{"gene":"ATP9A","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2025,"finding":"Cryo-EM structures of human monomeric ATP9A at 2.2 Å resolution in the outward-facing E2P state revealed a unique gating mechanism: unlike canonical P-type ATPases where gating is driven by TM1/TM2 helix movement linked to the A domain, ATP9A outward gating is achieved by movement of TM6-10 helices, likely initiated by TM6 unwinding, creating a larger phospholipid binding cavity. ATP9A does not require the auxiliary subunit CDC50 protein. ATPase activity is significantly increased by negatively charged phospholipids (phosphatidylserine, phosphatidylinositol, and phosphorylated PI species) but not electroneutral phospholipids. Molecular simulations showed spontaneous binding of phosphorylated PI species in the translocation pathway.","method":"Cryo-EM structure determination, ATPase activity assay, molecular dynamics simulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure at 2.2 Å with functional ATPase assay and molecular simulation in a single rigorous study","pmids":["40876594"],"is_preprint":false},{"year":2016,"finding":"ATP9A localizes to phosphatidylserine (PS)-positive early and recycling endosomes (but not late endosomes) in HeLa cells. Depletion of ATP9A delayed recycling of transferrin from endosomes to the plasma membrane and caused accumulation of glucose transporter 1 (GLUT1) in endosomes. ATP9A depletion did not affect early/late endosomal transport and degradation of EGF, nor transport of Shiga toxin B from early/recycling endosomes to the Golgi.","method":"siRNA knockdown, fluorescence microscopy/subcellular fractionation, transferrin recycling assay, GLUT1 localization assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean knockdown with multiple defined cargo readouts (transferrin, GLUT1, EGF, Shiga toxin) in a single focused study","pmids":["27733620"],"is_preprint":false},{"year":2018,"finding":"ATP9A is part of an evolutionarily conserved endosome-associated membrane remodelling complex composed of MON2, DOPEY2, and ATP9A (putative aminophospholipid translocase). This complex associates with SNX3-retromer to mediate endosome-to-Golgi transport of Wntless. In vivo suppression of Ce-tat-5 (C. elegans ATP9A ortholog) phenocopies loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and Wnt signaling defects. Overexpression of an ATPase-inhibited TAT-5(E246Q) mutant also perturbed Wnt signaling, implicating phospholipid flippase activity in SNX3-retromer-mediated Wntless sorting.","method":"Co-immunoprecipitation, in vivo RNAi (C. elegans), ATPase-dead mutant overexpression, Wnt phenotypic assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying complex, in vivo genetic suppression, and ATPase-dead mutant across two systems","pmids":["30213940"],"is_preprint":false},{"year":2019,"finding":"Knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in extracellular vesicle (EV) release. Pharmacological blocking of exosome release in ATP9A knockdown cells significantly reduced total EV numbers, supporting a role for ATP9A specifically in regulating exosome release. The increased EV release was independent of caspase-3 activation.","method":"siRNA knockdown, nanoparticle tracking analysis of EVs, pharmacological inhibition of exosome release","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KD with EV quantification and pharmacological validation, but single lab, single study","pmids":["30947313"],"is_preprint":false},{"year":2023,"finding":"ATP9A localizes mainly to endosomes and modulates RAB5 and RAB11 GTPase activation to regulate the endosomal recycling pathway. Atp9a null mice show decreased muscle strength, memory deficits, hyperkinetic movement disorder, abnormal neurite morphology, and impaired synaptic transmission. ATP9A pathogenic mutants (nonsense variants) have aberrant subcellular localization and cause abnormal endosomal recycling. ATP9A is required for maintaining neuronal neurite morphology and viability of neural cells in vitro.","method":"Atp9a knockout mouse model, primary neuron culture, subcellular localization assay, RAB5/RAB11 activation assay","journal":"Molecular psychiatry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — knockout mouse with multiple phenotypic readouts combined with mechanistic RAB GTPase activity assays and localization studies in a single focused study","pmids":["36604604"],"is_preprint":false},{"year":2023,"finding":"ATP9A interacts with ATP6V1A (a vacuolar ATPase subunit) and facilitates its transport to the plasma membrane, promoting plasma membrane cholesterol accumulation and driving RAC1-dependent macropinocytosis in hepatocellular carcinoma cells. ATP9A is critical for regulating