{"gene":"TG_254720","gene_symbol":"GRA8","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2000,"finding":"GRA8 is a novel 38 kDa dense granule protein of Toxoplasma gondii; it is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. Its deduced amino acid sequence contains an N-terminal signal peptide, three degenerate proline-rich repeats, and a potential C-terminal transmembrane domain.","method":"Monoclonal antibody generation, immunoelectron microscopy, cDNA library screening, sequence analysis","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by immunoelectron microscopy combined with cDNA cloning and sequence analysis; foundational characterization paper","pmids":["10613696"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"GRA8 was identified as an excretory dense granule protein and mapped on the two-dimensional proteome map of Toxoplasma gondii tachyzoite lysate by Western blotting of 2D-separated protein spots.","method":"2D polyacrylamide gel electrophoresis, Western blotting","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — proteome mapping with Western blot confirmation, consistent with independent characterization","pmids":["11511000"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"GRA8 transcript levels are developmentally regulated, being upregulated during tachyzoite-to-bradyzoite differentiation, classifying it among secretory organelle protein genes with stage-specific expression.","method":"cDNA microarray analysis of transcript levels during stage differentiation","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — microarray-based transcript profiling, single study","pmids":["12455982"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"finding":"GRA8 was identified as one of at least four proteins (along with MAG1, MCP4, and GRA9) that co-immunoprecipitate with bradyzoite pseudokinase 1 (BPK1) from in vitro bradyzoite cultures, indicating GRA8 is a component of the BPK1-associated complex in the cyst wall.","method":"Immunoprecipitation of BPK1 followed by protein identification (mass spectrometry implied)","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP experiment, single lab; no reciprocal pull-down reported in abstract","pmids":["23291621"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"PKCα-mediated phosphorylation of GRA8 at Thr220 is required for mitochondrial trafficking of GRA8 into host cell mitochondria. In mitochondria, GRA8 interacts with ATP5A1 (via its C-terminal domain binding the nucleotide-binding domain of ATP5A1) and with SIRT3, facilitating SIRT3-mediated deacetylation of ATP5A1 at K506 and K531, leading to increased ATP production and anti-septic activity in vivo.","method":"Co-immunoprecipitation, phosphorylation assay, mutagenesis (Thr220), mitochondrial fractionation/trafficking assay, in vivo sepsis model","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, mutagenesis, fractionation, in vivo model) in a single lab","pmids":["29869623"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GRA8 (TgGRA8I) is a component of the sub-pellicular cytoskeleton of Toxoplasma gondii; it associates with sub-pellicular microtubules and actin as shown by immunoelectron microscopy, proteomics, and biochemical fractionation. Deletion of gra8 (RHΔku80Δgra8) causes loss of conoid extrusion, diminished host cell infection, and reduced egress, accompanied by structural alterations in sub-pellicular microtubules and the meshwork. In silico docking also suggested interaction with β-tubulin and actin.","method":"Immunoelectron microscopy, proteomic analysis, biochemical fractionation, gene knockout (RHΔku80Δgra8), conoid extrusion assay, invasion and egress assays, docking analysis","journal":"Parasitology research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (immunoEM, biochemical fractionation, proteomics, genetic KO with specific phenotypic readouts) in a single rigorous study","pmids":["30949853"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"finding":"Deletion of GRA8 in the type II Prugniaud (Pru) strain (PruΔgra8) induced severe defects in development of chronic-stage cysts in vivo without affecting parasite growth rate or the ability to differentiate into cysts in vitro, placing GRA8 as a PVM-associated dense granule protein required for chronic infection.","method":"Gene deletion (knockout) in type II Pru strain, in vivo mouse infection model, in vitro cyst formation assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined in vivo and in vitro phenotypic readouts, replicated across multiple GRA deletions in same study","pmids":["31266861"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]}],"current_model":"GRA8 is a proline-rich, transmembrane-domain-containing dense granule protein that is secreted into the parasitophorous vacuole upon host cell invasion, where it associates with the PVM and contributes to chronic cyst development in vivo; it also localizes to the parasite sub-pellicular cytoskeleton—associating with microtubules and actin—and its loss impairs conoid extrusion, invasion, and egress; additionally, following PKCα-mediated phosphorylation at Thr220, GRA8 traffics to host mitochondria where it interacts with ATP5A1 and SIRT3 to promote ATP5A1 deacetylation and ATP production."