{"gene":"TG_228170","gene_symbol":"GRA44","run_date":"2026-09-06T08:22:39","timeline":{"discoveries":[{"year":2020,"finding":"GRA44 is required for translocation of the Toxoplasma effector protein GRA16 across the parasitophorous vacuole membrane into host cells, and for upregulation of host c-Myc and cyclin E1 in infected cells, placing it as an essential component of the effector translocation machinery alongside MYR1, MYR2, MYR3, ROP17, and ASP5.","method":"Genetic knockdown/disruption of GRA44 with phenotypic readouts (GRA16 translocation assay, host c-Myc and cyclin E1 upregulation)","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — independently replicated across two labs (Cygan et al. PMID:32075880 and Blakely et al. PMID:32075881) using conditional knockdown and genetic disruption with defined cellular phenotypes","pmids":["32075880","32075881"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA44 physically interacts with MYR1 and other secreted parasitophorous vacuole proteins, as demonstrated by co-immunoprecipitation and affinity purification of MYR1 identifying GRA44 as an interacting partner.","method":"Affinity purification/co-immunoprecipitation of MYR1 followed by mass spectrometry; reciprocal immunoprecipitation of GRA44","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP experiments performed independently in two separate studies (PMID:32075880 and PMID:32075881)","pmids":["32075880","32075881"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA44 is secreted into the parasitophorous vacuole and contains an ASP5 processing motif; it is proteolytically processed (likely by ASP5 or a related protozoan protease of the secretory pathway), generating two major cleavage fragments that are both secreted into the parasitophorous vacuole. However, processing at this site appears nonessential for GRA44's role in protein translocation.","method":"Biochemical fractionation, immunoblotting to detect processed fragments; ASP5-processing motif analysis; genetic experiments showing nonessential nature of processing","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent labs report processing and vacuole localization (PMID:32075880, PMID:32075881), but functional nonessentiality of processing site established in single study","pmids":["32075880","32075881"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA44 contains a putative acid phosphatase domain, suggesting an enzymatic function within the parasitophorous vacuole, though direct phosphatase activity was not experimentally confirmed in these studies.","method":"Sequence/domain analysis; domain described as 'putative'","journal":"mSphere","confidence":"Low","confidence_rationale":"Tier 4 / Weak — domain identified by computational prediction only; no in vitro enzymatic assay reported","pmids":["32075881"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA44 is a dense granule protein secreted into the parasitophorous vacuole that physically associates with the MYR1-containing effector translocation complex and is essential for translocating parasite effectors (e.g., GRA16) across the vacuole membrane into the host cell, thereby enabling downstream host cell reprogramming events such as c-Myc and cyclin E1 upregulation; it contains a putative acid phosphatase domain and is proteolytically processed by a secretory pathway protease, though this processing is not required for its translocation function."},"narrative":{"mechanistic_narrative":"GRA44 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole that functions as an essential component of the effector translocation machinery [PMID:32075880, PMID:32075881]. It physically associates with the MYR1-containing translocation complex and other secreted vacuolar proteins [PMID:32075880, PMID:32075881], and is required for translocating parasite effectors such as GRA16 across the parasitophorous vacuole membrane into the host cell, enabling downstream host reprogramming including upregulation of c-Myc and cyclin E1 [PMID:32075880, PMID:32075881]. Within the vacuole GRA44 is proteolytically processed at an ASP5 motif into two secreted fragments, but cleavage at this site is dispensable for its translocation function [PMID:32075880, PMID:32075881]. Beyond these findings, GRA44 carries a computationally predicted acid phosphatase domain whose enzymatic activity has not been demonstrated in the available corpus [PMID:32075881].","teleology":[{"year":2020,"claim":"Establishing that GRA44 is a functional component of the Toxoplasma effector export system answered whether the MYR1 translocon requires additional partners to move effectors into host cells.","evidence":"Conditional knockdown and genetic disruption of GRA44 with GRA16 translocation and host c-Myc/cyclin E1 readouts, replicated across two labs","pmids":["32075880","32075881"],"confidence":"High","gaps":["The precise step of translocation GRA44 mediates (cargo recognition, membrane pore formation, or complex stability) is not resolved","Whether GRA44 is required for translocation of effectors beyond GRA16 was not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Reciprocal co-immunoprecipitation placed GRA44 physically within the MYR1 translocation complex, connecting its functional phenotype to a defined molecular assembly.","evidence":"Affinity purification/Co-IP of MYR1 with mass spectrometry and reciprocal IP of GRA44, in two independent studies","pmids":["32075880","32075881"],"confidence":"High","gaps":["Direct binding versus indirect association within the complex is not distinguished","The stoichiometry and architecture of the GRA44-MYR1 complex are unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Characterizing