 binding, resolving the order of assembly: self-association first, then partner recruitment.","evidence":"Yeast two-hybrid, co-IP, vacuole morphology and hyperosmotic shock assays, site-directed mutagenesis","pmids":["23389034"],"confidence":"High","gaps":["Whether the multimer is a dimer, trimer, or higher-order oligomer remained unclear"]},{"year":2016,"claim":"Identification of biallelic VAC14 mutations in human patients with striatal neurodegeneration and vacuolar pathology, rescued by wild-type VAC14 cDNA, established VAC14 deficiency as a human Mendelian neurodegenerative disease.","evidence":"Exome sequencing, fibroblast vacuolation assay, wild-type VAC14 rescue transfection","pmids":["27292112"],"confidence":"Medium","gaps":["Small number of families reported","Genotype–phenotype correlation across different VAC14 domains not established"]},{"year":2023,"claim":"Genetic epistasis placing the lysosomal chloride transporter CLCN7 downstream of PI(3,5)P₂ identified a key effector: CLCN7 knockout rescued the enlarged lysosome phenotype of FIG4/VAC14 loss and improved neurological function in Fig4-null mice.","evidence":"CLCN7 KO in FIG4-null cells, lysosome morphology and pH measurement, dominant-negative CLCN7 in Fig4-null mouse with neurological and lifespan readouts","pmids":["37363915"],"confidence":"High","gaps":["Whether CLCN7 inhibition is a direct effect of PI(3,5)P₂ binding or indirect is not fully resolved","Therapeutic window for CLCN7 modulation unknown"]},{"year":2025,"claim":"A medium-resolution cryo-EM structure revealed VAC14 as a star-shaped pentamer, with two legs binding FIG4 and one additionally occupied by PIKfyve, and showed that patient mutations at VAC14–VAC14 interfaces disrupt oligomerization, complex assembly, and colocalization with retromer-positive endosomes.","evidence":"Cryo-EM, AlphaFold2 prediction, yeast interface mutants, PI(3,5)P₂ measurement, FSEC, human VAC14 KO cell complementation","pmids":["40305106"],"confidence":"High","gaps":["High-resolution atomic model not yet available","How the pentameric architecture positions PIKfyve for catalysis on the membrane is not resolved","Structural basis for VAC14-mediated FIG4 stabilization remains unknown"]},{"year":null,"claim":"Major unresolved questions include how VAC14 pentamer geometry mechanistically activates PIKfyve kinase activity, which E3 ubiquitin ligase targets FIG4 for degradation in the absence of VAC14, and whether the VAC14 complex directly senses upstream signals to dynamically regulate PI(3,5)P₂ pools.","evidence":"","pmids":[],"confidence":"Low","gaps":["No reconstituted in vitro activation assay with defined stoichiometry","E3 ligase for FIG4 not identified","Signal-dependent regulation of complex assembly/disassembly not characterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6,9,17]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,5,8]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,10,12,17]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[2,20]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,12]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,2,5,12]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,10,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,13]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[10,18]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,4,14]}],"complexes":["PIKfyve-ArPIKfyve-Sac3 (PAS) complex"],"partners":["PIKFYVE","FIG4","VAC7","ATG18","CLCN7","TBC1D15","RAB9A"],"other_free_text":[]},"mechanistic_narrative":"VAC14 is a HEAT-repeat scaffold protein that assembles a star-shaped pentameric complex with the lipid kinase PIKfyve/FAB1 and the PI(3,5)P₂ phosphatase FIG4/Sac3, thereby controlling the dynamic interconversion of PI3P and PI(3,5)P₂ on endolysosomal membranes essential for membrane trafficking and neuronal survival. VAC14 oligomerizes through conserved C-terminal motifs—a prerequisite for PIKfyve binding, FIG4 recruitment to vacuolar/endosomal membranes, and activation of PIKfyve kinase activity—and additionally stabilizes FIG4/Sac3 protein by protecting it from proteasomal degradation [PMID:19037259, PMID:23389034, PMID:20630877, PMID:40305106]. Loss of VAC14 causes massive neuronal vacuolation and neurodegeneration in mice, defective endosome-to-TGN retrograde trafficking, impaired AMPA receptor endocytosis at synapses with elevated surface GluA2 and enhanced excitatory transmission, and reduced insulin-stimulated GLUT4 translocation [PMID:17956977, PMID:22842785, PMID:15546865]. Biallelic VAC14 mutations that disrupt the dimerization domain cause PI(3,5)P₂ deficiency in human patients, establishing VAC14 loss-of-function as a cause of a neurodegenerative vacuolar disorder [PMID:27292112, PMID:40305106]."