{"gene":"ATP6V1D","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1995,"finding":"Subunit D of bovine V-ATPase (ATP6V1D) was identified by partial proteolysis and amino acid sequencing of chromaffin granule V-ATPase. A cDNA encoding a 247-amino acid protein (MW ~28 kDa) was cloned. The yeast ortholog VMA8 shares 55% identity, and deletion of VMA8 abolished V-ATPase activity and vacuolar acidification (loss of quinacrine accumulation), establishing subunit D as an integral and essential subunit of the V1 catalytic sector. Structural analysis suggested analogy to the gamma subunit of F-ATPases.","method":"Protein sequencing, cDNA cloning, yeast gene disruption (null mutant), quinacrine fluorescence assay for vacuolar acidification, complementation rescue","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (sequencing, KO, functional assay, complementation), foundational study replicated by independent lab","pmids":["7831318"],"is_preprint":false},{"year":1995,"finding":"Yeast VMA8 (ATP6V1D ortholog) encodes the 32-kDa V1 subunit of V-ATPase. Disruption of VMA8 resulted in complete loss of V-ATPase ATPase activity in vacuolar membranes. Immunoblot and density gradient fractionation showed that in vma8Δ cells, V1 subunits failed to associate with the vacuolar membrane while Vo subunits remained stable there, demonstrating that subunit D is required for assembly of the V1 sector onto the membrane. Vma8p was found only in the fully assembled V-ATPase and could not assemble onto vacuolar membranes in the absence of other V1 subunits.","method":"Gene disruption, ATPase activity assay on isolated vacuolar membranes, immunoblot analysis, density gradient fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal biochemical and genetic methods, independent confirmation of PMID:7831318","pmids":["7797485"],"is_preprint":false},{"year":2001,"finding":"The mouse V-ATPase subunit D gene (Atp6m, ortholog of human ATP6V1D) was cloned and mapped to chromosome 12 in a region syntenic with human chromosome 14q24. Evolutionary analysis across multiple species showed that subunit D is highly conserved and under strong negative selection, consistent with an essential, multi-functional role in V-ATPase-dependent cellular processes including pH regulation, vesicular transport, endocytosis, secretion, and apoptosis.","method":"cDNA cloning, chromosomal mapping, evolutionary/phylogenetic analysis","journal":"Cytogenetics and cell genetics","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct cloning and chromosomal localization with evolutionary context; single lab, no functional perturbation","pmids":["11435709"],"is_preprint":false},{"year":2024,"finding":"ATP6V1D was identified as a key metabolic regulator of hepatocellular carcinoma (HCC) stemness via a metabolic CRISPR-Cas9 knockout screen. Mechanistically, ATP6V1D maintains autophagic flux by two mechanisms: (1) promoting lysosomal acidification, and (2) enhancing the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, thereby facilitating autophagosome-lysosome fusion. Knockdown of ATP6V1D inhibited HCC stemness and malignant progression in vitro and in vivo. Silencing CHMP4B or IST1 phenocopied ATP6V1D loss.","method":"CRISPR-Cas9 knockout screen, shRNA knockdown, co-immunoprecipitation (co-IP), immunofluorescence, in vitro and in vivo tumor models, autophagic flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including CRISPR screen, co-IP, in vivo models, and epistasis via CHMP4B/IST1 silencing","pmids":["39316516"],"is_preprint":false},{"year":2026,"finding":"ATP6V1D (lysosomal V-ATPase subunit D) is upregulated in T. gondii-resistant Lewis rats in response to infection. CRISPR/Cas9 disruption of ATP6V1D in rat macrophages (NR8383) impaired v-ATPase function, leading to defective lysosomal acidification and increased intracellular T. gondii growth. Overexpression of GIMAP5 or GIMAP6 in ATP6V1D-knockout cells still reduced parasite growth (2.5- to 5-fold), but less robustly than in wild-type cells, establishing that ATP6V1D and GIMAPs act additively: GIMAPs drive lysosome translocation to the parasitophorous vacuole membrane while ATP6V1D enables lysosomal acidification to activate hydrolytic enzymes for parasite degradation.","method":"CRISPR/Cas9 knockout, transgene overexpression, lysosomal acidification assays, intracellular parasite growth quantification, genetic epistasis","journal":"Microbiology spectrum","confidence":"High","confidence_rationale":"Tier 2 — CRISPR KO with defined functional phenotype, epistasis with GIMAP overexpression, multiple orthogonal readouts","pmids":["41972776"],"is_preprint":false}],"current_model":"ATP6V1D encodes the D subunit of the V1 catalytic sector of the vacuolar H+-ATPase; it is essential for V1 assembly onto the membrane and for ATP hydrolysis-coupled proton pumping that acidifies lysosomes and vacuoles, and in cancer and immune contexts it additionally promotes autophagic flux by facilitating ESCRT-III (CHMP4B–IST1) complex assembly for autophagosome–lysosome fusion, and enables lysosomal killing of intracellular pathogens by acidification-dependent activation of hydrolytic enzymes."