macropinocytosis under nutrient starvation.","method":"Co-immunoprecipitation, subcellular fractionation, cholesterol staining, RAC1 activation assay, macropinocytosis assay","journal":"The Journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP identifying ATP9A–ATP6V1A interaction with functional macropinocytosis and RAC1 readouts, but single lab, single study","pmids":["36715683"],"is_preprint":false},{"year":2025,"finding":"ATP9A and ATP9B form homomeric and/or heteromeric complexes. Both are required for transport of VSVG from the Golgi to the plasma membrane in the exocytic pathway. Flippase activities of both ATP9A and ATP9B are crucial for this transport process. Heteromeric complex formation between ATP9A and ATP9B contributes to retention of ATP9A in the Golgi.","method":"Co-immunoprecipitation, VSVG transport assay, flippase-activity mutants, subcellular localization","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, flippase-dead mutant, and VSVG transport assay in a single focused study","pmids":["40234049"],"is_preprint":false},{"year":2025,"finding":"Overexpression of four selected missense mutant forms of Atp9a in HeLa cells and primary neuronal cultures led to loss of mature dendritic spines. Three missense variants retained endosomal localization while one remained blocked in the endoplasmic reticulum. shRNA-mediated knockdown of ATP9A in neurons decreased the number of dendrites per neuron, demonstrating a role for ATP9A in neuronal arborization and dendritic spine maturation.","method":"Overexpression of mutant ATP9A in HeLa cells and primary neuronal cultures, shRNA knockdown, confocal microscopy of dendritic spines and arborization","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function (mutant OE) and loss-of-function (shRNA KD) with defined neuronal morphology readouts, single lab","pmids":["40226306"],"is_preprint":false},{"year":2025,"finding":"Knockdown of ATP9A in human cells causes exposure of phosphatidylinositol-4-phosphate (PI4P) in the extracellular leaflet of the plasma membrane, and ATP9A expression level correlates with neomycin sensitivity. This implicates ATP9A (ortholog of yeast Neo1) in maintaining PI4P membrane asymmetry at the plasma membrane.","method":"siRNA knockdown, PI4P membrane asymmetry assay (extracellular leaflet detection), neomycin sensitivity assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — knockdown with PI4P asymmetry assay in human cells, single lab, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.03.03.641220"],"is_preprint":true}],"current_model":"ATP9A is a CDC50-independent P4-ATPase phospholipid flippase that localizes to early and recycling endosomes and the trans-Golgi network, where it translocates phospholipids (including phosphatidylserine and phosphatidylinositol species) from the luminal/exoplasmic to the cytoplasmic leaflet via a unique TM6-10-driven gating mechanism; it operates within a MON2–DOPEY2–ATP9A endosomal complex to support SNX3-retromer-mediated cargo sorting, promotes recycling of transferrin and GLUT1 from endosomes to the plasma membrane by modulating RAB5 and RAB11 GTPase activity, forms homo- and heteromeric complexes with ATP9B to drive exocytic VSVG transport from the Golgi, regulates exosome release and macropinocytosis, maintains plasma membrane PI4P asymmetry, and is required for neuronal dendritic arborization and spine maturation."},"narrative":{"mechanistic_narrative":"ATP9A is a CDC50-independent P4-ATPase phospholipid flippase that operates across the endosomal recycling system and trans-Golgi network to translocate phospholipids and thereby govern membrane trafficking [PMID:40876594, PMID:27733620]. Cryo-EM of the outward-facing E2P state shows that, unlike canonical P-type ATPases, ATP9A achieves outward gating through movement of the TM6-10 helices to enlarge a phospholipid-binding cavity, and its ATPase activity is selectively stimulated by negatively charged phospholipids including phosphatidylserine and phosphorylated phosphatidylinositol species [PMID:40876594]. ATP9A localizes to PS-positive early and recycling endosomes and supports recycling of transferrin and GLUT1 to the plasma membrane, while modulating RAB5 and RAB11 GTPase activation to drive endosomal recycling [PMID:27733620, PMID:36604604]. It functions within an evolutionarily conserved MON2-DOPEY2-ATP9A complex that associates with the SNX3-retromer to direct endosome-to-Golgi cargo sorting, a role requiring its flippase activity [PMID:30213940]. ATP9A also forms homomeric and heteromeric complexes with ATP9B that support exocytic VSVG transport from the Golgi and retain ATP9A in the Golgi [PMID:40234049], and it regulates exosome release and RAC1-dependent