},"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":193,"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":162,"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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it mediates three distinct mechanisms for rapid interconversion of PI3P and PI(3,5)P2, and the ingls missense mutation in Vac14 prevents association with Fab1, generating a partial complex\",\n      \"method\": \"Structural prediction, yeast genetics, direct binding assays, mouse mutant analysis, epistasis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (structural prediction, binding assays, genetic epistasis, mouse mutant phenotyping) in a highly-cited study\",\n      \"pmids\": [\"19037259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In yeast, Vac14 both positively regulates Fab1 kinase activity and directs the vacuolar localization and activation of the Fig4 PI(3,5)P2 phosphatase; Fig4 physically associates with Vac14 in a common membrane-associated complex, and in the absence of Vac14, Fig4 no longer localizes to the vacuole\",\n      \"method\": \"GFP localization, co-immunoprecipitation, in vitro phosphatase assay, genetic deletion analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro phosphatase assay, co-IP, and direct localization experiments with functional consequence, replicated across multiple genetic backgrounds\",\n      \"pmids\": [\"14528018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Loss of Vac14 in mice results in massive neurodegeneration, vacuolation of neurons and fibroblasts, and defective endosome-to-TGN retrograde trafficking, establishing VAC14 as essential for PI(3,5)P2-dependent membrane trafficking in neural cells\",\n      \"method\": \"Vac14 knockout mouse, cellular vacuolation assay, membrane trafficking assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined cellular and trafficking phenotypes, highly cited\",\n      \"pmids\": [\"17956977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human VAC14 (ArPIKfyve/hVac14) forms a stable ternary complex with PIKfyve and Sac3 (the mammalian FIG4 ortholog) on endosomal membranes; Sac3 preferentially hydrolyzes PI(3,5)P2 in vitro, and siRNA knockdown of Sac3 elevates PI(3,5)P2 levels, while depletion of ArPIKfyve or PIKfyve reduces PI(3,5)P2\",\n      \"method\": \"Co-immunoprecipitation, co-fractionation, co-localization, in vitro phosphatase assay, siRNA knockdown with lipid measurements, in vitro carrier vesicle reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstituted complex, in vitro enzymatic assay, siRNA functional validation, replicated with multiple orthogonal methods\",\n      \"pmids\": [\"17556371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human VAC14 (hVac14/ArPIKfyve) physically associates with PIKfyve, co-fractionates with it on intracellular membranes, and positively regulates PIKfyve lipid kinase activity; siRNA depletion of hVac14 reduces in vitro PIKfyve kinase activity and intracellular PI(3,5)P2, while ectopic expression increases PIKfyve activity and PI(3,5)P2 production\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, in vitro kinase assay, intracellular lipid labeling\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus co-IP plus siRNA functional validation with lipid measurements\",\n      \"pmids\": [\"15542851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In yeast, Vac14 is an upstream activator of Fab1-catalyzed PI(3,5)P2 synthesis required for regulated PI(3,5)P2 production in response to hyperosmotic shock; vac14Δ cells make very little PI(3,5)P2 and fail to sort proteins to the multivesicular body; FAB1 overexpression suppresses vac14Δ phenotypes, placing Vac14 upstream of Fab1\",\n      \"method\": \"Genetic deletion, lipid measurements, protein trafficking assays, FAB1 overexpression epistasis, visual screening\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis experiment places Vac14 upstream of Fab1, supported by lipid measurements and trafficking assays\",\n      \"pmids\": [\"12062051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ArPIKfyve (human VAC14) scaffolds the PIKfyve-ArPIKfyve-Sac3 (PAS) ternary complex through homomeric interactions mediated via its conserved C-terminal domain; ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve; disruption of ArPIKfyve-ArPIKfyve contact sites with a C-terminal peptide disassembles the PAS complex, reduces PIKfyve lipid kinase activity in vitro, and inhibits insulin-stimulated GLUT4 translocation\",\n      \"method\": \"Co-immunoprecipitation in transfected mammalian cells with truncation/point mutants, in vitro lipid kinase assay, GLUT4 translocation assay in 3T3-L1 adipocytes\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis of interaction domains, in vitro kinase assay, functional readout in adipocytes\",\n      \"pmids\": [\"18950639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Within the PAS (PIKfyve-ArPIKfyve-Sac3) complex, the Cpn60_TCP1 domain of PIKfyve is the major determinant for associating the ArPIKfyve-Sac3 subcomplex; Sac3 assembled in the PAS complex retains active PI(3,5)P2 phosphatase activity; neither Sac3 nor PIKfyve enzymatic activities affect PAS complex formation or stability\",\n      \"method\": \"Co-immunoprecipitation with domain truncation/point mutants in triple-transfected COS cells, vacuolation assay as functional readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic domain mapping with mutagenesis and functional phenotypic readout\",\n      \"pmids\": [\"19840946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ArPIKfyve stabilizes Sac3 protein by attenuating proteasome-dependent degradation; ArPIKfyve elevates Sac3 steady-state levels and extends its half-life through direct association, without altering Sac3 mRNA levels; the CMT4J pathogenic Sac3(I41T) mutation prevents ArPIKfyve from extending Sac3 half-life, identifying failure of this stabilization mechanism as a molecular defect in CMT4J\",\n      \"method\": \"Cycloheximide chase, proteasome inhibitor treatment, co-expression experiments, half-life measurements, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including cycloheximide chase, proteasome inhibition, and mutant analysis establishing a novel regulatory mechanism\",\n      \"pmids\": [\"20630877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Vac14 forms a dimer/multimer via conserved C-terminal motifs; multimerization is a prerequisite for Fab1 complex assembly, as monomeric Vac14 mutants cannot interact with Fab1 or Fig4; cells expressing monomeric Vac14 mutants have enlarged vacuoles that fail to fragment after hyperosmotic shock, indicating severely reduced PI(3,5)P2 levels\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, vacuole morphology assay, hyperosmotic shock assay, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding assays with mutagenesis, functional phenotypic validation in yeast\",\n      \"pmids\": [\"23389034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Endogenous 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 AMPA receptor (GluA2) levels due to diminished regulated AMPA receptor endocytosis; re-introduction of VAC14 into postsynaptic Vac14-/- cells reverses these effects\",\n      \"method\": \"Immunofluorescence localization, electrophysiology (mEPSC recording), surface receptor labeling, rescue by VAC14 re-expression in Vac14-/- neurons\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional consequence, electrophysiology, and rescue experiment establishing postsynaptic role\",\n      \"pmids\": [\"22842785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"VAC14 interacts with the PDZ domain of neuronal nitric oxide synthase (nNOS) via a novel internal PDZ-recognition motif that is beta-finger independent; mutagenesis defined essential residues within this motif\",\n      \"method\": \"Binding assays with Vac14 deletion constructs and nNOS PDZ domain, mutational analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab pulldown/binding assay with mutagenesis, single method\",\n      \"pmids\": [\"17161399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VAC14 interacts with Rab9 and the Rab7 GAP TBC1D15 as part of its protein interaction network, linking the VAC14 complex to regulation of vesicular transport; overexpression of wild-type Vac14 or PIKfyve-binding deficient Vac14 L156R causes late