},"narrative":{"teleology":[{"year":1995,"claim":"Identification of subunit D as an integral, essential component of the V1 sector resolved how the catalytic domain achieves its stoichiometric assembly and established its requirement for V-ATPase-dependent vacuolar acidification.","evidence":"Protein sequencing of bovine chromaffin granule V-ATPase, cDNA cloning, yeast VMA8 disruption with quinacrine acidification assay and complementation rescue (PMID:7831318); independent yeast gene disruption with ATPase activity assay and immunoblot/density gradient fractionation showing V1 failure to associate with vacuolar membranes (PMID:7797485)","pmids":["7831318","7797485"],"confidence":"High","gaps":["Atomic-resolution structure of subunit D within the V1 complex was not determined","Mechanism by which subunit D nucleates V1 assembly onto Vo was not defined","Mammalian loss-of-function data were lacking"]},{"year":2001,"claim":"Cloning and chromosomal mapping of the mouse ortholog, together with deep evolutionary conservation analysis, established that subunit D is under strong purifying selection, consistent with an indispensable role across eukaryotes.","evidence":"cDNA cloning, chromosomal mapping to mouse chromosome 12 (syntenic with human 14q24), phylogenetic analysis","pmids":["11435709"],"confidence":"Medium","gaps":["No functional perturbation was performed in mammalian cells","Tissue-specific expression or regulation was not characterized"]},{"year":2024,"claim":"Discovery that ATP6V1D sustains autophagic flux not only through lysosomal acidification but also by promoting CHMP4B–IST1 interaction and ESCRT-III assembly for autophagosome–lysosome fusion revealed a non-canonical, acidification-independent arm of V-ATPase subunit function in cancer stemness.","evidence":"CRISPR-Cas9 metabolic knockout screen in HCC cells, shRNA knockdown, co-immunoprecipitation of CHMP4B–IST1, immunofluorescence, in vitro and in vivo tumor models, epistasis via CHMP4B/IST1 silencing","pmids":["39316516"],"confidence":"High","gaps":["Direct physical interaction between ATP6V1D and the CHMP4B–IST1 complex was not shown; the mechanism of enhancement is unclear","Whether the ESCRT-III-promoting function operates outside of HCC or cancer contexts is untested","Structural basis for subunit D engagement with ESCRT machinery is unknown"]},{"year":2026,"claim":"Demonstration that ATP6V1D-dependent lysosomal acidification is required for macrophage killing of intracellular Toxoplasma gondii, acting additively with GIMAP-mediated lysosome translocation, established a cell-autonomous innate immune role for the V-ATPase D subunit.","evidence":"CRISPR/Cas9 knockout in rat NR8383 macrophages, lysosomal acidification assays, intracellular parasite growth quantification, genetic epistasis with GIMAP5/6 overexpression","pmids":["41972776"],"confidence":"High","gaps":["Whether this innate immune role extends to human macrophages or other intracellular pathogens is untested","The molecular interface between ATP6V1D loss and impaired hydrolytic enzyme activation has not been dissected"]},{"year":null,"claim":"Key unresolved questions include the structural basis of subunit D within the assembled mammalian V-ATPase, the direct molecular mechanism by which ATP6V1D promotes ESCRT-III assembly independently of acidification, and whether ATP6V1D mutations cause human Mendelian disease.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of mammalian V-ATPase with subunit D resolved in mechanistic detail","No direct binding interface mapped between ATP6V1D and ESCRT-III components","No human genetic disease attributed to ATP6V1D loss-of-function"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005773","term_label":"vacuole","supporting_discovery_ids":[0,1]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4]}],"complexes":["V-ATPase (V1 sector)"],"partners":["CHMP4B","IST1","GIMAP5","GIMAP6"],"other_free_text":[]},"mechanistic_narrative":"ATP6V1D encodes the D subunit of the V1 catalytic sector of the vacuolar H+-ATPase (V-ATPase), functioning as an essential structural component required