macropinocytosis [PMID:30947313, PMID:36715683]. Loss of ATP9A produces neuronal phenotypes: Atp9a-null mice show movement, memory, and synaptic defects, and ATP9A is required for dendritic arborization and spine maturation, with pathogenic missense and nonsense variants causing mislocalization and aberrant endosomal recycling [PMID:36604604, PMID:40226306].","teleology":[{"year":2016,"claim":"Established ATP9A's cellular site of action by showing it resides on PS-positive early and recycling endosomes and is selectively required for recycling of specific cargoes rather than general endosomal transport.","evidence":"siRNA knockdown with transferrin recycling, GLUT1, EGF, and Shiga toxin readouts in HeLa cells","pmids":["27733620"],"confidence":"High","gaps":["Did not demonstrate flippase activity biochemically","Mechanism linking flipping to cargo recycling not defined"]},{"year":2018,"claim":"Placed ATP9A in a defined molecular complex by identifying the conserved MON2-DOPEY2-ATP9A assembly that cooperates with SNX3-retromer, linking flippase activity to endosome-to-Golgi cargo sorting.","evidence":"Reciprocal Co-IP, C. elegans RNAi suppression, and ATPase-dead TAT-5(E246Q) mutant in Wnt signaling assays","pmids":["30213940"],"confidence":"High","gaps":["Stoichiometry and architecture of the MON2-DOPEY2-ATP9A complex unresolved","How retromer recruits the complex not defined"]},{"year":2019,"claim":"Extended ATP9A function to vesicle secretion by showing its depletion increases extracellular vesicle and exosome release.","evidence":"siRNA knockdown with nanoparticle tracking analysis and pharmacological exosome inhibition in hepatoma cells","pmids":["30947313"],"confidence":"Medium","gaps":["Single lab, single study","Molecular mechanism connecting flippase activity to exosome release not established"]},{"year":2023,"claim":"Connected the endosomal recycling defect to RAB GTPase regulation and to organismal phenotypes, defining ATP9A as required for neuronal morphology and viability.","evidence":"Atp9a knockout mouse with behavioral/synaptic phenotyping, primary neuron culture, and RAB5/RAB11 activation assays","pmids":["36604604"],"confidence":"High","gaps":["How flippase activity modulates RAB GTPase activation mechanistically unclear","Direct disease causation in patients not established here"]},{"year":2023,"claim":"Revealed a role in nutrient-stress signaling by linking ATP9A to ATP6V1A trafficking, plasma membrane cholesterol, and RAC1-dependent macropinocytosis.","evidence":"Co-IP, subcellular fractionation, cholesterol staining, and macropinocytosis/RAC1 assays in hepatocellular carcinoma cells","pmids":["36715683"],"confidence":"Medium","gaps":["Single lab, single study","Whether ATP6V1A is a direct flippase-dependent client unconfirmed"]},{"year":2025,"claim":"Resolved the structural basis of transport, showing CDC50-independence and a non-canonical TM6-10 gating mechanism activated by anionic phospholipid substrates.","evidence":"2.2 Å cryo-EM of the E2P state, ATPase activity assays, and molecular dynamics simulations","pmids":["40876594"],"confidence":"High","gaps":["Inward-facing states and full transport cycle not captured","Physiological substrate preference in cells not directly mapped to structure"]},{"year":2025,"claim":"Defined a Golgi exocytic function by showing ATP9A-ATP9B homo/heteromeric complexes and their flippase activities drive VSVG transport to the plasma membrane.","evidence":"Reciprocal Co-IP, flippase-dead mutants, and VSVG transport assays with localization analysis","pmids":["40234049"],"confidence":"Medium","gaps":["Functional division of labor between ATP9A and ATP9B not separated","Complex stoichiometry unresolved"]},{"year":2025,"claim":"Linked ATP9A variants directly to neuronal morphological deficits, establishing its requirement for dendritic arborization and spine maturation.","evidence":"Mutant overexpression and shRNA knockdown in HeLa cells and primary neurons with confocal spine/arborization analysis","pmids":["40226306"],"confidence":"Medium","gaps":["Single lab","Causal link between specific recycling cargoes and spine phenotype not established"]},{"year":2025,"claim":"Implicated ATP9A in maintaining plasma membrane PI4P asymmetry, broadening its substrate scope beyond PS.","evidence":"siRNA knockdown with extracellular-leaflet PI4P asymmetry and neomycin sensitivity assays (preprint)","pmids":["bio_10.1101_2025.03.03.641220"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Whether ATP9A directly flips PI4P versus indirectly maintaining asymmetry not resolved"]},{"year":null,"claim":"How ATP9A's phospholipid translocation is mechanistically coupled to RAB GTPase regulation, retromer cargo selection, and