endosomal/lysosomal vacuolation with late endosomal marker proteins on the vacuole membranes\",\n      \"method\": \"Protein affinity purification combined with MudPIT mass spectrometry, co-immunoprecipitation validation, immunofluorescence\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/pulldown validation of interactors identified by proteomics\",\n      \"pmids\": [\"24578385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ArPIKfyve (VAC14) and PIKfyve physically associate in 3T3-L1 adipocytes in an insulin-independent manner; the ArPIKfyve-PIKfyve-PI(3,5)P2 pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport, with siRNA depletion of ArPIKfyve or PIKfyve reducing PI(3,5)P2, inhibiting insulin-stimulated Akt phosphorylation, and reducing GLUT4 surface accumulation\",\n      \"method\": \"Co-immunoprecipitation in 3T3-L1 adipocytes, siRNA knockdown, in vitro lipid labeling of membranes, glucose uptake assay, GLUT4 surface labeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus siRNA knockdown with multiple functional readouts (lipid measurements, glucose uptake, GLUT4 surface levels)\",\n      \"pmids\": [\"15546865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Decreased VAC14 expression increases plasma membrane cholesterol, facilitating Salmonella docking and invasion; VAC14 therefore regulates susceptibility to Salmonella infection through modulation of cholesterol levels at the plasma membrane\",\n      \"method\": \"siRNA knockdown of VAC14, cholesterol measurement, Salmonella invasion assay, zebrafish infection model with ezetimibe treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with mechanistic validation (cholesterol measurement, invasion assay) and in vivo zebrafish model\",\n      \"pmids\": [\"28827342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ArPIKfyve-Sac3 heterodimer interacts with synphilin-1 (Sph1) in brain tissue; the ArPIKfyve-Sac3 complex prevents Sph1 aggregation through mechanisms involving increased cytosolic partitioning and basal autophagy-dependent removal of Sph1 aggregates; this effect requires active Sac3 phosphatase activity\",\n      \"method\": \"Mass spectrometry of brain-derived interactors, co-immunoprecipitation, modulation of ArPIKfyve/Sac3 levels by RNA silencing or overexpression, GFP-tagged Sph1 aggregation assay in multiple cell lines including primary neurons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP/mass spectrometry identification plus functional validation in multiple cell types, but single laboratory\",\n      \"pmids\": [\"26405034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"BioID proximity labeling of Vac14 and Fig4 identified COPI subunit COPB1 and the GTPase Arf1 (required for COPI assembly) as proximity interactors of Vac14, validated by proximity ligation assays, suggesting a novel link between the VAC14-PIKfyve-FIG4 complex and COPI-dependent endosomal dynamics\",\n      \"method\": \"BioID proximity labeling, mass spectrometry, proximity ligation assay validation\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — proximity labeling with limited validation (PLA only), single lab\",\n      \"pmids\": [\"34554760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"VAC14 forms a star-shaped pentamer scaffold (medium-resolution structure); two legs of VAC14 bind FIG4, with one leg also occupied by PIKfyve; VAC14 oligomerization is essential for Fab1/PIKfyve function, PI(3,5)P2 generation, VAC14 endosomal localization, and PIKfyve-VAC14-FIG4 complex formation; patient mutations in VAC14-VAC14 interfaces disrupt oligomerization, complex assembly, and colocalization with VPS35-containing endosomes\",\n      \"method\": \"CryoEM structure, AlphaFold2 prediction, yeast genetics with interface mutations, PI(3,5)P2 measurement, pull-down assays, fluorescence-detection size-exclusion chromatography (FSEC) of cell lysates, human VAC14 KO cell complementation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure combined with mutagenesis, in vivo functional assays, and human KO cell validation\",\n      \"pmids\": [\"40305106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"siRNA knockdown of VAC14 in stem cell-derived peripheral neuronal cells increases docetaxel sensitivity, measured by decreased neurite processes and branches; VAC14 heterozygous mice have greater nociceptive sensitivity prior to docetaxel