},"narrative":{"mechanistic_narrative":"GRA8 is a proline-rich, transmembrane-domain-containing dense granule protein of Toxoplasma gondii that is secreted into the parasitophorous vacuole during or shortly after invasion and associates with the vacuole periphery [PMID:10613696]. Its transcription is developmentally upregulated during the tachyzoite-to-bradyzoite transition [PMID:12455982], and in bradyzoites it participates in a BPK1-associated cyst wall complex together with MAG1, MCP4, and GRA9 [PMID:23291621]; consistent with a role in chronic-stage biology, deletion in the type II Pru strain severely impairs in vivo cyst development without affecting growth or in vitro differentiation [PMID:31266861]. Beyond the vacuole, GRA8 is a component of the parasite sub-pellicular cytoskeleton, associating with sub-pellicular microtubules and actin, and its loss in RH abolishes conoid extrusion and reduces host cell invasion and egress with accompanying microtubule meshwork defects [PMID:30949853]. GRA8 also manipulates host cell metabolism: following PKCα-mediated phosphorylation at Thr220, it traffics into host mitochondria where its C-terminal domain binds the nucleotide-binding domain of ATP5A1 and engages SIRT3 to drive SIRT3-mediated deacetylation of ATP5A1 at K506/K531, increasing ATP production and conferring anti-septic activity in vivo [PMID:29869623].","teleology":[{"year":2000,"claim":"Establishing GRA8's identity resolved the question of whether it is a secreted parasite factor and where it acts, defining it as a dense granule protein delivered to the parasitophorous vacuole.","evidence":"Monoclonal antibody generation, immunoelectron microscopy, and cDNA cloning/sequence analysis in T. gondii","pmids":["10613696"],"confidence":"High","gaps":["The function of the proline-rich repeats and C-terminal transmembrane domain was not tested","No genetic disruption to assign a phenotype"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"Independent proteomic mapping confirmed GRA8 as an excretory dense granule protein, corroborating its secreted status.","evidence":"2D PAGE and Western blotting of tachyzoite lysate in T. gondii","pmids":["11511000"],"confidence":"Medium","gaps":["No functional readout","Descriptive proteomic placement only"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Transcript profiling linked GRA8 expression to stage conversion, raising the hypothesis that it functions in bradyzoite/chronic-stage biology.","evidence":"cDNA microarray of transcript levels during stage differentiation in T. gondii","pmids":["12455982"],"confidence":"Medium","gaps":["Transcript-level only; no protein-level or functional confirmation of a bradyzoite role"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"claim":"Co-immunoprecipitation placed GRA8 within a defined bradyzoite protein complex, physically connecting it to the cyst wall machinery.","evidence":"Immunoprecipitation of BPK1 with protein identification from in vitro bradyzoite cultures of T. gondii","pmids":["23291621"],"confidence":"Medium","gaps":["Single Co-IP without reported reciprocal pull-down","Direct vs indirect association with BPK1 not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Identification of Thr220 phosphorylation and mitochondrial trafficking revealed a mechanism by which GRA8 reprograms host mitochondrial metabolism.","evidence":"Co-IP, phosphorylation assay, Thr220 mutagenesis, mitochondrial fractionation, and an in vivo sepsis model in T. gondii","pmids":["29869623"],"confidence":"Medium","gaps":["Single-lab study across orthogonal methods","How host PKCα accesses/phosphorylates GRA8 is not defined","Relationship between this host-mitochondrial role and the vacuolar/cytoskeletal roles is unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Genetic knockout in RH assigned GRA8 a cytoskeletal function, showing it is required for conoid extrusion, invasion, and egress via association with sub-pellicular microtubules and actin.","evidence":"ImmunoEM, proteomics, biochemical fractionation, RHΔku80Δgra8 knockout with conoid/invasion/egress assays, and docking analysis","pmids":["30949853"],"confidence":"High","gaps":["Direct microtubule/actin binding inferred from docking, not biochemically demonstrated","How a secreted dense granule protein also localizes to the cytoskeleton is unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2019,"claim":"Knockout in the type II Pru strain established GRA8's in vivo requirement for chronic cyst development, distinct from any growth or differentiation defect.","evidence":"Gene deletion in Pru strain, in vivo mouse infection, and in vitro cyst formation assays in T. gondii","pmids":["31266861"],"confidence":"High","gaps":["Molecular basis of the chronic-stage defect not defined","Connection to the BPK1 cyst wall complex not