{"gene":"TG_202620","gene_symbol":"GRA64","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2022,"finding":"GRA64 (TGME49_202620) is a transmembrane dense granule protein localized to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite vacuoles, where it is partially exposed to the host cell cytoplasm.","method":"Direct localization by immunofluorescence/ultrastructural analysis; subcellular fractionation","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional context, single lab, multiple imaging approaches","pmids":["35730903"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2022,"finding":"GRA64 interacts with components of the host endosomal sorting complexes required for transport (ESCRT) at the PVM, as demonstrated by co-immunoprecipitation and proximity-based biotinylation.","method":"Co-immunoprecipitation and proximity-based biotinylation (BioID)","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal interaction methods (Co-IP and proximity biotinylation), single lab","pmids":["35730903"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2022,"finding":"Genetic disruption of GRA64 (Δgra64) leads to enlarged vesicular structures underneath the cyst membrane in tissue cysts, as revealed by electron tomography, indicating a role for GRA64 in organizing ESCRT recruitment and intracystic vesicle formation during chronic infection.","method":"Genetic knockout (Δgra64) with electron tomography ultrastructural analysis","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with defined ultrastructural phenotype, single lab","pmids":["35730903"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"finding":"GRA64 cooperates with GRA14 to mediate recruitment of host ESCRT-III (CHMP4B) to the PVM; parasites lacking GRA64 showed reduced CHMP4B-dominant-negative-induced PVM invaginations and reduced accumulation of intra-PV host organelles, with greater defects in the GRA14/GRA64 double knockout, establishing GRA64's role in ESCRT-dependent scavenging of host organelles into the parasitophorous vacuole.","method":"Single and double genetic knockouts (ΔTgGRA64, ΔTgGRA14, double KO) with dominant-negative CHMP4B and Vps4A expression; fluorescence microscopy; electron microscopy","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic epistasis using single and double KO with dominant-negative alleles, multiple orthogonal methods, replicated across two labs","pmids":["36718630"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA64 is a transmembrane dense granule protein that localizes to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite stages, where it is exposed to the host cytoplasm and physically interacts with host ESCRT components (including CHMP4B) to recruit ESCRT-III machinery to the PVM; together with GRA14, GRA64 enables ESCRT-dependent formation of PVM invaginations and intra-vacuolar vesicles that facilitate scavenging of host organelles into the parasitophorous vacuole, and its loss causes structural defects in intracystic vesicle formation during chronic infection."},"narrative":{"mechanistic_narrative":"GRA64 is a Toxoplasma gondii transmembrane dense granule protein that functions at the host-parasite interface to co-opt host membrane-remodeling machinery [PMID:35730903, PMID:36718630]. It localizes to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite vacuoles, with a domain partially exposed to the host cytoplasm where it engages host endosomal sorting complexes required for transport (ESCRT) [PMID:35730903]. Through this exposed region, GRA64 cooperates with GRA14 to recruit host ESCRT-III (CHMP4B) to the PVM, driving ESCRT-dependent PVM invaginations and the scavenging of host organelles into the parasitophorous vacuole; loss of GRA64 reduces these invaginations and organelle accumulation, with the strongest defect in the GRA14/GRA64 double knockout [PMID:36718630]. During chronic infection, GRA64 disruption produces enlarged vesicular structures beneath the cyst membrane, implicating it in organizing intracystic vesicle formation [PMID:35730903].","teleology":[{"year":2022,"claim":"Establishing where GRA64 acts and how it is oriented was the first step in defining its role at the host-parasite interface; localizing it to the PVM with host-cytoplasm exposure positioned it to interact with host factors.","evidence":"Immunofluorescence, ultrastructural analysis, and subcellular fractionation of tachyzoite and bradyzoite vacuoles in T. gondii ME49","pmids":["35730903"],"confidence":"Medium","gaps":["Precise topology and which residues face the host cytoplasm not mapped","Membrane insertion/trafficking mechanism to the PVM not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2022,"claim":"Identifying host binding partners answered what GRA64's cytoplasm-exposed domain does; two orthogonal methods showed it engages host ESCRT components at the PVM.","evidence":"Co-immunoprecipitation and proximity-based biotinylation (BioID) in T. gondii ME49","pmids":["35730903"],"confidence":"Medium","gaps":["Direct versus indirect binding to specific ESCRT subunits not resolved","Interaction interface not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2022,"claim":"Genetic loss-of-function connected GRA64 to a chronic-stage structural phenotype, showing it is required for proper intracystic vesicle organization during bradyzoite/cyst development.","evidence":"Δgra64 knockout with electron tomography of tissue cysts in T. gondii ME49","pmids":["35730903"],"confidence":"Medium","gaps":["Causal link between altered vesicles and parasite fitness/persistence not established","Whether the phenotype reflects ESCRT