treatment, demonstrating a neuroprotective role of VAC14 in peripheral neurons\",\n      \"method\": \"siRNA knockdown in neuronal cells, morphometric analysis of neurites, nociceptive behavioral testing in VAC14 heterozygous mice\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA KD with defined cellular phenotype plus in vivo mouse model, but mechanistic pathway not fully elucidated\",\n      \"pmids\": [\"27143689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Biallelic VAC14 mutations (splice-site and missense variants in the dimerization domain) cause PI(3,5)P2 deficiency in patient fibroblasts manifesting as vacuole accumulation, which is rescued by transfection of wild-type VAC14 cDNA, demonstrating that VAC14 dimerization domain integrity is essential for its function\",\n      \"method\": \"Exome sequencing, fibroblast vacuolation assay, wild-type VAC14 rescue transfection\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — rescue experiment in patient cells confirms functional significance of identified variants\",\n      \"pmids\": [\"27292112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PI(3,5)P2, produced by the VAC14-PIKfyve-FIG4 complex, inhibits the lysosomal chloride transporter ClC-7 (CLCN7); loss of FIG4 or VAC14 reduces PI(3,5)P2 and results in enlarged lysosomes that are rescued by CLCN7 knockout, and CLCN7 reduction improves neurological function in Fig4 null mice, placing CLCN7 downstream of VAC14/FIG4 signaling\",\n      \"method\": \"CLCN7 knockout in FIG4 null cells, lysosome morphology assay, lysosomal pH measurement, dominant-negative CLCN7 in Fig4 null mouse with neurological and lifespan readouts\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in both cell culture and mouse model with multiple functional readouts\",\n      \"pmids\": [\"37363915\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VAC14 (ArPIKfyve) is a HEAT-repeat scaffold protein that forms a star-shaped pentameric complex with the lipid kinase PIKfyve/FAB1 and the PI(3,5)P2 phosphatase FIG4/Sac3, nucleating this complex through homomeric oligomerization and direct contacts with all partners; within this complex VAC14 activates PIKfyve kinase activity, directs FIG4 to endolysosomal membranes, stabilizes FIG4/Sac3 protein by protecting it from proteasomal degradation, and thereby controls the dynamic interconversion of PI3P and PI(3,5)P2 that is essential for endolysosomal trafficking, AMPA receptor endocytosis at synapses, GLUT4 translocation, and neuronal survival.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"VAC14 is a HEAT-repeat scaffold protein that assembles a star-shaped pentameric complex with the lipid kinase PIKfyve/FAB1 and the PI(3,5)P₂ phosphatase FIG4/Sac3, thereby controlling the dynamic interconversion of PI3P and PI(3,5)P₂ on endolysosomal membranes essential for membrane trafficking and neuronal survival. VAC14 oligomerizes through conserved C-terminal motifs—a prerequisite for PIKfyve binding, FIG4 recruitment to vacuolar/endosomal membranes, and activation of PIKfyve kinase activity—and additionally stabilizes FIG4/Sac3 protein by protecting it from proteasomal degradation [PMID:19037259, PMID:23389034, PMID:20630877, PMID:40305106]. Loss of VAC14 causes massive neuronal vacuolation and neurodegeneration in mice, defective endosome-to-TGN retrograde trafficking, impaired AMPA receptor endocytosis at synapses with elevated surface GluA2 and enhanced excitatory transmission, and reduced insulin-stimulated GLUT4 translocation [PMID:17956977, PMID:22842785, PMID:15546865]. Biallelic VAC14 mutations that disrupt the dimerization domain cause PI(3,5)P₂ deficiency in human patients, establishing VAC14 loss-of-function as a cause of a neurodegenerative vacuolar disorder [PMID:27292112, PMID:40305106].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing VAC14 as an upstream activator of PI(3,5)P₂ synthesis resolved how Fab1 kinase activity is regulated: Vac14 deletion nearly abolished PI(3,5)P₂ production in yeast, and FAB1 overexpression suppressed the phenotype, placing Vac14 genetically upstream.