the diverse downstream trafficking outputs (recycling, exosome release, macropinocytosis, exocytosis) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking flippase substrate specificity to specific trafficking outcomes","Direct substrates in each pathway not individually validated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[6]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,2,6]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,5]}],"complexes":["MON2-DOPEY2-ATP9A complex","ATP9A-ATP9B complex"],"partners":["MON2","DOPEY2","ATP9B","ATP6V1A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75110","full_name":"Probable phospholipid-transporting ATPase IIA","aliases":["ATPase class II type 9A"],"length_aa":1047,"mass_kda":118.6,"function":"Plays a role in regulating membrane trafficking of cargo proteins, namely endosome to plasma membrane recycling, probably acting through RAB5 and RAB11 activation (PubMed:27733620, PubMed:30213940, PubMed:36604604). Also involved in endosome to trans-Golgi network retrograde transport (PubMed:27733620, PubMed:30213940). In complex with MON2 and DOP1B, regulates SNX3 retromer-mediated endosomal sorting of WLS, a transporter of Wnt morphogens in developing tissues. Participates in the formation of endosomal carriers that direct WLS trafficking back to Golgi, away from lysosomal degradation (PubMed:30213940). Appears to be implicated in intercellular communication by negatively regulating the release of exosomes (PubMed:30947313). The flippase activity towards membrane lipids and its role in membrane asymmetry remains to be proved (PubMed:30947313). Required for the maintenance of neurite morphology and synaptic transmission (By similarity)","subcellular_location":"Early endosome membrane; Recycling endosome membrane; Late endosome membrane; Golgi apparatus, trans-Golgi network membrane; Cell membrane","url":"https://www.uniprot.org/uniprotkb/O75110/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP9A","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"STX12","stoichiometry":0.2},{"gene":"VAMP3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ATP9A","total_profiled":1310},"omim":[{"mim_id":"620242","title":"NEURODEVELOPMENTAL DISORDER WITH POOR GROWTH AND BEHAVIORAL ABNORMALITIES; NEDGBA","url":"https://www.omim.org/entry/620242"},{"mim_id":"614446","title":"ATPase, CLASS II, TYPE 9B; ATP9B","url":"https://www.omim.org/entry/614446"},{"mim_id":"609126","title":"ATPase, CLASS II, TYPE 9A; ATP9A","url":"https://www.omim.org/entry/609126"},{"mim_id":"605868","title":"ATPase, PHOSPHOLIPID TRANSPORTING, 11A; ATP11A","url":"https://www.omim.org/entry/605868"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":87.5}],"url":"https://www.proteinatlas.org/search/ATP9A"},"hgnc":{"alias_symbol":["KIAA0611","ATPIIA"],"prev_symbol":[]},"alphafold":{"accession":"O75110","domains":[{"cath_id":"2.70.150.10","chopping":"156-286","consensus_level":"high","plddt":87.0191,"start":156,"end":286},{"cath_id":"-","chopping":"343-368_826-1045","consensus_level":"high","plddt":87.9726,"start":343,"end":1045},{"cath_id":"3.40.50.1000","chopping":"388-397_658-819","consensus_level":"high","plddt":89.2391,"start":388,"end":819},{"cath_id":"3.40.1110.10","chopping":"400-429_451-474_493-653","consensus_level":"high","plddt":88.6182,"start":400,"end":653},{"cath_id":"1.10.287","chopping":"68-135","consensus_level":"high","plddt":85.2456,"start":68,"end":135}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75110","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75110-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75110-F1-predicted_aligned_error_v6.png","plddt_mean":84.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP9A","jax_strain_url":"https://www.jax.org/strain/search?query=ATP9A"},"sequence":{"accession":"O75110","fasta_url":"https://rest.uniprot.org/uniprotkb/O75110.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75110/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75110"}},"corpus_meta":[{"pmid":"30213940","id":"PMC_30213940","title":"SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30213940","citation_count":71,"is_preprint":false},{"pmid":"27733620","id":"PMC_27733620","title":"The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane.","date":"2016","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/27733620","citation_count":56,"is_preprint":false},{"pmid":"30947313","id":"PMC_30947313","title":"The P4-ATPase ATP9A is a novel determinant of exosome release.