for V1 assembly onto the Vo membrane sector and for ATP hydrolysis-driven proton pumping that acidifies lysosomes and vacuoles [PMID:7831318, PMID:7797485]. Beyond canonical acidification, ATP6V1D maintains autophagic flux in hepatocellular carcinoma cells by promoting ESCRT-III complex assembly through enhancement of the CHMP4B–IST1 interaction, thereby facilitating autophagosome–lysosome fusion and sustaining cancer cell stemness [PMID:39316516]. In macrophages, ATP6V1D-dependent lysosomal acidification activates hydrolytic enzymes required for degradation of intracellular Toxoplasma gondii, acting additively with GIMAP-mediated lysosome translocation to restrict parasite growth [PMID:41972776]."},"prefetch_data":{"uniprot":{"accession":"Q9Y5K8","full_name":"V-type proton ATPase subunit D","aliases":["V-ATPase 28 kDa accessory protein","Vacuolar proton pump subunit D"],"length_aa":247,"mass_kda":28.3,"function":"Subunit of the V1 complex of vacuolar(H+)-ATPase (V-ATPase), a multisubunit enzyme composed of a peripheral complex (V1) that hydrolyzes ATP and a membrane integral complex (V0) that translocates protons (PubMed:33065002). V-ATPase is responsible for acidifying and maintaining the pH of intracellular compartments and in some cell types, is targeted to the plasma membrane, where it is responsible for acidifying the extracellular environment (By similarity). May play a role in cilium biogenesis through regulation of the transport and the localization of proteins to the cilium (PubMed:21844891)","subcellular_location":"Membrane; Cytoplasmic vesicle, clathrin-coated vesicle membrane; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cell projection, cilium","url":"https://www.uniprot.org/uniprotkb/Q9Y5K8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ATP6V1D","classification":"Common Essential","n_dependent_lines":1050,"n_total_lines":1208,"dependency_fraction":0.8692052980132451},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000100554","cell_line_id":"CID001924","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":2},{"compartment":"vesicles","grade":2}],"interactors":[{"gene":"ATP6AP2","stoichiometry":10.0},{"gene":"ATP6V1A","stoichiometry":10.0},{"gene":"ATP6V1B2","stoichiometry":10.0},{"gene":"ATP6V1H","stoichiometry":10.0},{"gene":"ATP6V1E1","stoichiometry":10.0},{"gene":"ATP6V1G1","stoichiometry":10.0},{"gene":"DAG1","stoichiometry":4.0},{"gene":"ATP6V0A1","stoichiometry":0.2},{"gene":"ATP6V0D1","stoichiometry":0.2},{"gene":"CEP135","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001924","total_profiled":1310},"omim":[{"mim_id":"614780","title":"SORTING NEXIN 10; SNX10","url":"https://www.omim.org/entry/614780"},{"mim_id":"609398","title":"ATPase, H+ TRANSPORTING, LYSOSOMAL, 34-KD, V1 SUBUNIT D; ATP6V1D","url":"https://www.omim.org/entry/609398"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ATP6V1D"},"hgnc":{"alias_symbol":["VATD","VMA8"],"prev_symbol":["ATP6M"]},"alphafold":{"accession":"Q9Y5K8","domains":[{"cath_id":"1.10.287.3240","chopping":"12-184","consensus_level":"high","plddt":90.9329,"start":12,"end":184}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5K8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5K8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5K8-F1-predicted_aligned_error_v6.png","plddt_mean":86.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ATP6V1D","jax_strain_url":"https://www.jax.org/strain/search?query=ATP6V1D"},"sequence":{"accession":"Q9Y5K8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y5K8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y5K8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5K8"}},"corpus_meta":[{"pmid":"7831318","id":"PMC_7831318","title":"A bovine cDNA and a yeast gene (VMA8) encoding the subunit D of the vacuolar H(+)-ATPase.","date":"1995","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/7831318","citation_count":78,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10868806","id":"PMC_10868806","title":"Quinupristin/dalfopristin-resistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany.","date":"2000","source":"Microbial drug resistance (Larchmont, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/10868806","citation_count":57,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11841212","id":"PMC_11841212","title":"Crystal structure of Vat(D): an acetyltransferase that inactivates streptogramin group A antibiotics.","date":"2002","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11841212","citation_count":52,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"7797485","id":"PMC_7797485","title":"VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase required for function and assembly of the enzyme complex.