mis-recruitment not directly tested"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"claim":"Reciprocal genetic epistasis with GRA14 and dominant-negative ESCRT perturbation established GRA64's mechanistic role: it drives ESCRT-III-dependent PVM invagination and scavenging of host organelles into the vacuole.","evidence":"Single and double knockouts (ΔGRA64, ΔGRA14, double KO) with dominant-negative CHMP4B and Vps4A expression, fluorescence and electron microscopy in T. gondii","pmids":["36718630"],"confidence":"High","gaps":["Molecular basis of cooperativity between GRA64 and GRA14 not defined","Functional consequence of host organelle scavenging for parasite metabolism not established","Order of ESCRT subunit recruitment relative to GRA64 not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How GRA64 physically nucleates ESCRT-III assembly and whether its chronic-stage vesicle role is mechanistically identical to its tachyzoite scavenging function remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the GRA64-ESCRT interaction","Direct binding partner among ESCRT subunits not identified","Contribution to virulence or persistence in vivo not quantified"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Host ESCRT-III machinery (CHMP4B) recruited to the PVM"],"partners":["HOST ESCRT COMPONENTS (INCLUDING CHMP4B)","GRA14 (PARASITE DENSE GRANULE PROTEIN)"],"other_free_text":["Transmembrane dense granule protein exposed to the host cytoplasm at the PVM","Required for ESCRT-dependent PVM invaginations and scavenging of host organelles into the parasitophorous vacuole","Loss produces enlarged vesicular structures beneath the cyst membrane during chronic infection"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"35730903","id":"PMC_35730903","title":"Dense Granule Protein GRA64 Interacts with Host Cell ESCRT Proteins during Toxoplasma gondii Infection.","date":"2022","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/35730903","citation_count":26,"is_preprint":false},{"pmid":"36718630","id":"PMC_36718630","title":"Toxoplasma gondii scavenges mammalian host organelles through the usurpation of host ESCRT-III and Vps4A.","date":"2023","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/36718630","citation_count":11,"is_preprint":false},{"pmid":"41296383","id":"PMC_41296383","title":"Development and Evaluation of Six Novel Recombinant GRA Proteins in Serodiagnosis of Human Toxoplasmosis.","date":"2025","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41296383","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4044,"output_tokens":1123,"usd":0.014488,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6084,"output_tokens":2250,"usd":0.043335,"stage2_stop_reason":"end_turn"},"total_usd":0.057823,"stage1_batch_id":"msgbatch_018xTxyBPcQFK9jdgubtxgsj","stage2_batch_id":"msgbatch_01UiG5BTaJP7BvKKL6RLeyv2","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"GRA64 (TGME49_202620) is a transmembrane dense granule protein localized to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite vacuoles, where it is partially exposed to the host cell cytoplasm.\",\n      \"method\": \"Direct localization by immunofluorescence/ultrastructural analysis; subcellular fractionation\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional context, single lab, multiple imaging approaches\",\n      \"pmids\": [\"35730903\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GRA64 interacts with components of the host endosomal sorting complexes required for transport (ESCRT) at the PVM, as demonstrated by co-immunoprecipitation and proximity-based biotinylation.\",\n      \"method\": \"Co-immunoprecipitation and proximity-based biotinylation (BioID)\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal interaction methods (Co-IP and proximity biotinylation), single lab\",\n      \"pmids\": [\"35730903\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Genetic disruption of GRA64 (Δgra64) leads to enlarged vesicular structures underneath the cyst membrane in tissue cysts, as revealed by electron tomography, indicating a role for GRA64 in organizing ESCRT recruitment and intracystic vesicle formation during chronic infection.\",\n      \"method\": \"Genetic knockout (Δgra64) with electron tomography ultrastructural analysis\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with defined ultrastructural phenotype, single lab\",\n      \"pmids\": [\"35730903\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GRA64 cooperates with GRA14 to mediate recruitment of host ESCRT-III (CHMP4B) to the PVM; parasites lacking GRA64 showed reduced CHMP4B-dominant-negative-induced PVM invaginations and reduced accumulation of intra-PV host organelles, with greater defects in the GRA14/GRA64 double knockout, establishing GRA64's role in ESCRT-dependent scavenging of host organelles into the parasitophorous vacuole.\",\n      \"method\": \"Single and double genetic knockouts (ΔTgGRA64, ΔTgGRA14, double KO) with dominant-negative CHMP4B and Vps4A expression; fluorescence microscopy; electron microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic epistasis using single and double KO with dominant-negative alleles, multiple orthogonal methods, replicated across two labs\",\n      \"pmids\": [\"36718630\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"GRA64 is a transmembrane dense granule protein that localizes to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite stages, where it is exposed to the host cytoplasm and physically interacts with host ESCRT components (including CHMP4B) to recruit ESCRT-III machinery to the PVM; together with GRA14, GRA64 enables ESCRT-dependent formation of PVM invaginations and intra-vacuolar vesicles that facilitate scavenging of host organelles into the parasitophorous vacuole, and its loss causes structural defects in intracystic vesicle formation during chronic infection.