GRA44 processing addressed whether maturation by a secretory-pathway protease is required for its function, distinguishing biogenesis from activity.","evidence":"Biochemical fractionation and immunoblotting of cleavage fragments plus ASP5-motif analysis, with genetic testing of processing-site nonessentiality (single study)","pmids":["32075880","32075881"],"confidence":"Medium","gaps":["Nonessentiality of the processing site established in a single study","Identity of the responsible protease (ASP5 versus another) not definitively assigned","Function of the two secreted fragments individually is unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Identification of a putative acid phosphatase domain raised the possibility of an enzymatic role for GRA44 in the vacuole.","evidence":"Sequence/domain prediction; domain described as putative with no enzymatic assay","pmids":["32075881"],"confidence":"Low","gaps":["No in vitro or in situ phosphatase activity was demonstrated","No substrate identified","Whether any predicted enzymatic activity contributes to translocation is untested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"Whether GRA44 possesses catalytic phosphatase activity and how it mechanistically contributes to effector membrane translocation remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No enzymatic assay confirming or refuting phosphatase activity","No structural model of GRA44 within the translocation complex","Mechanism by which the complex moves effectors across the PVM is undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MYR1-containing effector translocation complex"],"partners":["MYR1"],"other_free_text":["Required for GRA16 effector translocation across the parasitophorous vacuole membrane and for host c-Myc and cyclin E1 upregulation [#0]","Proteolytically processed at an ASP5 motif into two secreted fragments; processing dispensable for translocation function [#2]","Contains a computationally predicted acid phosphatase domain with no demonstrated enzymatic activity [#3]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"32075880","id":"PMC_32075880","title":"Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32075880","citation_count":53,"is_preprint":false},{"pmid":"32075881","id":"PMC_32075881","title":"The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32075881","citation_count":23,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3788,"output_tokens":1175,"usd":0.014495,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6091,"output_tokens":2083,"usd":0.041265,"stage2_stop_reason":"end_turn"},"total_usd":0.05576,"stage1_batch_id":"msgbatch_01WgHz6kceqm573AGLyz57at","stage2_batch_id":"msgbatch_01LK4wDYj5zYquahv7gi9893","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA44 is required for translocation of the Toxoplasma effector protein GRA16 across the parasitophorous vacuole membrane into host cells, and for upregulation of host c-Myc and cyclin E1 in infected cells, placing it as an essential component of the effector translocation machinery alongside MYR1, MYR2, MYR3, ROP17, and ASP5.\",\n      \"method\": \"Genetic knockdown/disruption of GRA44 with phenotypic readouts (GRA16 translocation assay, host c-Myc and cyclin E1 upregulation)\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independently replicated across two labs (Cygan et al. PMID:32075880 and Blakely et al. PMID:32075881) using conditional knockdown and genetic disruption with defined cellular phenotypes\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA44 physically interacts with MYR1 and other secreted parasitophorous vacuole proteins, as demonstrated by co-immunoprecipitation and affinity purification of MYR1 identifying GRA44 as an interacting partner.\",\n      \"method\": \"Affinity purification/co-immunoprecipitation of MYR1 followed by mass spectrometry; reciprocal immunoprecipitation of GRA44\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP experiments performed independently in two separate studies (PMID:32075880 and PMID:32075881)\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA44 is secreted into the parasitophorous vacuole and contains an ASP5 processing motif; it is proteolytically processed (likely by ASP5 or a related protozoan protease of the secretory pathway), generating two major cleavage fragments that are both secreted into the parasitophorous vacuole. However, processing at this site appears nonessential for GRA44's role in protein translocation.\",\n      \"method\": \"Biochemical fractionation, immunoblotting to detect processed fragments; ASP5-processing motif analysis; genetic experiments showing nonessential nature of processing\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent labs report processing and vacuole localization (PMID:32075880, PMID:32075881), but functional nonessentiality of processing site established in single study\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA44 contains a putative acid phosphatase domain, suggesting an enzymatic function within the parasitophorous vacuole, though direct phosphatase activity was not experimentally confirmed in these studies.\",\n      \"method\": \"Sequence/domain analysis; domain described as 'putative'\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — domain identified by computational prediction only; no in vitro enzymatic assay reported\",\n      \"pmids\": [\"32075881\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"GRA44 is a dense granule protein secreted into the parasitophorous vacuole that physically associates with the MYR1-containing effector translocation complex and is essential for translocating parasite effectors (e.g., GRA16) across the vacuole membrane into the host cell, thereby enabling downstream host cell reprogramming events such as c-Myc and cyclin E1 upregulation; it contains a putative acid phosphatase domain and is proteolytically processed by a secretory pathway protease, though this processing is not required for its translocation function.