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7797485","citation_count":48,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11600385","id":"PMC_11600385","title":"Indication of transposition of a mobile DNA element containing the vat(D) and erm(B) genes in Enterococcus faecium.","date":"2001","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/11600385","citation_count":27,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24135791","id":"PMC_24135791","title":"Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.","date":"2013","source":"Microbial pathogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/24135791","citation_count":9,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39316516","id":"PMC_39316516","title":"ATP6V1D drives hepatocellular carcinoma stemness and progression via both lysosome acidification-dependent and -independent mechanisms.","date":"2024","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/39316516","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11435709","id":"PMC_11435709","title":"cDNA cloning, chromosomal localization and evolutionary analysis of mouse vacuolar ATPase subunit D, Atp6m.","date":"2001","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11435709","citation_count":4,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"40668679","id":"PMC_40668679","title":"Arabidopsis phospholipase Dζ2 facilitates vacuolar acidification and autophagy under phosphorus starvation by interacting with VATD.","date":"2025","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40668679","citation_count":1,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26249703","id":"PMC_26249703","title":"Expression, purification, crystallization and X-ray crystallographic analysis of the periplasmic binding protein VatD from Vibrio vulnificus M2799.","date":"2015","source":"Acta crystallographica. 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32001091","citation_count":168,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8934,"output_tokens":1388,"usd":0.023811},"stage2":{"model":"claude-opus-4-6","input_tokens":4614,"output_tokens":1456,"usd":0.089205},"total_usd":0.26606,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":20499,"output_tokens":2481,"usd":0.049356},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":5200,"output_tokens":1725,"usd":0.103688}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"Subunit D (ATP6V1D/VMA8) is an integral subunit of the catalytic V1 sector of vacuolar H+-ATPase; its structural analysis suggests analogy to the gamma subunit of F-ATPases. Disruption of VMA8 in yeast abolished vacuolar H+-ATPase activity and prevented V1 subunits from associating with the vacuolar membrane, while Vo subunits were transported normally.\",\n      \"method\": \"Protein sequencing, cDNA cloning, yeast null mutant (delta vma8::URA3) phenotypic rescue, quinacrine accumulation assay, Northern blot\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including genetic disruption, complementation rescue, and functional assay; replicated by independent lab (PMID:7797485)\",\n      \"pmids\": [\"7831318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"VMA8 (yeast ortholog of ATP6V1D) encodes a 32-kDa V1 subunit required for both V-ATPase function and assembly; in vma8Δ cells V1 subunits fail to associate with the vacuolar membrane but Vo subunits are stable there. Vma8p associates only with the fully assembled V-ATPase and cannot assemble onto vacuolar membranes in the absence of other V1 subunits.\",\n      \"method\": \"VMA8 gene disruption, vacuolar membrane isolation and ATPase activity assay, immunoblot, density gradient fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined biochemical phenotype, multiple orthogonal methods, independently consistent with PMID:7831318\",\n      \"pmids\": [\"7797485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ATP6V1D promotes lysosomal acidification and autophagic flux in hepatocellular carcinoma cells. Mechanistically, ATP6V1D not only acidifies lysosomes but also enhances the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, thereby facilitating autophagosome-lysosome fusion.