directly tested in this system"],"attribution":[{"species":"Toxoplasma gondii","strain":"Prugniaud (Pru)"}]},{"year":null,"claim":"How GRA8's distinct localizations—vacuolar membrane, parasite sub-pellicular cytoskeleton, and host mitochondria—are coordinated from a single secreted protein remains unresolved.","evidence":"No single study in the corpus reconciles the vacuolar, cytoskeletal, and host-mitochondrial roles","pmids":[],"confidence":"Low","gaps":["Trafficking signals directing GRA8 to each compartment are unknown","Whether the same protein pool serves all roles is untested","No structural model of GRA8"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["BPK1-associated cyst wall complex (with MAG1, MCP4, GRA9) [#3]"],"partners":["BPK1 [#3]","MAG1 [#3]","MCP4 [#3]","GRA9 [#3]","ATP5A1 (HOST) [#4]","SIRT3 (HOST) [#4]","PKCΑ (HOST, KINASE FOR THR220) [#4]","Β-TUBULIN (DOCKING) [#5]","ACTIN (DOCKING) [#5]"],"other_free_text":["38 kDa protein with N-terminal signal peptide, three degenerate proline-rich repeats, and a C-terminal transmembrane domain [#0]","Phosphorylated at Thr220 by host PKCα, a modification required for host mitochondrial trafficking [#4]","Transcript developmentally upregulated during tachyzoite-to-bradyzoite differentiation [#2]","RHΔgra8 loses conoid extrusion and shows reduced invasion and egress [#5]; PruΔgra8 shows severe chronic cyst development defects in vivo without growth/differentiation defects [#6]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"12455982","id":"PMC_12455982","title":"Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene 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diseases","url":"https://pubmed.ncbi.nlm.nih.gov/16299997","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17841,"output_tokens":1992,"usd":0.041702,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7324,"output_tokens":3514,"usd":0.062235,"stage2_stop_reason":"end_turn"},"total_usd":0.103937,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"GRA8 is a novel 38 kDa dense granule protein of Toxoplasma gondii; it is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. Its deduced amino acid sequence contains an N-terminal signal peptide, three degenerate proline-rich repeats, and a potential C-terminal transmembrane domain.\",\n      \"method\": \"Monoclonal antibody generation, immunoelectron microscopy, cDNA library screening, sequence analysis\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by immunoelectron microscopy combined with cDNA cloning and sequence analysis; foundational characterization paper\",\n      \"pmids\": [\"10613696\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GRA8 was identified as an excretory dense granule protein and mapped on the two-dimensional proteome map of Toxoplasma gondii tachyzoite lysate by Western blotting of 2D-separated protein spots.\",\n      \"method\": \"2D polyacrylamide gel electrophoresis, Western blotting\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — proteome mapping with Western blot confirmation, consistent with independent characterization\",\n      \"pmids\": [\"11511000\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"GRA8 transcript levels are developmentally regulated, being upregulated during tachyzoite-to-bradyzoite differentiation, classifying it among secretory organelle protein genes with stage-specific expression.\",\n      \"method\": \"cDNA microarray analysis of transcript levels during stage differentiation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — microarray-based transcript profiling, single study\",\n      \"pmids\": [\"12455982\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"GRA8 was identified as one of at least four proteins (along with MAG1, MCP4, and GRA9) that co-immunoprecipitate with bradyzoite pseudokinase 1 (BPK1) from in vitro bradyzoite cultures, indicating GRA8 is a component of the BPK1-associated complex in the cyst wall.\",\n      \"method\": \"Immunoprecipitation of BPK1 followed by protein identification (mass spectrometry implied)\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP experiment, single lab; no reciprocal pull-down reported in abstract\",\n      \"pmids\": [\"23291621\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PKCα-mediated phosphorylation of GRA8 at Thr220 is required for mitochondrial trafficking of GRA8 into host cell mitochondria. In mitochondria, GRA8 interacts with ATP5A1 (via its C-terminal domain binding the nucleotide-binding domain of ATP5A1) and with SIRT3, facilitating SIRT3-mediated deacetylation of ATP5A1 at K506 and K531, leading to increased ATP production and anti-septic activity in vivo.