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"GRA64 is a Toxoplasma gondii transmembrane dense granule protein that functions at the host-parasite interface to co-opt host membrane-remodeling machinery [#0, #3]. It localizes to the parasitophorous vacuolar membrane (PVM) in both tachyzoite and bradyzoite vacuoles, with a domain partially exposed to the host cytoplasm where it engages host endosomal sorting complexes required for transport (ESCRT) [#0, #1]. Through this exposed region, GRA64 cooperates with GRA14 to recruit host ESCRT-III (CHMP4B) to the PVM, driving ESCRT-dependent PVM invaginations and the scavenging of host organelles into the parasitophorous vacuole; loss of GRA64 reduces these invaginations and organelle accumulation, with the strongest defect in the GRA14/GRA64 double knockout [#3]. During chronic infection, GRA64 disruption produces enlarged vesicular structures beneath the cyst membrane, implicating it in organizing intracystic vesicle formation [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2022,\n      \"claim\": \"Establishing where GRA64 acts and how it is oriented was the first step in defining its role at the host-parasite interface; localizing it to the PVM with host-cytoplasm exposure positioned it to interact with host factors.\",\n      \"evidence\": \"Immunofluorescence, ultrastructural analysis, and subcellular fractionation of tachyzoite and bradyzoite vacuoles in T. gondii ME49\",\n      \"pmids\": [\"35730903\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Precise topology and which residues face the host cytoplasm not mapped\", \"Membrane insertion/trafficking mechanism to the PVM not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying host binding partners answered what GRA64's cytoplasm-exposed domain does; two orthogonal methods showed it engages host ESCRT components at the PVM.\",\n      \"evidence\": \"Co-immunoprecipitation and proximity-based biotinylation (BioID) in T. gondii ME49\",\n      \"pmids\": [\"35730903\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect binding to specific ESCRT subunits not resolved\", \"Interaction interface not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic loss-of-function connected GRA64 to a chronic-stage structural phenotype, showing it is required for proper intracystic vesicle organization during bradyzoite/cyst development.\",\n      \"evidence\": \"\\u0394gra64 knockout with electron tomography of tissue cysts in T. gondii ME49\",\n      \"pmids\": [\"35730903\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between altered vesicles and parasite fitness/persistence not established\", \"Whether the phenotype reflects ESCRT mis-recruitment not directly tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Reciprocal genetic epistasis with GRA14 and dominant-negative ESCRT perturbation established GRA64's mechanistic role: it drives ESCRT-III-dependent PVM invagination and scavenging of host organelles into the vacuole.\",\n      \"evidence\": \"Single and double knockouts (\\u0394GRA64, \\u0394GRA14, double KO) with dominant-negative CHMP4B and Vps4A expression, fluorescence and electron microscopy in T. gondii\",\n      \"pmids\": [\"36718630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of cooperativity between GRA64 and GRA14 not defined\", \"Functional consequence of host organelle scavenging for parasite metabolism not established\", \"Order of ESCRT subunit recruitment relative to GRA64 not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA64 physically nucleates ESCRT-III assembly and whether its chronic-stage vesicle role is mechanistically identical to its tachyzoite scavenging function remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the GRA64-ESCRT interaction\", \"Direct binding partner among ESCRT subunits not identified\", \"Contribution to virulence or persistence in vivo not quantified\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005515\", \"supporting_discovery_ids\": [1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]},\n      {\"term_id\": \"GO:0016020\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0061912\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Host ESCRT-III machinery (CHMP4B) recruited to the PVM\"\n    ],\n    \"partners\": [\n      \"Host ESCRT components (including CHMP4B)\",\n      \"GRA14 (parasite dense granule protein)\"\n    ],\n    \"other_free_text\": [\n      \"Transmembrane dense granule protein exposed to the host cytoplasm at the PVM\",\n      \"Required for ESCRT-dependent PVM invaginations and scavenging of host organelles into the parasitophorous vacuole\",\n      \"Loss produces enlarged vesicular structures beneath the cyst membrane during chronic infection\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_202620","name":"GRA64","product":"dense granule protein GRA64","uniprot_acc":"S8GNR6","chromosome":"TGME49_chrVIIa","start_pos":3164569},{"strain":"GT1","gene_id":"TGGT1_202620","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrVIIa","start_pos":3063678},{"strain":"RH88","gene_id":"TGRH88_036390","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023088","start_pos":3258561},{"strain":"VEG","gene_id":"TGVEG_202620","name":null,"product":"putative transmembrane protein","uniprot_acc":"B9QFV0","chromosome":"TGVEG_chrVIIa","start_pos":3253136}]}