\",\n      \"method\": \"CRISPR-Cas9 metabolic screen, ATP6V1D knockdown in vitro and in vivo, co-immunoprecipitation (CHMP4B–IST1 interaction), autophagic flux assays, bafilomycin A1 treatment\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — single lab with multiple orthogonal methods (CRISPR screen, KD, co-IP, functional assays) but not independently replicated\",\n      \"pmids\": [\"39316516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ATP6V1D is required for lysosomal acidification in macrophages; CRISPR/Cas9 disruption of ATP6V1D impaired v-ATPase function, leading to defective lysosomal acidification and increased intracellular Toxoplasma gondii growth. ATP6V1D acts additively with GIMAP-driven lysosome-PVM fusion to activate hydrolytic enzymes.\",\n      \"method\": \"CRISPR/Cas9 knockout in rat macrophage (NR8383) cell line, GIMAP transgene overexpression, parasite growth assay, lysosomal acidification assay\",\n      \"journal\": \"Microbiology spectrum\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype and epistasis via GIMAP overexpression; single lab\",\n      \"pmids\": [\"41972776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mouse Atp6m (Atp6v1d ortholog) maps to chromosome 12 in a region syntenic with human chromosome 14q24; evolutionary analysis across species reveals it is highly conserved and under strong negative selection, consistent with an essential role in multiple cellular processes including cytosolic pH regulation, vesicular transport, endocytosis, secretion, and apoptosis.\",\n      \"method\": \"cDNA cloning, chromosomal mapping, evolutionary sequence analysis\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational/evolutionary analysis only; no direct functional experiment\",\n      \"pmids\": [\"11435709\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP6V1D (subunit D of V-ATPase) is an integral component of the V1 catalytic sector required for complex assembly and proton-pumping activity; it drives lysosomal acidification which sustains autophagic flux, and additionally promotes autophagosome-lysosome fusion by enhancing ESCRT-III (CHMP4B–IST1) assembly, while in immune contexts it cooperates with GIMAP-mediated lysosome trafficking to activate hydrolytic killing of intracellular pathogens.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEPT papers.\n\n**Classification:**\n\n- [1] PMID:7831318 — KEEP (bovine/yeast V-ATPase subunit D = ATP6V1D ortholog)\n- [2] PMID:10868806 — EXCLUDE (alias collision: vatD = streptogramin acetyltransferase in Enterococcus, unrelated)\n- [3] PMID:11841212 — EXCLUDE (alias collision: Vat(D) = acetyltransferase in E. faecium)\n- [4] PMID:7797485 — KEEP (yeast VMA8 = ATP6V1D ortholog, V-ATPase subunit D)\n- [5] PMID:11600385 — EXCLUDE (alias collision: vat(D) streptogramin resistance gene)\n- [6] PMID:24135791 — EXCLUDE (alias collision: VatD = periplasmic siderophore-binding protein in Vibrio)\n- [7] PMID:39316516 — KEEP (ATP6V1D in HCC, direct mechanistic study)\n- [8] PMID:11435709 — KEEP (mouse Atp6m = ATP6V1D ortholog, cloning/mapping)\n- [9] PMID:40668679 — EXCLUDE (symbol collision: VATD in Arabidopsis — plant organism, fundamentally different cellular context)\n- [10] PMID:26249703 — EXCLUDE (alias collision: VatD = Vibrio periplasmic binding protein)\n- [11] PMID:40911183 — EXCLUDE (biomarker/expression study, no direct mechanism)\n- [12] PMID:40693774 — EXCLUDE (alias collision: vatD = streptogramin resistance gene in C. botulinum)\n- [13] PMID:41972776 — KEEP (ATP6V1D in Toxoplasma restriction, direct functional study)\n- [14] PMID:40309610 — EXCLUDE (SNP association study, no direct mechanism)\n- [15] bio_10.1101_2025.01.31.635826 — EXCLUDE (alias collision: VatD acetyltransferase)\n\n**Additional curated papers:**\n- PMID:22053050 — KEEP (v-ATPase mechanism with amino acid sensing/mTORC1; general V-ATPase but relevant)\n- PMID:11836511, 9442887, 10221984, 10224039, 9210392, 32001091 — General V-ATPase reviews; only include if they contain specific mechanistic findings about subunit D\n- PMID:26186194, 28514442, 26496610, 25416956, 32296183, 33961781, 22939629, 26344197 — Interactome studies (BioPlex etc.); KEEP if ATP6V1D interactions specifically described\n- Others (cDNA collections, genomics) — largely infrastructure, no specific ATP6V1D mechanism\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"Subunit D of bovine V-ATPase (ATP6V1D) was identified by partial proteolysis and amino acid sequencing of chromaffin granule V-ATPase. A cDNA encoding a 247-amino acid protein (MW ~28 kDa) was cloned. The yeast ortholog VMA8 shares 55% identity, and deletion of VMA8 abolished V-ATPase activity and vacuolar acidification (loss of quinacrine accumulation), establishing subunit D as an integral and essential subunit of the V1 catalytic sector. Structural analysis suggested analogy to the gamma subunit of F-ATPases.