{"gene":"TG_237820","gene_symbol":"ISP2","run_date":"2026-09-11T11:01:00","timeline":{"discoveries":[{"year":2010,"finding":"ISP2 localizes to the central sub-compartment of the Toxoplasma gondii IMC, and its targeting is dependent on N-terminal residues predicted for coordinated myristoylation and palmitoylation.","method":"Fluorescence microscopy, N-terminal targeting sequence analysis, site-directed mutagenesis of lipid modification sites","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiments combined with mutagenesis of lipid modification sites, replicated across multiple ISP family members in a single rigorous study","pmids":["20844581"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Disruption of ISP1 causes dramatic relocalization of ISP2 (and ISP3) from the central IMC region to the apical cap, demonstrating that ISP1 acts as a gatekeeper that hierarchically restricts ISP2 to its sub-compartment through its C-terminal region (beyond the N-terminal targeting domain).","method":"Genetic knockout of ISP1 followed by fluorescence microscopy of ISP2 localization; deletion analysis of ISP1 domains","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with direct localization readout, domain dissection confirming N-terminal sufficiency for apical cap targeting but requirement for C-terminal domain in ISP2 exclusion","pmids":["20844581"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, establishing a functional role for ISP2 in coordinating Toxoplasma replication.","method":"Gene disruption (knockout) of ISP2 with phenotypic analysis of daughter cell formation by fluorescence and electron microscopy","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with defined cellular phenotype (endodyogeny defect), replicated in the same study with multiple controls","pmids":["20844581"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"ISP2 is an IMC sub-compartment protein that is anchored to the central region of the Toxoplasma gondii IMC via coordinated N-terminal myristoylation and palmitoylation (likely executed by the PAT TgDHHC2); its sub-compartment identity is enforced hierarchically by ISP1 (which gates ISP2 away from the apical cap through its C-terminal domain); structurally, ISP family members adopt a repurposed pleckstrin homology fold that mediates protein–protein interactions rather than phospholipid binding; and loss of ISP2 causes severe daughter-cell formation defects during endodyogeny, indicating it plays an essential scaffolding role in parasite replication."},"narrative":{"mechanistic_narrative":"ISP2 is an inner membrane complex (IMC) sub-compartment protein of Toxoplasma gondii that acts as a scaffolding factor required for daughter-cell formation during endodyogeny [PMID:20844581]. It localizes specifically to the central sub-compartment of the IMC through an N-terminal targeting signal that depends on coordinated myristoylation and palmitoylation for membrane anchoring [PMID:20844581]. Its sub-compartment identity is enforced hierarchically: the related protein ISP1 gates ISP2 away from the apical cap, and disruption of ISP1 relocalizes ISP2 to the apical cap through a mechanism requiring the ISP1 C-terminal domain [PMID:20844581]. Loss of ISP2 produces severe defects in daughter-cell formation, establishing its essential role in coordinating parasite replication [PMID:20844581].","teleology":[{"year":2010,"claim":"Establishing where ISP2 resides and how it is anchored addressed how the IMC is partitioned into sub-compartments and defined a lipid-dependent targeting mechanism.","evidence":"Fluorescence microscopy with N-terminal targeting analysis and site-directed mutagenesis of predicted myristoylation/palmitoylation sites in T. gondii","pmids":["20844581"],"confidence":"High","gaps":["The specific enzymes executing the lipid modifications were not identified in this study","Direct binding partners in the central sub-compartment were not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Testing whether sub-compartment identity is imposed by other ISP proteins revealed a hierarchical gatekeeping system in which ISP1 restricts ISP2 to the central region.","evidence":"Genetic knockout of ISP1 with fluorescence readout of ISP2 localization plus ISP1 domain-deletion analysis in T. gondii","pmids":["20844581"],"confidence":"High","gaps":["Molecular basis of ISP1-mediated exclusion (direct interaction vs indirect crowding) not resolved","Which ISP1 C-terminal residues mediate ISP2 exclusion not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Determining the consequence of ISP2 loss assigned a functional role in replication, showing ISP2 is required for proper daughter-cell formation during endodyogeny.","evidence":"Gene disruption of ISP2 with phenotypic analysis by fluorescence and electron microscopy in T. gondii","pmids":["20844581"],"confidence":"High","gaps":["The molecular scaffolding activity underlying the defect was not identified","Interacting components of the daughter-cell machinery not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["ISP1","ISP3"],"other_free_text":["N-terminally myristoylated and palmitoylated for IMC membrane anchoring [#0]","Targeted to the central IMC sub-compartment; excluded from the apical cap by ISP1 gatekeeping [#0, #1]","Adopts a pleckstrin homology fold repurposed for protein–protein interactions rather than phospholipid binding [#3, #4]","Required for daughter-cell formation during endodyogeny [#2]","Membrane association depends on the palmitoyl acyltransferase TgDHHC2 (inferred by family membership) [#5]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"20844581","id":"PMC_20844581","title":"A novel family of Toxoplasma IMC proteins displays a hierarchical organization and functions in coordinating parasite division.","date":"2010","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/20844581","citation_count":192,"is_preprint":false},{"pmid":"22659420","id":"PMC_22659420","title":"Toxoplasma ISP4 is a central IMC sub-compartment protein whose localization depends on palmitoylation but not myristoylation.","date":"2012","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/22659420","citation_count":44,"is_preprint":false},{"pmid":"27548677","id":"PMC_27548677","title":"Virus-Like Nanoparticle Vaccine Confers Protection against Toxoplasma gondii.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27548677","citation_count":33,"is_preprint":false},{"pmid":"21829561","id":"PMC_21829561","title":"Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21829561","citation_count":33,"is_preprint":false},{"pmid":"25088010","id":"PMC_25088010","title":"Identification and characterization of Toxoplasma SIP, a conserved apicomplexan cytoskeleton protein involved in maintaining the shape, motility and virulence of the parasite.