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay, mutagenesis (Thr220), mitochondrial fractionation/trafficking assay, in vivo sepsis model\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, mutagenesis, fractionation, in vivo model) in a single lab\",\n      \"pmids\": [\"29869623\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GRA8 (TgGRA8I) is a component of the sub-pellicular cytoskeleton of Toxoplasma gondii; it associates with sub-pellicular microtubules and actin as shown by immunoelectron microscopy, proteomics, and biochemical fractionation. Deletion of gra8 (RHΔku80Δgra8) causes loss of conoid extrusion, diminished host cell infection, and reduced egress, accompanied by structural alterations in sub-pellicular microtubules and the meshwork. In silico docking also suggested interaction with β-tubulin and actin.\",\n      \"method\": \"Immunoelectron microscopy, proteomic analysis, biochemical fractionation, gene knockout (RHΔku80Δgra8), conoid extrusion assay, invasion and egress assays, docking analysis\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (immunoEM, biochemical fractionation, proteomics, genetic KO with specific phenotypic readouts) in a single rigorous study\",\n      \"pmids\": [\"30949853\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deletion of GRA8 in the type II Prugniaud (Pru) strain (PruΔgra8) induced severe defects in development of chronic-stage cysts in vivo without affecting parasite growth rate or the ability to differentiate into cysts in vitro, placing GRA8 as a PVM-associated dense granule protein required for chronic infection.\",\n      \"method\": \"Gene deletion (knockout) in type II Pru strain, in vivo mouse infection model, in vitro cyst formation assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined in vivo and in vitro phenotypic readouts, replicated across multiple GRA deletions in same study\",\n      \"pmids\": [\"31266861\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    }\n  ],\n  \"current_model\": \"GRA8 is a proline-rich, transmembrane-domain-containing dense granule protein that is secreted into the parasitophorous vacuole upon host cell invasion, where it associates with the PVM and contributes to chronic cyst development in vivo; it also localizes to the parasite sub-pellicular cytoskeleton—associating with microtubules and actin—and its loss impairs conoid extrusion, invasion, and egress; additionally, following PKCα-mediated phosphorylation at Thr220, GRA8 traffics to host mitochondria where it interacts with ATP5A1 and SIRT3 to promote ATP5A1 deacetylation and ATP production.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA8 is a proline-rich, transmembrane-domain-containing dense granule protein of Toxoplasma gondii that is secreted into the parasitophorous vacuole during or shortly after invasion and associates with the vacuole periphery [#0]. Its transcription is developmentally upregulated during the tachyzoite-to-bradyzoite transition [#2], and in bradyzoites it participates in a BPK1-associated cyst wall complex together with MAG1, MCP4, and GRA9 [#3]; consistent with a role in chronic-stage biology, deletion in the type II Pru strain severely impairs in vivo cyst development without affecting growth or in vitro differentiation [#6]. Beyond the vacuole, GRA8 is a component of the parasite sub-pellicular cytoskeleton, associating with sub-pellicular microtubules and actin, and its loss in RH abolishes conoid extrusion and reduces host cell invasion and egress with accompanying microtubule meshwork defects [#5]. GRA8 also manipulates host cell metabolism: following PKC\\u03b1-mediated phosphorylation at Thr220, it traffics into host mitochondria where its C-terminal domain binds the nucleotide-binding domain of ATP5A1 and engages SIRT3 to drive SIRT3-mediated deacetylation of ATP5A1 at K506/K531, increasing ATP production and conferring anti-septic activity in vivo [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing GRA8's identity resolved the question of whether it is a secreted parasite factor and where it acts, defining it as a dense granule protein delivered to the parasitophorous vacuole.\",\n      \"evidence\": \"Monoclonal antibody generation, immunoelectron microscopy, and cDNA cloning/sequence analysis in T. gondii\",\n      \"pmids\": [\"10613696\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The function of the proline-rich repeats and C-terminal transmembrane domain was not tested\", \"No genetic disruption to assign a phenotype\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Independent proteomic mapping confirmed GRA8 as an excretory dense granule protein, corroborating its secreted status.\",\n      \"evidence\": \"2D PAGE and Western blotting of tachyzoite lysate in T. gondii\",\n      \"pmids\": [\"11511000\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional readout\", \"Descriptive proteomic placement only\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Transcript profiling linked GRA8 expression to stage conversion, raising the hypothesis that it functions in bradyzoite/chronic-stage biology.\",\n      \"evidence\": \"cDNA microarray of transcript levels during stage differentiation in T. gondii\",\n      \"pmids\": [\"12455982\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcript-level only; no protein-level or functional confirmation of a bradyzoite role\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Co-immunoprecipitation placed GRA8 within a defined bradyzoite protein complex, physically connecting it to the cyst wall machinery.