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30947313","citation_count":31,"is_preprint":false},{"pmid":"36604604","id":"PMC_36604604","title":"ATP9A deficiency causes ADHD and aberrant endosomal recycling via modulating RAB5 and RAB11 activity.","date":"2023","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/36604604","citation_count":22,"is_preprint":false},{"pmid":"34379057","id":"PMC_34379057","title":"Biallelic truncating variants in ATP9A cause a novel neurodevelopmental disorder involving postnatal microcephaly and failure to thrive.","date":"2021","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34379057","citation_count":20,"is_preprint":false},{"pmid":"36715683","id":"PMC_36715683","title":"The phospholipid flippase ATP9A enhances macropinocytosis to promote nutrient starvation tolerance in hepatocellular carcinoma.","date":"2023","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/36715683","citation_count":12,"is_preprint":false},{"pmid":"40234049","id":"PMC_40234049","title":"Lipid flippases ATP9A and ATP9B form a complex and contribute to the exocytic pathway from the Golgi.","date":"2025","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/40234049","citation_count":5,"is_preprint":false},{"pmid":"39832103","id":"PMC_39832103","title":"Circular RNA ATP9A Stimulates Non-small Cell Lung Cancer Progression via MicroRNA-582-3p/Ribosomal Protein Large P0 Axis and Activating Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway.","date":"2025","source":"Applied biochemistry and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/39832103","citation_count":3,"is_preprint":false},{"pmid":"40226306","id":"PMC_40226306","title":"Heterozygous Missense Variants in the ATPase Phospholipid Transporting 9A Gene, ATP9A, Alter Dendritic Spine Maturation and Cause Dominantly Inherited Nonsyndromic Intellectual Disability.","date":"2025","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/40226306","citation_count":2,"is_preprint":false},{"pmid":"40876594","id":"PMC_40876594","title":"A unique gating mechanism revealed by the cryo-EM structure of monomeric ATP9A flippase.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40876594","citation_count":1,"is_preprint":false},{"pmid":"39871218","id":"PMC_39871218","title":"Integration of Mendelian Randomization to explore the genetic influences of pediatric sepsis: a focus on RGL4, ATP9A, MAP3K7CL, and DDX11L2.","date":"2025","source":"BMC pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/39871218","citation_count":1,"is_preprint":false},{"pmid":"41604004","id":"PMC_41604004","title":"The role of ATP9A (c.1091G > C; p.(Arg364Thr)) variant in cognitive impairment: diagnostic insight from whole exome sequencing.","date":"2026","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/41604004","citation_count":1,"is_preprint":false},{"pmid":"41581045","id":"PMC_41581045","title":"Identification of ATP9A-NFATC2 gene fusion transcript in Behcet's disease, a subtype of uveitis.","date":"2026","source":"Indian journal of ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/41581045","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.03.641220","title":"P4-ATPase control over phosphoinositide membrane asymmetry and neomycin resistance","date":"2025-03-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.03.641220","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.11.13.623339","title":"Lipid flippases ATP9A and ATP9B form a complex and contribute to the secretory pathway from the Golgi apparatus","date":"2024-11-15","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.13.623339","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9875,"output_tokens":2720,"usd":0.035213,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9951,"output_tokens":3242,"usd":0.065403,"stage2_stop_reason":"end_turn"},"total_usd":0.100616,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of human monomeric ATP9A at 2.2 Å resolution in the outward-facing E2P state revealed a unique gating mechanism: unlike canonical P-type ATPases where gating is driven by TM1/TM2 helix movement linked to the A domain, ATP9A outward gating is achieved by movement of TM6-10 helices, likely initiated by TM6 unwinding, creating a larger phospholipid binding cavity. ATP9A does not require the auxiliary subunit CDC50 protein. ATPase activity is significantly increased by negatively charged phospholipids (phosphatidylserine, phosphatidylinositol, and phosphorylated PI species) but not electroneutral phospholipids. Molecular simulations showed spontaneous binding of phosphorylated PI species in the translocation pathway.