{"gene":"VMA12","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":1993,"finding":"VMA12 (Vma12p) is essential for assembly of V-ATPase subunits onto the vacuolar membrane in S. cerevisiae. In vma12 null mutants, peripheral membrane V1 subunits (69, 60, 42, 27 kDa) fail to localize to the vacuolar membrane despite normal cellular levels, and integral membrane V0 subunits (100 and 17 kDa) are absent or greatly reduced from vacuolar membrane fractions. Vma12p itself is not a component of the purified active V-ATPase complex, indicating it acts as an assembly/targeting factor rather than a structural subunit.","method":"Chromosomal deletion (null mutant construction), Western blotting of whole-cell and vacuolar membrane fractions, glycerol gradient centrifugation purification of V-ATPase complex, anti-Vma12p antibody immunodetection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined biochemical phenotype, subcellular fractionation, multiple orthogonal methods, replicated by independent lab (PMID:8465604)","pmids":["8419376"],"is_preprint":false},{"year":1993,"finding":"VPH2 (identical to VMA12) is required for vacuolar H+-ATPase activity: vph2 mutants have greatly reduced vacuolar proton pumping and ATPase activity, and V1 nucleotide-binding subunits fail to be correctly targeted to the vacuolar membrane.","method":"Genetic complementation, deletion analysis, biochemical assay of vacuolar proton pumping and ATPase activity, subcellular fractionation","journal":"Yeast (Chichester, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — independent replication of VMA12/VPH2 function with biochemical assays; authors confirm identity with VMA12","pmids":["8465604"],"is_preprint":false},{"year":1997,"finding":"Vma12p (VMA12) is an integral ER membrane protein with both N- and C-termini oriented toward the cytosol. In cells lacking Vma12p, the 100-kDa V0 subunit (correctly inserted into the ER membrane) is rapidly degraded (t1/2 ~30 min), indicating Vma12p functions in the ER after V0 subunit insertion to promote assembly of V0 subunits into a stable complex, which is required for their stability and transport out of the ER.","method":"Biochemical membrane fractionation, protease protection assay, immunolocalization to ER, pulse-chase degradation assay in vma12Δ cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal biochemical methods (fractionation, protease protection, pulse-chase, immunolocalization) in a single rigorous study establishing ER localization and mechanistic role","pmids":["9325326"],"is_preprint":false},{"year":2016,"finding":"Human TMEM199 (C17orf32), identified as the human homolog of yeast Vma12p/Vph2p, is required for Golgi homeostasis. Loss-of-function mutations in TMEM199 cause deficient Golgi glycosylation (reduced N- and O-glycosylation, specifically reduced galactose and sialic acid incorporation). V5-tagged TMEM199 localizes with ERGIC and COPI markers in HeLa cells. Lentiviral transduction with wild-type TMEM199 restores Golgi glycosylation in patient fibroblasts.","method":"Exome sequencing of CDG patients, metabolic labeling of sialic acids in fibroblasts, lentiviral rescue with wild-type TMEM199, V5-tagged TMEM199 localization by immunofluorescence with ERGIC/COPI markers, electron microscopy of liver biopsy, mass spectrometry of glycans","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional rescue experiment, multiple patient mutations, metabolic labeling, subcellular localization, and EM across multiple orthogonal methods","pmids":["26833330"],"is_preprint":false},{"year":2017,"finding":"TMEM199 (and CCDC115) function as V-ATPase assembly factors: genetic disruption of TMEM199 impairs V-ATPase activity, leading to intracellular iron depletion, which in turn reduces PHD (prolyl hydroxylase) catalytic activity and stabilizes HIF1α under aerobic conditions. Iron supplementation directly restores PHD activity following V-ATPase inhibition, establishing the mechanistic link: TMEM199 → V-ATPase → endo-lysosomal acidification → iron homeostasis → PHD activity → HIF1α stability.","method":"Genome-wide genetic screen in near-haploid human cells, genetic disruption of TMEM199 and CCDC115, HIF1α stabilization assays, iron supplementation rescue of PHD activity","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased genome-wide screen followed by mechanistic rescue experiment (iron supplementation restoring PHD activity), multiple orthogonal approaches in a single rigorous study","pmids":["28296633"],"is_preprint":false},{"year":2021,"finding":"TMEM199 deficiency impairs lysosomal acidification and autophagic (lipophagic) capacity in hepatocytes, leading to defective lipid droplet-lysosome