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA44 is a Toxoplasma gondii dense granule protein secreted into the parasitophorous vacuole that functions as an essential component of the effector translocation machinery [#0]. It physically associates with the MYR1-containing translocation complex and other secreted vacuolar proteins [#1], and is required for translocating parasite effectors such as GRA16 across the parasitophorous vacuole membrane into the host cell, enabling downstream host reprogramming including upregulation of c-Myc and cyclin E1 [#0]. Within the vacuole GRA44 is proteolytically processed at an ASP5 motif into two secreted fragments, but cleavage at this site is dispensable for its translocation function [#2]. Beyond these findings, GRA44 carries a computationally predicted acid phosphatase domain whose enzymatic activity has not been demonstrated in the available corpus [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2020,\n      \"claim\": \"Establishing that GRA44 is a functional component of the Toxoplasma effector export system answered whether the MYR1 translocon requires additional partners to move effectors into host cells.\",\n      \"evidence\": \"Conditional knockdown and genetic disruption of GRA44 with GRA16 translocation and host c-Myc/cyclin E1 readouts, replicated across two labs\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The precise step of translocation GRA44 mediates (cargo recognition, membrane pore formation, or complex stability) is not resolved\",\n        \"Whether GRA44 is required for translocation of effectors beyond GRA16 was not tested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Reciprocal co-immunoprecipitation placed GRA44 physically within the MYR1 translocation complex, connecting its functional phenotype to a defined molecular assembly.\",\n      \"evidence\": \"Affinity purification/Co-IP of MYR1 with mass spectrometry and reciprocal IP of GRA44, in two independent studies\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct binding versus indirect association within the complex is not distinguished\",\n        \"The stoichiometry and architecture of the GRA44-MYR1 complex are unknown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Characterizing GRA44 processing addressed whether maturation by a secretory-pathway protease is required for its function, distinguishing biogenesis from activity.\",\n      \"evidence\": \"Biochemical fractionation and immunoblotting of cleavage fragments plus ASP5-motif analysis, with genetic testing of processing-site nonessentiality (single study)\",\n      \"pmids\": [\"32075880\", \"32075881\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Nonessentiality of the processing site established in a single study\",\n        \"Identity of the responsible protease (ASP5 versus another) not definitively assigned\",\n        \"Function of the two secreted fragments individually is unknown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of a putative acid phosphatase domain raised the possibility of an enzymatic role for GRA44 in the vacuole.\",\n      \"evidence\": \"Sequence/domain prediction; domain described as putative with no enzymatic assay\",\n      \"pmids\": [\"32075881\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No in vitro or in situ phosphatase activity was demonstrated\",\n        \"No substrate identified\",\n        \"Whether any predicted enzymatic activity contributes to translocation is untested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether GRA44 possesses catalytic phosphatase activity and how it mechanistically contributes to effector membrane translocation remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No enzymatic assay confirming or refuting phosphatase activity\",\n        \"No structural model of GRA44 within the translocation complex\",\n        \"Mechanism by which the complex moves effectors across the PVM is undefined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140318\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006810\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\"MYR1-containing effector translocation complex\"],\n    \"partners\": [\"MYR1\"],\n    \"other_free_text\": [\n      \"Required for GRA16 effector translocation across the parasitophorous vacuole membrane and for host c-Myc and cyclin E1 upregulation [#0]\",\n      \"Proteolytically processed at an ASP5 motif into two secreted fragments; processing dispensable for translocation function [#2]\",\n      \"Contains a computationally predicted acid phosphatase domain with no demonstrated enzymatic activity [#3]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_228170","name":"GRA44","product":"dense granule protein GRA44","uniprot_acc":"S8G6H2","chromosome":"TGME49_chrX","start_pos":520205},{"strain":"GT1","gene_id":"TGGT1_228170","name":null,"product":"inner membrane complex protein IMC2A","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":431147},{"strain":"RH88","gene_id":"TGRH88_047510","name":null,"product":"inner membrane complex protein IMC2A","uniprot_acc":null,"chromosome":"CM023091","start_pos":7045151},{"strain":"VEG","gene_id":"TGVEG_228170","name":null,"product":"inner membrane complex protein IMC2A","uniprot_acc":"B9Q8E3","chromosome":"TGVEG_chrX","start_pos":411345}]}