\",\n      \"method\": \"Cryo-EM structure determination, ATPase activity assay, molecular dynamics simulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure at 2.2 Å with functional ATPase assay and molecular simulation in a single rigorous study\",\n      \"pmids\": [\"40876594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ATP9A localizes to phosphatidylserine (PS)-positive early and recycling endosomes (but not late endosomes) in HeLa cells. Depletion of ATP9A delayed recycling of transferrin from endosomes to the plasma membrane and caused accumulation of glucose transporter 1 (GLUT1) in endosomes. ATP9A depletion did not affect early/late endosomal transport and degradation of EGF, nor transport of Shiga toxin B from early/recycling endosomes to the Golgi.\",\n      \"method\": \"siRNA knockdown, fluorescence microscopy/subcellular fractionation, transferrin recycling assay, GLUT1 localization assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockdown with multiple defined cargo readouts (transferrin, GLUT1, EGF, Shiga toxin) in a single focused study\",\n      \"pmids\": [\"27733620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ATP9A is part of an evolutionarily conserved endosome-associated membrane remodelling complex composed of MON2, DOPEY2, and ATP9A (putative aminophospholipid translocase). This complex associates with SNX3-retromer to mediate endosome-to-Golgi transport of Wntless. In vivo suppression of Ce-tat-5 (C. elegans ATP9A ortholog) phenocopies loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and Wnt signaling defects. Overexpression of an ATPase-inhibited TAT-5(E246Q) mutant also perturbed Wnt signaling, implicating phospholipid flippase activity in SNX3-retromer-mediated Wntless sorting.\",\n      \"method\": \"Co-immunoprecipitation, in vivo RNAi (C. elegans), ATPase-dead mutant overexpression, Wnt phenotypic assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying complex, in vivo genetic suppression, and ATPase-dead mutant across two systems\",\n      \"pmids\": [\"30213940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in extracellular vesicle (EV) release. Pharmacological blocking of exosome release in ATP9A knockdown cells significantly reduced total EV numbers, supporting a role for ATP9A specifically in regulating exosome release. The increased EV release was independent of caspase-3 activation.\",\n      \"method\": \"siRNA knockdown, nanoparticle tracking analysis of EVs, pharmacological inhibition of exosome release\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KD with EV quantification and pharmacological validation, but single lab, single study\",\n      \"pmids\": [\"30947313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP9A localizes mainly to endosomes and modulates RAB5 and RAB11 GTPase activation to regulate the endosomal recycling pathway. Atp9a null mice show decreased muscle strength, memory deficits, hyperkinetic movement disorder, abnormal neurite morphology, and impaired synaptic transmission. ATP9A pathogenic mutants (nonsense variants) have aberrant subcellular localization and cause abnormal endosomal recycling. ATP9A is required for maintaining neuronal neurite morphology and viability of neural cells in vitro.\",\n      \"method\": \"Atp9a knockout mouse model, primary neuron culture, subcellular localization assay, RAB5/RAB11 activation assay\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with multiple phenotypic readouts combined with mechanistic RAB GTPase activity assays and localization studies in a single focused study\",\n      \"pmids\": [\"36604604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ATP9A interacts with ATP6V1A (a vacuolar ATPase subunit) and facilitates its transport to the plasma membrane, promoting plasma membrane cholesterol accumulation and driving RAC1-dependent macropinocytosis in hepatocellular carcinoma cells. ATP9A is critical for regulating macropinocytosis under nutrient starvation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, cholesterol staining, RAC1 activation assay, macropinocytosis assay\",\n      \"journal\": \"The Journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP identifying ATP9A–ATP6V1A interaction with functional macropinocytosis and RAC1 readouts, but single lab, single study\",\n      \"pmids\": [\"36715683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ATP9A and ATP9B form homomeric and/or heteromeric complexes. Both are required for transport of VSVG from the Golgi to the plasma membrane in the exocytic pathway. Flippase activities of both ATP9A and ATP9B are crucial for this transport process. Heteromeric complex formation between ATP9A and ATP9B contributes to retention of ATP9A in the Golgi.