interaction and hepatic steatosis. Silencing of TMEM199 in HepG2 cells results in increased numbers and size of lipid droplets that co-localize with lysosomes, and increased secretion of apoB-containing particles. Excessive de novo lipogenesis, failing oxidative capacity, and elevated lipid uptake were not observed as causal mechanisms.","method":"siRNA knockdown of TMEM199 in HepG2 cells, CRISPR/Cas9 knock-in mouse model, iPSC-derived hepatocyte-like cells from patients, lipid droplet/lysosome co-localization imaging, apoB secretion assay, lysosomal acidification assay, autophagic flux assay","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell and animal models with functional assays, but mechanistic pathway (lipophagy impairment) established by single lab","pmids":["34626841"],"is_preprint":false},{"year":2018,"finding":"In Candida albicans, Vph2 (ortholog of VMA12) localizes to the endoplasmic reticulum (consistent with S. cerevisiae), and is required for correct localization of V-ATPase subunits Vph1 (V0) and Tfp1 (V1), vacuolar acidification, endocytosis, cell wall integrity (activating the CWI pathway), and hyphal development.","method":"Deletion mutant construction, fluorescence microscopy of V-ATPase subunit localization, vacuolar acidification assays, endocytosis assays, cell wall stress assays, CWI pathway analysis, hyphal induction assays, mouse model of systemic candidiasis","journal":"Fungal genetics and biology : FG & B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple defined phenotypic readouts in a fungal ortholog, single lab","pmids":["29522815"],"is_preprint":false},{"year":2019,"finding":"Vph2 in Candida albicans localizes to the endoplasmic reticulum (around the nucleus and in patches close to the cell periphery, consistent with S. cerevisiae). Loss of VPH2 leads to increased intracellular GSH levels, which induces the unfolded protein response (UPR) and causes hypersensitivity to reductive stress (DTT, β-mercaptoethanol). The GSH-specific scavenger CDNB partially alleviates UPR induction and growth defects, mechanistically linking Vph2 to ER redox homeostasis.","method":"Fluorescence microscopy for ER localization, sensitivity assays with DTT/β-ME, GSH quantification, UPR gene expression analysis (PRB1, PMT4), chemical rescue with CDNB","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — ER localization confirmed, multiple phenotypic and chemical rescue experiments, single lab in fungal ortholog","pmids":["30928095"],"is_preprint":false},{"year":2025,"finding":"TMEM199, together with its partner CCDC115, interacts with IFNGR1/2 (IFN-γ receptors) and facilitates their trafficking to RAB11A-positive recycling endosomes. TMEM199/CCDC115 also recruits TRAPP II complex to recycling endosomes and activates RAB11A, enhancing IFNGR1/2 recycling and downstream JAK-STAT signaling, leading to increased PD-L1 transcription in tumor cells.","method":"Co-immunoprecipitation of TMEM199/CCDC115 with IFNGR1/2, fluorescence microscopy of IFNGR1/2 trafficking to RAB11A-positive endosomes, TRAPP II recruitment assay, RAB11A activation assay, PD-L1 expression assay following TMEM199 manipulation","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2-3 / Weak — Co-IP and trafficking experiments from single lab, single paper, novel finding not yet replicated","pmids":["41319859"],"is_preprint":false}],"current_model":"TMEM199 (VMA12/VPH2) is an integral ER membrane protein that functions as a V-ATPase assembly factor, promoting assembly of V0 subunits in the ER (thereby preventing their degradation), enabling V-ATPase-dependent endo-lysosomal acidification required for Golgi homeostasis, cellular iron homeostasis (and thus PHD/HIF1α regulation), and lipophagy; more recently, TMEM199 and its partner CCDC115 have been found to facilitate recycling of IFN-γ receptors (IFNGR1/2) to RAB11A-positive endosomes via TRAPP II-mediated RAB11A activation, thereby amplifying IFN-γ–PD-L1 signaling."},"narrative":{"mechanistic_narrative":"VMA12 (yeast Vma12p/Vph2p; human TMEM199) is an integral endoplasmic reticulum membrane protein that acts as a dedicated assembly factor for the vacuolar/lysosomal H+-ATPase (V-ATPase) rather than as a structural subunit of the active enzyme [PMID:8419376, PMID:9325326]. In yeast lacking Vma12p, peripheral V1 subunits fail to localize to the vacuolar membrane and the membrane-embedded V0 subunit is rapidly degraded after its insertion into the ER, demonstrating that Vma12p promotes assembly and stabilization of V0 in the ER and is required for vacuolar proton pumping and ATPase activity [PMID:8419376, PMID:8465604, PMID:9325326]. This function is conserved in the human homolog: loss-of-function mutations in TMEM199 cause a congenital disorder of glycosylation, with deficient Golgi N- and O-glycosylation that is restored by re-expression of wild-type TMEM199, and the protein localizes to the ERGIC/COPI compartment [PMID:26833330]. Through its control of V-ATPase-dependent endo-lysosomal acidification, TMEM199 governs cellular iron homeostasis, such that its loss depletes intracellular iron, reduces prolyl hydroxylase activity, and stabilizes HIF1α, while iron supplementation restores PHD activity [PMID:28296633]. TMEM199 is similarly required for lysosomal acidification and lipophagy, and its deficiency drives lipid droplet accumulation and hepatic steatosis [PMID:34626841]. Together with its partner CCDC115, TMEM199 also interacts with the IFN-γ receptors IFNGR1/2 and recruits the TRAPP II complex to activate RAB11A, promoting receptor recycling, JAK-STAT signaling, and PD-L1 expression [PMID:41319859].","teleology":[{"year":1993,"claim":"Established that Vma12p is needed to build the V-ATPase but is not itself part of the enzyme, defining it as an assembly/targeting factor rather than a structural subunit.","evidence":"Chromosomal null mutant with Western blotting of vacuolar membrane fractions and glycerol-gradient purification of the V-ATPase complex in S. cerevisiae","pmids":["8419376","8465604"],"confidence":"High","gaps":["Did not localize where in the cell Vma12p acts","Did not define the molecular step of assembly it catalyzes"]},{"year":1997,"claim":"Localized Vma12p function to the ER and showed it stabilizes the V0 subunit after membrane insertion, pinpointing the assembly step as ER V0 maturation.","evidence":"Protease protection, membrane fractionation, immunolocalization, and pulse-chase degradation assays in vma12Δ yeast","pmids":["9325326"],"confidence":"High","gaps":["No direct V0 subunit-binding interaction defined","Whether other subunits depend on Vma12p for ER stability not resolved"]},{"year":2016,"claim":"Connected the human homolog TMEM199 to disease, showing its loss causes a glycosylation disorder via disrupted Golgi homeostasis.","evidence":"Exome sequencing of CDG patients, glycan mass spectrometry, lentiviral rescue, and ERGIC/COPI co-localization in HeLa cells","pmids":["26833330"],"confidence":"High","gaps":["Did not directly demonstrate V-ATPase assembly defect in human cells","Link between acidification and glycosylation not fully mechanistic in this study"]},{"year":2017,"claim":"Defined a mechanistic chain from TMEM199 to HIF1α by showing V-ATPase-dependent acidification controls iron homeostasis and thereby PHD activity.","evidence":"Genome-wide haploid genetic screen, TMEM199/CCDC115 disruption, HIF1α stabilization assays, and iron supplementation rescue of PHD activity","pmids":["28296633"],"confidence":"High","gaps":["Mechanism by which V-ATPase loss depletes iron not fully detailed","Did not address tissue-specific consequences"]},{"year":2018,"claim":"Extended the ER-localized V-ATPase assembly role to a pathogenic fungal ortholog, linking it to vacuolar acidification, endocytosis, cell wall integrity, and virulence.","evidence":"Deletion mutants, V-ATPase subunit localization microscopy, acidification/endocytosis/CWI assays, and a candidiasis mouse model in C. albicans","pmids":["29522815"],"confidence":"Medium","gaps":["Single lab in a fungal ortholog","Direct subunit interactions not mapped"]},{"year":2019,"claim":"Implicated Vph2 in ER redox balance by showing its loss elevates GSH and induces the UPR, broadening its role beyond V-ATPase assembly.","evidence":"ER localization microscopy, reductive stress sensitivity assays, GSH quantification, UPR gene expression, and CDNB chemical rescue in C. albicans","pmids":["30928095"],"confidence":"Medium","gaps":["Causal connection between V-ATPase function and GSH levels unresolved","Single lab, fungal ortholog"]},{"year":2021,"claim":"Showed that loss of TMEM199-dependent acidification blocks lipophagy and drives hepatic steatosis, providing a physiological consequence of impaired lysosomal function.","evidence":"siRNA knockdown in HepG2, CRISPR knock-in mouse, patient iPSC-derived hepatocytes, lipid droplet/lysosome imaging, and autophagic flux assays","pmids":["34626841"],"confidence":"Medium","gaps":["Lipophagy mechanism established by a single lab","Molecular link from acidification defect to lipid droplet retention not fully detailed"]},{"year":2025,"claim":"Revealed a trafficking function in which TMEM199/CCDC115 recruits TRAPP II to activate RAB11A and recycle IFN-γ receptors, coupling the protein to immune signaling and PD-L1 expression.","evidence":"Co-immunoprecipitation with IFNGR1/2, trafficking