\",\n      \"evidence\": \"Yeast genetic deletion, lipid measurements, epistasis via FAB1 overexpression, and MVB sorting assays\",\n      \"pmids\": [\"12062051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Vac14-mediated Fab1 activation unknown at this stage\", \"Whether the relationship is via direct physical interaction was not demonstrated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating that Vac14 physically associates with Fig4 and recruits it to the vacuolar membrane revealed that the same scaffold protein controls both PI(3,5)P₂ synthesis and turnover, establishing the concept of a coordinated regulatory complex.\",\n      \"evidence\": \"Co-immunoprecipitation, GFP localization, in vitro phosphatase assay, and genetic deletion analysis in yeast\",\n      \"pmids\": [\"14528018\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the complex unknown\", \"Whether the interaction is conserved in mammals was not yet tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of human VAC14 (ArPIKfyve) as a direct PIKfyve-binding partner that positively regulates its kinase activity extended the yeast findings to mammals and linked the pathway to insulin-stimulated GLUT4 translocation and glucose uptake in adipocytes.\",\n      \"evidence\": \"Co-IP in mammalian cells and 3T3-L1 adipocytes, siRNA knockdown with in vitro kinase assay and lipid measurements, GLUT4 surface labeling\",\n      \"pmids\": [\"15542851\", \"15546865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How VAC14 stimulates PIKfyve kinase activity mechanistically was not resolved\", \"Whether the mammalian complex also includes a phosphatase subunit was not yet shown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Generation of Vac14 knockout mice revealed that VAC14 is essential for neuronal survival: loss causes massive neurodegeneration and cellular vacuolation, and the ternary PIKfyve-ArPIKfyve-Sac3 (PAS) complex was biochemically reconstituted in mammalian cells, confirming that kinase and phosphatase coexist on the same scaffold.\",\n      \"evidence\": \"Vac14 KO mouse phenotyping, membrane trafficking assays, co-IP/co-fractionation/co-localization of endogenous mammalian complex, in vitro phosphatase assay\",\n      \"pmids\": [\"17956977\", \"17556371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific neuronal cell types and circuits affected were not defined\", \"Whether Sac3/FIG4 stability depends on VAC14 was unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapping the HEAT-repeat architecture of VAC14 and demonstrating that C-terminal-mediated homomeric interaction is required for PAS complex assembly established VAC14 as the obligate scaffold organizing both enzymatic partners.\",\n      \"evidence\": \"Structural prediction, truncation/point mutant co-IP, in vitro kinase assay, GLUT4 translocation in adipocytes, yeast epistasis and mouse mutant analysis\",\n      \"pmids\": [\"19037259\", \"18950639\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of the multimer was lacking\", \"Precise oligomeric state (dimer vs. higher order) was debated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying the PIKfyve Cpn60_TCP1 domain as the determinant for docking with the ArPIKfyve-Sac3 subcomplex, and showing that enzymatic activities of neither partner affect complex stability, clarified that scaffold assembly precedes and is independent of catalysis.\",\n      \"evidence\": \"Domain truncation/point mutant co-IP in triple-transfected COS cells, vacuolation assay\",\n      \"pmids\": [\"19840946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether post-translational modifications regulate assembly was not tested\", \"No structural data for the PIKfyve-VAC14 interface\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovering that VAC14 stabilizes FIG4/Sac3 by attenuating its proteasomal degradation added a non-catalytic regulatory function to the scaffold and explained why the CMT4J-associated Sac3(I41T) mutation reduces Sac3 levels—it disrupts VAC14-mediated stabilization.\",\n      \"evidence\": \"Cycloheximide chase, proteasome inhibitor treatment, co-expression experiments, half-life measurements, siRNA knockdown\",\n      \"pmids\": [\"20630877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether VAC14 shields a specific Sac3 degron was not determined\", \"Ubiquitin ligase responsible for Sac3 turnover was not identified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that VAC14 localizes to synapses and that its loss elevates surface AMPA receptors by impairing regulated endocytosis established a postsynaptic function for PI(3,5)P₂ signaling in excitatory neurotransmission.