\",\n      \"method\": \"Protein sequencing, cDNA cloning, yeast gene disruption (null mutant), quinacrine fluorescence assay for vacuolar acidification, complementation rescue\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (sequencing, KO, functional assay, complementation), foundational study replicated by independent lab\",\n      \"pmids\": [\"7831318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Yeast VMA8 (ATP6V1D ortholog) encodes the 32-kDa V1 subunit of V-ATPase. Disruption of VMA8 resulted in complete loss of V-ATPase ATPase activity in vacuolar membranes. Immunoblot and density gradient fractionation showed that in vma8Δ cells, V1 subunits failed to associate with the vacuolar membrane while Vo subunits remained stable there, demonstrating that subunit D is required for assembly of the V1 sector onto the membrane. Vma8p was found only in the fully assembled V-ATPase and could not assemble onto vacuolar membranes in the absence of other V1 subunits.\",\n      \"method\": \"Gene disruption, ATPase activity assay on isolated vacuolar membranes, immunoblot analysis, density gradient fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal biochemical and genetic methods, independent confirmation of PMID:7831318\",\n      \"pmids\": [\"7797485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The mouse V-ATPase subunit D gene (Atp6m, ortholog of human ATP6V1D) was cloned and mapped to chromosome 12 in a region syntenic with human chromosome 14q24. Evolutionary analysis across multiple species showed that subunit D is highly conserved and under strong negative selection, consistent with an essential, multi-functional role in V-ATPase-dependent cellular processes including pH regulation, vesicular transport, endocytosis, secretion, and apoptosis.\",\n      \"method\": \"cDNA cloning, chromosomal mapping, evolutionary/phylogenetic analysis\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct cloning and chromosomal localization with evolutionary context; single lab, no functional perturbation\",\n      \"pmids\": [\"11435709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ATP6V1D was identified as a key metabolic regulator of hepatocellular carcinoma (HCC) stemness via a metabolic CRISPR-Cas9 knockout screen. Mechanistically, ATP6V1D maintains autophagic flux by two mechanisms: (1) promoting lysosomal acidification, and (2) enhancing the interaction between CHMP4B and IST1 to foster ESCRT-III complex assembly, thereby facilitating autophagosome-lysosome fusion. Knockdown of ATP6V1D inhibited HCC stemness and malignant progression in vitro and in vivo. Silencing CHMP4B or IST1 phenocopied ATP6V1D loss.\",\n      \"method\": \"CRISPR-Cas9 knockout screen, shRNA knockdown, co-immunoprecipitation (co-IP), immunofluorescence, in vitro and in vivo tumor models, autophagic flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including CRISPR screen, co-IP, in vivo models, and epistasis via CHMP4B/IST1 silencing\",\n      \"pmids\": [\"39316516\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ATP6V1D (lysosomal V-ATPase subunit D) is upregulated in T. gondii-resistant Lewis rats in response to infection. CRISPR/Cas9 disruption of ATP6V1D in rat macrophages (NR8383) impaired v-ATPase function, leading to defective lysosomal acidification and increased intracellular T. gondii growth. Overexpression of GIMAP5 or GIMAP6 in ATP6V1D-knockout cells still reduced parasite growth (2.5- to 5-fold), but less robustly than in wild-type cells, establishing that ATP6V1D and GIMAPs act additively: GIMAPs drive lysosome translocation to the parasitophorous vacuole membrane while ATP6V1D enables lysosomal acidification to activate hydrolytic enzymes for parasite degradation.\",\n      \"method\": \"CRISPR/Cas9 knockout, transgene overexpression, lysosomal acidification assays, intracellular parasite growth quantification, genetic epistasis\",\n      \"journal\": \"Microbiology spectrum\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with defined functional phenotype, epistasis with GIMAP overexpression, multiple orthogonal readouts\",\n      \"pmids\": [\"41972776\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ATP6V1D encodes the D subunit of the V1 catalytic sector of the vacuolar H+-ATPase; it is essential for V1 assembly onto the membrane and for ATP hydrolysis-coupled proton pumping that acidifies lysosomes and vacuoles, and in cancer and immune contexts it additionally promotes autophagic flux by facilitating ESCRT-III (CHMP4B–IST1) complex assembly for autophagosome–lysosome fusion, and enables lysosomal killing of intracellular pathogens by acidification-dependent activation of hydrolytic enzymes.