microscopy to RAB11A endosomes, TRAPP II recruitment and RAB11A activation assays, and PD-L1 expression readouts","pmids":["41319859"],"confidence":"Medium","gaps":["Single Co-IP-based study not yet independently replicated","Whether this function is independent of V-ATPase assembly role unclear","Direct vs. indirect IFNGR1/2 interaction not resolved"]},{"year":null,"claim":"How TMEM199 physically engages V0 subunits and whether its endosomal trafficking/RAB11A role is mechanistically separable from its canonical V-ATPase assembly function remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of TMEM199 bound to V-ATPase subunits","Direct substrate/subunit contacts unmapped","Relationship between assembly-factor and trafficking roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[2,6,7]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3]}],"complexes":[],"partners":["CCDC115","IFNGR1","IFNGR2","RAB11A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N511","full_name":"Vacuolar ATPase assembly protein VMA12","aliases":["Transmembrane protein 199"],"length_aa":208,"mass_kda":23.1,"function":"Accessory component of the proton-transporting vacuolar (V)-ATPase protein pump involved in intracellular iron homeostasis. In aerobic conditions, required for intracellular iron homeostasis, thus triggering the activity of Fe(2+) prolyl hydroxylase (PHD) enzymes, and leading to HIF1A hydroxylation and subsequent proteasomal degradation. Necessary for endolysosomal acidification and lysosomal degradation (PubMed:28296633). May be involved in Golgi homeostasis (PubMed:26833330). Binds 20(S)-hydroxycholesterol (20(S)-OHC) (By similarity)","subcellular_location":"Cytoplasmic vesicle, COPI-coated vesicle membrane; Endoplasmic reticulum-Golgi intermediate compartment membrane; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q8N511/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/VMA12","classification":"Common Essential","n_dependent_lines":955,"n_total_lines":1208,"dependency_fraction":0.7905629139072847},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ATP6V0A2","stoichiometry":10.0},{"gene":"ATP6V1F","stoichiometry":0.2},{"gene":"CCDC47","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/VMA12","total_profiled":1310},"omim":[{"mim_id":"616829","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE IIp; CDG2P","url":"https://www.omim.org/entry/616829"},{"mim_id":"616815","title":"VACUOLAR ATPase ASSEMBLY FACTOR VMA12; VMA12","url":"https://www.omim.org/entry/616815"}],"hpa":{"profiled":true,"resolved_as":"TMEM199","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TMEM199"},"hgnc":{"alias_symbol":["VPH2","MGC45714"],"prev_symbol":["TMEM199","C17orf32"]},"alphafold":{"accession":"Q8N511","domains":[{"cath_id":"-","chopping":"3-85","consensus_level":"high","plddt":80.9304,"start":3,"end":85},{"cath_id":"1.20.5","chopping":"95-129","consensus_level":"medium","plddt":83.3414,"start":95,"end":129},{"cath_id":"1.10.287","chopping":"130-208","consensus_level":"medium","plddt":80.9767,"start":130,"end":208}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N511","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N511-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N511-F1-predicted_aligned_error_v6.png","plddt_mean":80.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VMA12","jax_strain_url":"https://www.jax.org/strain/search?query=VMA12"},"sequence":{"accession":"Q8N511","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N511.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N511/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N511"}},"corpus_meta":[{"pmid":"28296633","id":"PMC_28296633","title":"The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.","date":"2017","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/28296633","citation_count":83,"is_preprint":false},{"pmid":"26833330","id":"PMC_26833330","title":"TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation.","date":"2016","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26833330","citation_count":75,"is_preprint":false},{"pmid":"8419376","id":"PMC_8419376","title":"VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.","date":"1993","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8419376","citation_count":63,"is_preprint":false},{"pmid":"9325326","id":"PMC_9325326","title":"VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H+-ATPase assembly.