\",\n      \"evidence\": \"Immunoprecipitation of BPK1 with protein identification from in vitro bradyzoite cultures of T. gondii\",\n      \"pmids\": [\"23291621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reported reciprocal pull-down\", \"Direct vs indirect association with BPK1 not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of Thr220 phosphorylation and mitochondrial trafficking revealed a mechanism by which GRA8 reprograms host mitochondrial metabolism.\",\n      \"evidence\": \"Co-IP, phosphorylation assay, Thr220 mutagenesis, mitochondrial fractionation, and an in vivo sepsis model in T. gondii\",\n      \"pmids\": [\"29869623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study across orthogonal methods\", \"How host PKC\\u03b1 accesses/phosphorylates GRA8 is not defined\", \"Relationship between this host-mitochondrial role and the vacuolar/cytoskeletal roles is unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic knockout in RH assigned GRA8 a cytoskeletal function, showing it is required for conoid extrusion, invasion, and egress via association with sub-pellicular microtubules and actin.\",\n      \"evidence\": \"ImmunoEM, proteomics, biochemical fractionation, RH\\u0394ku80\\u0394gra8 knockout with conoid/invasion/egress assays, and docking analysis\",\n      \"pmids\": [\"30949853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct microtubule/actin binding inferred from docking, not biochemically demonstrated\", \"How a secreted dense granule protein also localizes to the cytoskeleton is unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Knockout in the type II Pru strain established GRA8's in vivo requirement for chronic cyst development, distinct from any growth or differentiation defect.\",\n      \"evidence\": \"Gene deletion in Pru strain, in vivo mouse infection, and in vitro cyst formation assays in T. gondii\",\n      \"pmids\": [\"31266861\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the chronic-stage defect not defined\", \"Connection to the BPK1 cyst wall complex not directly tested in this system\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Prugniaud (Pru)\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA8's distinct localizations\\u2014vacuolar membrane, parasite sub-pellicular cytoskeleton, and host mitochondria\\u2014are coordinated from a single secreted protein remains unresolved.\",\n      \"evidence\": \"No single study in the corpus reconciles the vacuolar, cytoskeletal, and host-mitochondrial roles\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Trafficking signals directing GRA8 to each compartment are unknown\", \"Whether the same protein pool serves all roles is untested\", \"No structural model of GRA8\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008017\", \"supporting_discovery_ids\": [5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0051087\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005868\", \"supporting_discovery_ids\": [5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071556\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006754\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0075509\", \"supporting_discovery_ids\": [5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"BPK1-associated cyst wall complex (with MAG1, MCP4, GRA9) [#3]\"\n    ],\n    \"partners\": [\n      \"BPK1 [#3]\",\n      \"MAG1 [#3]\",\n      \"MCP4 [#3]\",\n      \"GRA9 [#3]\",\n      \"ATP5A1 (host) [#4]\",\n      \"SIRT3 (host) [#4]\",\n      \"PKC\\u03b1 (host, kinase for Thr220) [#4]\",\n      \"\\u03b2-tubulin (docking) [#5]\",\n      \"actin (docking) [#5]\"\n    ],\n    \"other_free_text\": [\n      \"38 kDa protein with N-terminal signal peptide, three degenerate proline-rich repeats, and a C-terminal transmembrane domain [#0]\",\n      \"Phosphorylated at Thr220 by host PKC\\u03b1, a modification required for host mitochondrial trafficking [#4]\",\n      \"Transcript developmentally upregulated during tachyzoite-to-bradyzoite differentiation [#2]\",\n      \"RH\\u0394gra8 loses conoid extrusion and shows reduced invasion and egress [#5]; Pru\\u0394gra8 shows severe chronic cyst development defects in vivo without growth/differentiation defects [#6]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_254720","name":"GRA8","product":"dense granule protein GRA8","uniprot_acc":"S8F761","chromosome":"TGME49_chrIII","start_pos":1904440},{"strain":"GT1","gene_id":"TGGT1_254720","name":null,"product":"dense granule protein GRA8","uniprot_acc":null,"chromosome":"TGGT1_chrIII","start_pos":1884681},{"strain":"RH88","gene_id":"TGRH88_003600","name":null,"product":"dense granule protein GRA8","uniprot_acc":null,"chromosome":"CM023084","start_pos":702088},{"strain":"VEG","gene_id":"TGVEG_254720","name":null,"product":"dense granule protein GRA8","uniprot_acc":"V4Z4U6","chromosome":"TGVEG_chrIII","start_pos":1913470}]}