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ATP6V1D is an essential subunit of the V1 catalytic sector of the vacuolar H⁺-ATPase (V-ATPase), required for both complex assembly and proton-pumping activity. Genetic disruption in yeast demonstrated that loss of ATP6V1D (VMA8) prevents V1 subunits from associating with the vacuolar membrane while Vo subunits are transported normally, establishing its role as a critical assembly factor for the holoenzyme [PMID:7831318, PMID:7797485]. Beyond its canonical role in lysosomal acidification, ATP6V1D promotes autophagosome–lysosome fusion by enhancing ESCRT-III complex assembly through facilitation of the CHMP4B–IST1 interaction in hepatocellular carcinoma cells [PMID:39316516]. In macrophages, ATP6V1D-dependent lysosomal acidification cooperates with GIMAP-mediated lysosome trafficking to activate hydrolytic killing of intracellular pathogens such as Toxoplasma gondii [PMID:41972776].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"The identity of V-ATPase subunit D as an integral V1-sector component essential for both holoenzyme assembly and proton-pump function was established, resolving whether individual V1 subunits are independently required for membrane association of the catalytic sector.\",\n      \"evidence\": \"Yeast VMA8 gene disruption, complementation rescue, quinacrine accumulation assay, vacuolar membrane fractionation, and immunoblotting in two independent laboratories\",\n      \"pmids\": [\"7831318\", \"7797485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of subunit D contacts with other V1 and Vo subunits was not resolved\",\n        \"Whether subunit D has regulatory or catalytic roles beyond scaffolding was untested\",\n        \"All data in yeast; mammalian function was inferred by homology\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"ATP6V1D was shown to have a non-canonical function in promoting autophagosome–lysosome fusion through ESCRT-III assembly, answering whether V-ATPase subunits contribute to autophagy beyond acidification alone.\",\n      \"evidence\": \"CRISPR-Cas9 metabolic screen, ATP6V1D knockdown in hepatocellular carcinoma cells in vitro and in vivo, co-immunoprecipitation of CHMP4B–IST1, autophagic flux assays with bafilomycin A1\",\n      \"pmids\": [\"39316516\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Not independently replicated; single-lab finding\",\n        \"Whether ATP6V1D directly binds CHMP4B or IST1, or acts indirectly, is unresolved\",\n        \"Generalizability beyond hepatocellular carcinoma cells is untested\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"ATP6V1D-dependent lysosomal acidification was demonstrated to be functionally required in macrophages for intracellular pathogen killing, establishing its role in innate immune defense and revealing epistatic cooperation with GIMAP-driven lysosome–parasitophorous vacuole fusion.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in rat macrophage NR8383 cells, GIMAP transgene overexpression, Toxoplasma gondii growth assay, lysosomal acidification assay\",\n      \"pmids\": [\"41972776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study in one macrophage cell line; in vivo immune relevance not demonstrated\",\n        \"Molecular mechanism of cooperation between ATP6V1D and GIMAP pathways is uncharacterized\",\n        \"Whether other V-ATPase subunits have similar non-redundant immune roles was not tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of ATP6V1D interactions within the V1 sector and its direct versus indirect role in ESCRT-III assembly remain unresolved, and no mammalian genetic disease has been linked to ATP6V1D mutations through direct evidence.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of mammalian ATP6V1D in the context of V-ATPase holoenzyme\",\n        \"Whether ATP6V1D directly contacts CHMP4B or IST1 is unknown\",\n        \"No in vivo mammalian knockout phenotype has been reported\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\n      \"V-ATPase (V1 sector)\"\n    ],\n    \"partners\": [\n      \"CHMP4B\",\n      \"IST1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ATP6V1D encodes the D subunit of the V1 catalytic sector of the vacuolar H+-ATPase (V-ATPase), functioning as an essential structural component required for V1 assembly onto the Vo membrane sector and for ATP hydrolysis-driven proton pumping that acidifies lysosomes and vacuoles [PMID:7831318, PMID:7797485]. Beyond canonical acidification, ATP6V1D maintains autophagic flux in hepatocellular carcinoma cells by promoting ESCRT-III complex assembly through enhancement of the CHMP4B–IST1 interaction, thereby facilitating autophagosome–lysosome fusion and sustaining cancer cell stemness [PMID:39316516]. In macrophages, ATP6V1D-dependent lysosomal acidification activates hydrolytic enzymes required for degradation of intracellular Toxoplasma gondii, acting additively with GIMAP-mediated lysosome translocation to restrict parasite growth [PMID:41972776].