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9325326","citation_count":58,"is_preprint":false},{"pmid":"8465604","id":"PMC_8465604","title":"The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H(+)-ATPase.","date":"1993","source":"Yeast (Chichester, England)","url":"https://pubmed.ncbi.nlm.nih.gov/8465604","citation_count":27,"is_preprint":false},{"pmid":"34626841","id":"PMC_34626841","title":"Defective Lipid Droplet-Lysosome Interaction Causes Fatty Liver Disease as Evidenced by Human Mutations in TMEM199 and CCDC115.","date":"2021","source":"Cellular and molecular gastroenterology and hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/34626841","citation_count":23,"is_preprint":false},{"pmid":"29522815","id":"PMC_29522815","title":"Roles of VPH2 and VMA6 in localization of V-ATPase subunits, cell wall functions and filamentous development in Candida albicans.","date":"2018","source":"Fungal genetics and biology : FG & B","url":"https://pubmed.ncbi.nlm.nih.gov/29522815","citation_count":20,"is_preprint":false},{"pmid":"29321044","id":"PMC_29321044","title":"Three unreported cases of TMEM199-CDG, a rare genetic liver disease with abnormal glycosylation.","date":"2018","source":"Orphanet journal of rare diseases","url":"https://pubmed.ncbi.nlm.nih.gov/29321044","citation_count":18,"is_preprint":false},{"pmid":"30928095","id":"PMC_30928095","title":"Vph2 is required for protection against a reductive stress in Candida albicans.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30928095","citation_count":7,"is_preprint":false},{"pmid":"32253532","id":"PMC_32253532","title":"Transgenic plant generated by RNAi-mediated knocking down of soybean Vma12 and soybean mosaic virus resistance evaluation.","date":"2020","source":"AMB Express","url":"https://pubmed.ncbi.nlm.nih.gov/32253532","citation_count":6,"is_preprint":false},{"pmid":"35401690","id":"PMC_35401690","title":"TMEM199-Congenital Disorder of Glycosylation With Novel Phenotype and Genotype in a Chinese Boy.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35401690","citation_count":4,"is_preprint":false},{"pmid":"36706865","id":"PMC_36706865","title":"Higher frequency of TMEM199-CDG in the southern mediterranean area is associated with c.92G>C (p.Arg31Pro) mutation.","date":"2023","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36706865","citation_count":2,"is_preprint":false},{"pmid":"41319859","id":"PMC_41319859","title":"TMEM199 promotes PD-L1 expression and tumor immune evasion by activating the recycling of IFNGR1/2.","date":"2025","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/41319859","citation_count":1,"is_preprint":false},{"pmid":"15487498","id":"PMC_15487498","title":"[Correction of five different types of errors of model REFSEQs appeared in NCBI human gene database only by using two novel human genes C17orf32 and ZNF362].","date":"2004","source":"Yi chuan xue bao = Acta genetica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/15487498","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10034,"output_tokens":2938,"usd":0.037086,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10256,"output_tokens":3006,"usd":0.063215,"stage2_stop_reason":"end_turn"},"total_usd":0.100301,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1993,\n      \"finding\": \"VMA12 (Vma12p) is essential for assembly of V-ATPase subunits onto the vacuolar membrane in S. cerevisiae. In vma12 null mutants, peripheral membrane V1 subunits (69, 60, 42, 27 kDa) fail to localize to the vacuolar membrane despite normal cellular levels, and integral membrane V0 subunits (100 and 17 kDa) are absent or greatly reduced from vacuolar membrane fractions. Vma12p itself is not a component of the purified active V-ATPase complex, indicating it acts as an assembly/targeting factor rather than a structural subunit.\",\n      \"method\": \"Chromosomal deletion (null mutant construction), Western blotting of whole-cell and vacuolar membrane fractions, glycerol gradient centrifugation purification of V-ATPase complex, anti-Vma12p antibody immunodetection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined biochemical phenotype, subcellular fractionation, multiple orthogonal methods, replicated by independent lab (PMID:8465604)\",\n      \"pmids\": [\"8419376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"VPH2 (identical to VMA12) is required for vacuolar H+-ATPase activity: vph2 mutants have greatly reduced vacuolar proton pumping and ATPase activity, and V1 nucleotide-binding subunits fail to be correctly targeted to the vacuolar membrane.