\",\n      \"method\": \"Co-immunoprecipitation, VSVG transport assay, flippase-activity mutants, subcellular localization\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, flippase-dead mutant, and VSVG transport assay in a single focused study\",\n      \"pmids\": [\"40234049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Overexpression of four selected missense mutant forms of Atp9a in HeLa cells and primary neuronal cultures led to loss of mature dendritic spines. Three missense variants retained endosomal localization while one remained blocked in the endoplasmic reticulum. shRNA-mediated knockdown of ATP9A in neurons decreased the number of dendrites per neuron, demonstrating a role for ATP9A in neuronal arborization and dendritic spine maturation.\",\n      \"method\": \"Overexpression of mutant ATP9A in HeLa cells and primary neuronal cultures, shRNA knockdown, confocal microscopy of dendritic spines and arborization\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function (mutant OE) and loss-of-function (shRNA KD) with defined neuronal morphology readouts, single lab\",\n      \"pmids\": [\"40226306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Knockdown of ATP9A in human cells causes exposure of phosphatidylinositol-4-phosphate (PI4P) in the extracellular leaflet of the plasma membrane, and ATP9A expression level correlates with neomycin sensitivity. This implicates ATP9A (ortholog of yeast Neo1) in maintaining PI4P membrane asymmetry at the plasma membrane.\",\n      \"method\": \"siRNA knockdown, PI4P membrane asymmetry assay (extracellular leaflet detection), neomycin sensitivity assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — knockdown with PI4P asymmetry assay in human cells, single lab, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.03.03.641220\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ATP9A is a CDC50-independent P4-ATPase phospholipid flippase that localizes to early and recycling endosomes and the trans-Golgi network, where it translocates phospholipids (including phosphatidylserine and phosphatidylinositol species) from the luminal/exoplasmic to the cytoplasmic leaflet via a unique TM6-10-driven gating mechanism; it operates within a MON2–DOPEY2–ATP9A endosomal complex to support SNX3-retromer-mediated cargo sorting, promotes recycling of transferrin and GLUT1 from endosomes to the plasma membrane by modulating RAB5 and RAB11 GTPase activity, forms homo- and heteromeric complexes with ATP9B to drive exocytic VSVG transport from the Golgi, regulates exosome release and macropinocytosis, maintains plasma membrane PI4P asymmetry, and is required for neuronal dendritic arborization and spine maturation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ATP9A is a CDC50-independent P4-ATPase phospholipid flippase that operates across the endosomal recycling system and trans-Golgi network to translocate phospholipids and thereby govern membrane trafficking [#0, #1]. Cryo-EM of the outward-facing E2P state shows that, unlike canonical P-type ATPases, ATP9A achieves outward gating through movement of the TM6-10 helices to enlarge a phospholipid-binding cavity, and its ATPase activity is selectively stimulated by negatively charged phospholipids including phosphatidylserine and phosphorylated phosphatidylinositol species [#0]. ATP9A localizes to PS-positive early and recycling endosomes and supports recycling of transferrin and GLUT1 to the plasma membrane, while modulating RAB5 and RAB11 GTPase activation to drive endosomal recycling [#1, #4]. It functions within an evolutionarily conserved MON2-DOPEY2-ATP9A complex that associates with the SNX3-retromer to direct endosome-to-Golgi cargo sorting, a role requiring its flippase activity [#2]. ATP9A also forms homomeric and heteromeric complexes with ATP9B that support exocytic VSVG transport from the Golgi and retain ATP9A in the Golgi [#6], and it regulates exosome release and RAC1-dependent macropinocytosis [#3, #5]. Loss of ATP9A produces neuronal phenotypes: Atp9a-null mice show movement, memory, and synaptic defects, and ATP9A is required for dendritic arborization and spine maturation, with pathogenic missense and nonsense variants causing mislocalization and aberrant endosomal recycling [#4, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established ATP9A's cellular site of action by showing it resides on PS-positive early and recycling endosomes and is selectively required for recycling of specific cargoes rather than general endosomal transport.\",\n      \"evidence\": \"siRNA knockdown with transferrin recycling, GLUT1, EGF, and Shiga toxin readouts in HeLa cells\",\n      \"pmids\": [\"27733620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not demonstrate flippase activity biochemically\", \"Mechanism linking flipping to cargo recycling not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed ATP9A in a defined molecular complex by identifying the conserved MON2-DOPEY2-ATP9A assembly that cooperates with SNX3-retromer, linking flippase activity to endosome-to-Golgi cargo sorting.