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of subunit D as an integral, essential component of the V1 sector resolved how the catalytic domain achieves its stoichiometric assembly and established its requirement for V-ATPase-dependent vacuolar acidification.\",\n      \"evidence\": \"Protein sequencing of bovine chromaffin granule V-ATPase, cDNA cloning, yeast VMA8 disruption with quinacrine acidification assay and complementation rescue (PMID:7831318); independent yeast gene disruption with ATPase activity assay and immunoblot/density gradient fractionation showing V1 failure to associate with vacuolar membranes (PMID:7797485)\",\n      \"pmids\": [\"7831318\", \"7797485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Atomic-resolution structure of subunit D within the V1 complex was not determined\",\n        \"Mechanism by which subunit D nucleates V1 assembly onto Vo was not defined\",\n        \"Mammalian loss-of-function data were lacking\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Cloning and chromosomal mapping of the mouse ortholog, together with deep evolutionary conservation analysis, established that subunit D is under strong purifying selection, consistent with an indispensable role across eukaryotes.\",\n      \"evidence\": \"cDNA cloning, chromosomal mapping to mouse chromosome 12 (syntenic with human 14q24), phylogenetic analysis\",\n      \"pmids\": [\"11435709\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No functional perturbation was performed in mammalian cells\",\n        \"Tissue-specific expression or regulation was not characterized\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that ATP6V1D sustains autophagic flux not only through lysosomal acidification but also by promoting CHMP4B–IST1 interaction and ESCRT-III assembly for autophagosome–lysosome fusion revealed a non-canonical, acidification-independent arm of V-ATPase subunit function in cancer stemness.\",\n      \"evidence\": \"CRISPR-Cas9 metabolic knockout screen in HCC cells, shRNA knockdown, co-immunoprecipitation of CHMP4B–IST1, immunofluorescence, in vitro and in vivo tumor models, epistasis via CHMP4B/IST1 silencing\",\n      \"pmids\": [\"39316516\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct physical interaction between ATP6V1D and the CHMP4B–IST1 complex was not shown; the mechanism of enhancement is unclear\",\n        \"Whether the ESCRT-III-promoting function operates outside of HCC or cancer contexts is untested\",\n        \"Structural basis for subunit D engagement with ESCRT machinery is unknown\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstration that ATP6V1D-dependent lysosomal acidification is required for macrophage killing of intracellular Toxoplasma gondii, acting additively with GIMAP-mediated lysosome translocation, established a cell-autonomous innate immune role for the V-ATPase D subunit.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in rat NR8383 macrophages, lysosomal acidification assays, intracellular parasite growth quantification, genetic epistasis with GIMAP5/6 overexpression\",\n      \"pmids\": [\"41972776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether this innate immune role extends to human macrophages or other intracellular pathogens is untested\",\n        \"The molecular interface between ATP6V1D loss and impaired hydrolytic enzyme activation has not been dissected\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of subunit D within the assembled mammalian V-ATPase, the direct molecular mechanism by which ATP6V1D promotes ESCRT-III assembly independently of acidification, and whether ATP6V1D mutations cause human Mendelian disease.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of mammalian V-ATPase with subunit D resolved in mechanistic detail\",\n        \"No direct binding interface mapped between ATP6V1D and ESCRT-III components\",\n        \"No human genetic disease attributed to ATP6V1D loss-of-function\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005773\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [\n      \"V-ATPase (V1 sector)\"\n    ],\n    \"partners\": [\n      \"CHMP4B\",\n      \"IST1\",\n      \"GIMAP5\",\n      \"GIMAP6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}