\",\n      \"method\": \"Genetic complementation, deletion analysis, biochemical assay of vacuolar proton pumping and ATPase activity, subcellular fractionation\",\n      \"journal\": \"Yeast (Chichester, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independent replication of VMA12/VPH2 function with biochemical assays; authors confirm identity with VMA12\",\n      \"pmids\": [\"8465604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Vma12p (VMA12) is an integral ER membrane protein with both N- and C-termini oriented toward the cytosol. In cells lacking Vma12p, the 100-kDa V0 subunit (correctly inserted into the ER membrane) is rapidly degraded (t1/2 ~30 min), indicating Vma12p functions in the ER after V0 subunit insertion to promote assembly of V0 subunits into a stable complex, which is required for their stability and transport out of the ER.\",\n      \"method\": \"Biochemical membrane fractionation, protease protection assay, immunolocalization to ER, pulse-chase degradation assay in vma12Δ cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal biochemical methods (fractionation, protease protection, pulse-chase, immunolocalization) in a single rigorous study establishing ER localization and mechanistic role\",\n      \"pmids\": [\"9325326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Human TMEM199 (C17orf32), identified as the human homolog of yeast Vma12p/Vph2p, is required for Golgi homeostasis. Loss-of-function mutations in TMEM199 cause deficient Golgi glycosylation (reduced N- and O-glycosylation, specifically reduced galactose and sialic acid incorporation). V5-tagged TMEM199 localizes with ERGIC and COPI markers in HeLa cells. Lentiviral transduction with wild-type TMEM199 restores Golgi glycosylation in patient fibroblasts.\",\n      \"method\": \"Exome sequencing of CDG patients, metabolic labeling of sialic acids in fibroblasts, lentiviral rescue with wild-type TMEM199, V5-tagged TMEM199 localization by immunofluorescence with ERGIC/COPI markers, electron microscopy of liver biopsy, mass spectrometry of glycans\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional rescue experiment, multiple patient mutations, metabolic labeling, subcellular localization, and EM across multiple orthogonal methods\",\n      \"pmids\": [\"26833330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TMEM199 (and CCDC115) function as V-ATPase assembly factors: genetic disruption of TMEM199 impairs V-ATPase activity, leading to intracellular iron depletion, which in turn reduces PHD (prolyl hydroxylase) catalytic activity and stabilizes HIF1α under aerobic conditions. Iron supplementation directly restores PHD activity following V-ATPase inhibition, establishing the mechanistic link: TMEM199 → V-ATPase → endo-lysosomal acidification → iron homeostasis → PHD activity → HIF1α stability.\",\n      \"method\": \"Genome-wide genetic screen in near-haploid human cells, genetic disruption of TMEM199 and CCDC115, HIF1α stabilization assays, iron supplementation rescue of PHD activity\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased genome-wide screen followed by mechanistic rescue experiment (iron supplementation restoring PHD activity), multiple orthogonal approaches in a single rigorous study\",\n      \"pmids\": [\"28296633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TMEM199 deficiency impairs lysosomal acidification and autophagic (lipophagic) capacity in hepatocytes, leading to defective lipid droplet-lysosome interaction and hepatic steatosis. Silencing of TMEM199 in HepG2 cells results in increased numbers and size of lipid droplets that co-localize with lysosomes, and increased secretion of apoB-containing particles. Excessive de novo lipogenesis, failing oxidative capacity, and elevated lipid uptake were not observed as causal mechanisms.\",\n      \"method\": \"siRNA knockdown of TMEM199 in HepG2 cells, CRISPR/Cas9 knock-in mouse model, iPSC-derived hepatocyte-like cells from patients, lipid droplet/lysosome co-localization imaging, apoB secretion assay, lysosomal acidification assay, autophagic flux assay\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell and animal models with functional assays, but mechanistic pathway (lipophagy impairment) established by single lab\",\n      \"pmids\": [\"34626841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Candida albicans, Vph2 (ortholog of VMA12) localizes to the endoplasmic reticulum (consistent with S. cerevisiae), and is required for correct localization of V-ATPase subunits Vph1 (V0) and Tfp1 (V1), vacuolar acidification, endocytosis, cell wall integrity (activating the CWI pathway), and hyphal development.