\",\n      \"evidence\": \"Reciprocal Co-IP, C. elegans RNAi suppression, and ATPase-dead TAT-5(E246Q) mutant in Wnt signaling assays\",\n      \"pmids\": [\"30213940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the MON2-DOPEY2-ATP9A complex unresolved\", \"How retromer recruits the complex not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended ATP9A function to vesicle secretion by showing its depletion increases extracellular vesicle and exosome release.\",\n      \"evidence\": \"siRNA knockdown with nanoparticle tracking analysis and pharmacological exosome inhibition in hepatoma cells\",\n      \"pmids\": [\"30947313\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single study\", \"Molecular mechanism connecting flippase activity to exosome release not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected the endosomal recycling defect to RAB GTPase regulation and to organismal phenotypes, defining ATP9A as required for neuronal morphology and viability.\",\n      \"evidence\": \"Atp9a knockout mouse with behavioral/synaptic phenotyping, primary neuron culture, and RAB5/RAB11 activation assays\",\n      \"pmids\": [\"36604604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How flippase activity modulates RAB GTPase activation mechanistically unclear\", \"Direct disease causation in patients not established here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a role in nutrient-stress signaling by linking ATP9A to ATP6V1A trafficking, plasma membrane cholesterol, and RAC1-dependent macropinocytosis.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, cholesterol staining, and macropinocytosis/RAC1 assays in hepatocellular carcinoma cells\",\n      \"pmids\": [\"36715683\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single study\", \"Whether ATP6V1A is a direct flippase-dependent client unconfirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the structural basis of transport, showing CDC50-independence and a non-canonical TM6-10 gating mechanism activated by anionic phospholipid substrates.\",\n      \"evidence\": \"2.2 Å cryo-EM of the E2P state, ATPase activity assays, and molecular dynamics simulations\",\n      \"pmids\": [\"40876594\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Inward-facing states and full transport cycle not captured\", \"Physiological substrate preference in cells not directly mapped to structure\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a Golgi exocytic function by showing ATP9A-ATP9B homo/heteromeric complexes and their flippase activities drive VSVG transport to the plasma membrane.\",\n      \"evidence\": \"Reciprocal Co-IP, flippase-dead mutants, and VSVG transport assays with localization analysis\",\n      \"pmids\": [\"40234049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional division of labor between ATP9A and ATP9B not separated\", \"Complex stoichiometry unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked ATP9A variants directly to neuronal morphological deficits, establishing its requirement for dendritic arborization and spine maturation.\",\n      \"evidence\": \"Mutant overexpression and shRNA knockdown in HeLa cells and primary neurons with confocal spine/arborization analysis\",\n      \"pmids\": [\"40226306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Causal link between specific recycling cargoes and spine phenotype not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated ATP9A in maintaining plasma membrane PI4P asymmetry, broadening its substrate scope beyond PS.\",\n      \"evidence\": \"siRNA knockdown with extracellular-leaflet PI4P asymmetry and neomycin sensitivity assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.03.641220\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Whether ATP9A directly flips PI4P versus indirectly maintaining asymmetry not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ATP9A's phospholipid translocation is mechanistically coupled to RAB GTPase regulation, retromer cargo selection, and the diverse downstream trafficking outputs (recycling, exosome release, macropinocytosis, exocytosis) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking flippase substrate specificity to specific trafficking outcomes\", \"Direct substrates in each pathway not individually validated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140359\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [\n      \"MON2-DOPEY2-ATP9A complex\",\n      \"ATP9A-ATP9B complex\"\n    ],\n    \"partners\": [\n      \"MON2\",\n      \"DOPEY2\",\n      \"ATP9B\",\n      \"ATP6V1A\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}