\",\n      \"evidence\": \"Immunofluorescence, mEPSC electrophysiology, surface GluA2 labeling, rescue by VAC14 re-expression in Vac14⁻/⁻ neurons\",\n      \"pmids\": [\"22842785\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PI(3,5)P₂ acts directly on endocytic machinery or via an effector at the synapse is unclear\", \"Behavioral consequences of enhanced AMPA signaling not characterized\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Precise mutagenesis in yeast confirmed that Vac14 multimerization through C-terminal motifs is an absolute prerequisite for Fab1 and Fig4 binding, resolving the order of assembly: self-association first, then partner recruitment.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, vacuole morphology and hyperosmotic shock assays, site-directed mutagenesis\",\n      \"pmids\": [\"23389034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the multimer is a dimer, trimer, or higher-order oligomer remained unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of biallelic VAC14 mutations in human patients with striatal neurodegeneration and vacuolar pathology, rescued by wild-type VAC14 cDNA, established VAC14 deficiency as a human Mendelian neurodegenerative disease.\",\n      \"evidence\": \"Exome sequencing, fibroblast vacuolation assay, wild-type VAC14 rescue transfection\",\n      \"pmids\": [\"27292112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Small number of families reported\", \"Genotype–phenotype correlation across different VAC14 domains not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Genetic epistasis placing the lysosomal chloride transporter CLCN7 downstream of PI(3,5)P₂ identified a key effector: CLCN7 knockout rescued the enlarged lysosome phenotype of FIG4/VAC14 loss and improved neurological function in Fig4-null mice.\",\n      \"evidence\": \"CLCN7 KO in FIG4-null cells, lysosome morphology and pH measurement, dominant-negative CLCN7 in Fig4-null mouse with neurological and lifespan readouts\",\n      \"pmids\": [\"37363915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLCN7 inhibition is a direct effect of PI(3,5)P₂ binding or indirect is not fully resolved\", \"Therapeutic window for CLCN7 modulation unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A medium-resolution cryo-EM structure revealed VAC14 as a star-shaped pentamer, with two legs binding FIG4 and one additionally occupied by PIKfyve, and showed that patient mutations at VAC14–VAC14 interfaces disrupt oligomerization, complex assembly, and colocalization with retromer-positive endosomes.\",\n      \"evidence\": \"Cryo-EM, AlphaFold2 prediction, yeast interface mutants, PI(3,5)P₂ measurement, FSEC, human VAC14 KO cell complementation\",\n      \"pmids\": [\"40305106\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution atomic model not yet available\", \"How the pentameric architecture positions PIKfyve for catalysis on the membrane is not resolved\", \"Structural basis for VAC14-mediated FIG4 stabilization remains unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include how VAC14 pentamer geometry mechanistically activates PIKfyve kinase activity, which E3 ubiquitin ligase targets FIG4 for degradation in the absence of VAC14, and whether the VAC14 complex directly senses upstream signals to dynamically regulate PI(3,5)P₂ pools.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No reconstituted in vitro activation assay with defined stoichiometry\", \"E3 ligase for FIG4 not identified\", \"Signal-dependent regulation of complex assembly/disassembly not characterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6, 9, 17]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 5, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 10, 12, 17]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2, 20]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 2, 5, 12]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 10, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 13]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [10, 18]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 4, 14]}\n    ],\n    \"complexes\": [\n      \"PIKfyve-ArPIKfyve-Sac3 (PAS) complex\"\n    ],\n    \"partners\": [\n      \"PIKfyve\",\n      \"FIG4\",\n      \"VAC7\",\n      \"ATG18\",\n      \"CLCN7\",\n      \"TBC1D15\",\n      \"RAB9A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}