\",\n      \"method\": \"Deletion mutant construction, fluorescence microscopy of V-ATPase subunit localization, vacuolar acidification assays, endocytosis assays, cell wall stress assays, CWI pathway analysis, hyphal induction assays, mouse model of systemic candidiasis\",\n      \"journal\": \"Fungal genetics and biology : FG & B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple defined phenotypic readouts in a fungal ortholog, single lab\",\n      \"pmids\": [\"29522815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Vph2 in Candida albicans localizes to the endoplasmic reticulum (around the nucleus and in patches close to the cell periphery, consistent with S. cerevisiae). Loss of VPH2 leads to increased intracellular GSH levels, which induces the unfolded protein response (UPR) and causes hypersensitivity to reductive stress (DTT, β-mercaptoethanol). The GSH-specific scavenger CDNB partially alleviates UPR induction and growth defects, mechanistically linking Vph2 to ER redox homeostasis.\",\n      \"method\": \"Fluorescence microscopy for ER localization, sensitivity assays with DTT/β-ME, GSH quantification, UPR gene expression analysis (PRB1, PMT4), chemical rescue with CDNB\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — ER localization confirmed, multiple phenotypic and chemical rescue experiments, single lab in fungal ortholog\",\n      \"pmids\": [\"30928095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TMEM199, together with its partner CCDC115, interacts with IFNGR1/2 (IFN-γ receptors) and facilitates their trafficking to RAB11A-positive recycling endosomes. TMEM199/CCDC115 also recruits TRAPP II complex to recycling endosomes and activates RAB11A, enhancing IFNGR1/2 recycling and downstream JAK-STAT signaling, leading to increased PD-L1 transcription in tumor cells.\",\n      \"method\": \"Co-immunoprecipitation of TMEM199/CCDC115 with IFNGR1/2, fluorescence microscopy of IFNGR1/2 trafficking to RAB11A-positive endosomes, TRAPP II recruitment assay, RAB11A activation assay, PD-L1 expression assay following TMEM199 manipulation\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Weak — Co-IP and trafficking experiments from single lab, single paper, novel finding not yet replicated\",\n      \"pmids\": [\"41319859\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TMEM199 (VMA12/VPH2) is an integral ER membrane protein that functions as a V-ATPase assembly factor, promoting assembly of V0 subunits in the ER (thereby preventing their degradation), enabling V-ATPase-dependent endo-lysosomal acidification required for Golgi homeostasis, cellular iron homeostasis (and thus PHD/HIF1α regulation), and lipophagy; more recently, TMEM199 and its partner CCDC115 have been found to facilitate recycling of IFN-γ receptors (IFNGR1/2) to RAB11A-positive endosomes via TRAPP II-mediated RAB11A activation, thereby amplifying IFN-γ–PD-L1 signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VMA12 (yeast Vma12p/Vph2p; human TMEM199) is an integral endoplasmic reticulum membrane protein that acts as a dedicated assembly factor for the vacuolar/lysosomal H+-ATPase (V-ATPase) rather than as a structural subunit of the active enzyme [#0, #2]. In yeast lacking Vma12p, peripheral V1 subunits fail to localize to the vacuolar membrane and the membrane-embedded V0 subunit is rapidly degraded after its insertion into the ER, demonstrating that Vma12p promotes assembly and stabilization of V0 in the ER and is required for vacuolar proton pumping and ATPase activity [#0, #1, #2]. This function is conserved in the human homolog: loss-of-function mutations in TMEM199 cause a congenital disorder of glycosylation, with deficient Golgi N- and O-glycosylation that is restored by re-expression of wild-type TMEM199, and the protein localizes to the ERGIC/COPI compartment [#3]. Through its control of V-ATPase-dependent endo-lysosomal acidification, TMEM199 governs cellular iron homeostasis, such that its loss depletes intracellular iron, reduces prolyl hydroxylase activity, and stabilizes HIF1\\u03b1, while iron supplementation restores PHD activity [#4]. TMEM199 is similarly required for lysosomal acidification and lipophagy, and its deficiency drives lipid droplet accumulation and hepatic steatosis [#5]. Together with its partner CCDC115, TMEM199 also interacts with the IFN-\\u03b3 receptors IFNGR1/2 and recruits the TRAPP II complex to activate RAB11A, promoting receptor recycling, JAK-STAT signaling, and PD-L1 expression [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established that Vma12p is needed to build the V-ATPase but is not itself part of the enzyme, defining it as an assembly/targeting factor rather than a structural subunit.\",\n      \"evidence\": \"Chromosomal null mutant with Western blotting of vacuolar membrane fractions and glycerol-gradient purification of the V-ATPase complex in S. cerevisiae\",\n      \"pmids\": [\"8419376\", \"8465604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not localize where in the cell Vma12p acts\", \"Did not define the molecular step of assembly it catalyzes\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Localized Vma12p function to the ER and showed it stabilizes the V0 subunit after membrane insertion, pinpointing the assembly step as ER V0 maturation.\",\n      \"evidence\": \"Protease protection, membrane fractionation, immunolocalization, and pulse-chase degradation assays in vma12\\u0394 yeast\",\n      \"pmids\": [\"9325326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct V0 subunit-binding interaction defined\", \"Whether other subunits depend on Vma12p for ER stability not resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected the human homolog TMEM199 to disease, showing its loss causes a glycosylation disorder via disrupted Golgi homeostasis.\",\n      \"evidence\": \"Exome sequencing of CDG patients, glycan mass spectrometry, lentiviral rescue, and ERGIC/COPI co-localization in HeLa cells\",\n      \"pmids\": [\"26833330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not directly demonstrate V-ATPase assembly defect in human cells\", \"Link between acidification and glycosylation not fully mechanistic in this study\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined a mechanistic chain from TMEM199 to HIF1\\u03b1 by showing V-ATPase-dependent acidification controls iron homeostasis and thereby PHD activity.\",\n      \"evidence\": \"Genome-wide haploid genetic screen, TMEM199/CCDC115 disruption, HIF1\\u03b1 stabilization assays, and iron supplementation rescue of PHD activity\",\n      \"pmids\": [\"28296633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which V-ATPase loss depletes iron not fully detailed\", \"Did not address tissue-specific consequences\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended the ER-localized V-ATPase assembly role to a pathogenic fungal ortholog, linking it to vacuolar acidification, endocytosis, cell wall integrity, and virulence.\",\n      \"evidence\": \"Deletion mutants, V-ATPase subunit localization microscopy, acidification/endocytosis/CWI assays, and a candidiasis mouse model in C. albicans\",\n      \"pmids\": [\"29522815\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab in a fungal ortholog\", \"Direct subunit interactions not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Implicated Vph2 in ER redox balance by showing its loss elevates GSH and induces the UPR, broadening its role beyond V-ATPase assembly.\",\n      \"evidence\": \"ER localization microscopy, reductive stress sensitivity assays, GSH quantification, UPR gene expression, and CDNB chemical rescue in C. albicans\",\n      \"pmids\": [\"30928095\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal connection between V-ATPase function and GSH levels unresolved\", \"Single lab, fungal ortholog\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed that loss of TMEM199-dependent acidification blocks lipophagy and drives hepatic steatosis, providing a physiological consequence of impaired lysosomal function.\",\n      \"evidence\": \"siRNA knockdown in HepG2, CRISPR knock-in mouse, patient iPSC-derived hepatocytes, lipid droplet/lysosome imaging, and autophagic flux assays\",\n      \"pmids\": [\"34626841\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lipophagy mechanism established by a single lab\", \"Molecular link from acidification defect to lipid droplet retention not fully detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a trafficking function in which TMEM199/CCDC115 recruits TRAPP II to activate RAB11A and recycle IFN-\\u03b3 receptors, coupling the protein to immune signaling and PD-L1 expression.\",\n      \"evidence\": \"Co-immunoprecipitation with IFNGR1/2, trafficking microscopy to RAB11A endosomes, TRAPP II recruitment and RAB11A activation assays, and PD-L1 expression readouts\",\n      \"pmids\": [\"41319859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP-based study not yet independently replicated\", \"Whether this function is independent of V-ATPase assembly role unclear\", \"Direct vs. indirect IFNGR1/2 interaction not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TMEM199 physically engages V0 subunits and whether its endosomal trafficking/RAB11A role is mechanistically separable from its canonical V-ATPase assembly function remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of TMEM199 bound to V-ATPase subunits\", \"Direct substrate/subunit contacts unmapped\", \"Relationship between assembly-factor and trafficking roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 6, 7]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CCDC115\", \"IFNGR1\", \"IFNGR2\", \"RAB11A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}