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/25088010","citation_count":28,"is_preprint":false},{"pmid":"27586039","id":"PMC_27586039","title":"Cross-protection induced by Toxoplasma gondii virus-like particle vaccine upon intraperitoneal route challenge.","date":"2016","source":"Acta tropica","url":"https://pubmed.ncbi.nlm.nih.gov/27586039","citation_count":15,"is_preprint":false},{"pmid":"34634933","id":"PMC_34634933","title":"A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery.","date":"2021","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/34634933","citation_count":12,"is_preprint":false},{"pmid":"40801525","id":"PMC_40801525","title":"Co-dependent formation of the Toxoplasma gondii subpellicular microtubules and inner membrane skeleton.","date":"2025","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/40801525","citation_count":11,"is_preprint":false},{"pmid":"19820829","id":"PMC_19820829","title":"Toxoplasma gondii: further studies on the subpellicular network.","date":"2009","source":"Memorias do Instituto Oswaldo Cruz","url":"https://pubmed.ncbi.nlm.nih.gov/19820829","citation_count":11,"is_preprint":false},{"pmid":"38270431","id":"PMC_38270431","title":"Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/38270431","citation_count":10,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":23438,"output_tokens":1695,"usd":0.04787,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6913,"output_tokens":2738,"usd":0.051507,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01WgHz6kceqm573AGLyz57at","stage2_batch_id":"msgbatch_01LK4wDYj5zYquahv7gi9893","note":"batch pricing = 50% of standard","skipped":true,"corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"summary amended","hand_review_patch":"2026-09-11 AK hand-review verdicts (handoff/tools_2026-09-11/patch)","hand_review_verdict":"edit"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"ISP2 localizes to the central sub-compartment of the Toxoplasma gondii IMC, and its targeting is dependent on N-terminal residues predicted for coordinated myristoylation and palmitoylation.\",\n      \"method\": \"Fluorescence microscopy, N-terminal targeting sequence analysis, site-directed mutagenesis of lipid modification sites\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiments combined with mutagenesis of lipid modification sites, replicated across multiple ISP family members in a single rigorous study\",\n      \"pmids\": [\"20844581\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Disruption of ISP1 causes dramatic relocalization of ISP2 (and ISP3) from the central IMC region to the apical cap, demonstrating that ISP1 acts as a gatekeeper that hierarchically restricts ISP2 to its sub-compartment through its C-terminal region (beyond the N-terminal targeting domain).\",\n      \"method\": \"Genetic knockout of ISP1 followed by fluorescence microscopy of ISP2 localization; deletion analysis of ISP1 domains\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with direct localization readout, domain dissection confirming N-terminal sufficiency for apical cap targeting but requirement for C-terminal domain in ISP2 exclusion\",\n      \"pmids\": [\"20844581\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, establishing a functional role for ISP2 in coordinating Toxoplasma replication.\",\n      \"method\": \"Gene disruption (knockout) of ISP2 with phenotypic analysis of daughter cell formation by fluorescence and electron microscopy\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with defined cellular phenotype (endodyogeny defect), replicated in the same study with multiple controls\",\n      \"pmids\": [\"20844581\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structures of TgISP1 and TgISP3 reveal that ISP family members adopt a pleckstrin homology (PH) fold. Despite the PH fold, biochemical assays showed no phospholipid-binding activity; instead, conserved surface residues and crystal packing suggest the PH-binding site has been repurposed for protein–protein interactions, with multimerization potentially enhancing avidity.\",\n      \"method\": \"X-ray crystallography (2.10 Å for ISP1; 2.32 Å for ISP3), phospholipid-binding biochemical assays, conserved surface residue mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of two family members with functional biochemical validation (negative phospholipid binding), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24675080\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ISP1 and ISP3 (and by extension ISP2 as a family member) do NOT bind phospholipids despite their pleckstrin homology fold; conserved surface residues overlay the canonical phospholipid-binding site and are proposed to mediate protein–protein interactions instead.\",\n      \"method\": \"Biochemical phospholipid-binding assays with purified recombinant ISP1 and ISP3\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro assay establishing a negative result for phospholipid binding, single lab, result directly informs ISP2 mechanism by family membership\",\n      \"pmids\": [\"24675080\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TgDHHC2 and TgDHHC14, two IMC-resident palmitoyl acyltransferases, act sequentially in IMC sub-compartment biogenesis; depletion of TgDHHC2 causes ISP1 and ISP3 (and their homolog ISP2 by family membership is implied) to lose membrane association, identifying them as likely substrates of TgDHHC2.\",\n      \"method\": \"Conditional knockdown of TgDHHC2/14, fluorescence microscopy of palmitoylated IMC proteins including ISP1/ISP3 as proxies for the ISP family\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockdown with direct localization readout for ISP family members; ISP2 inferred by family membership, not directly tested in this paper\",\n      \"pmids\": [\"32329212\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"ISP2 is an IMC sub-compartment protein that is anchored to the central region of the Toxoplasma gondii IMC via coordinated N-terminal myristoylation and palmitoylation (likely executed by the PAT TgDHHC2); its sub-compartment identity is enforced hierarchically by ISP1 (which gates ISP2 away from the apical cap through its C-terminal domain); structurally, ISP family members adopt a repurposed pleckstrin homology fold that mediates protein–protein interactions rather than phospholipid binding; and loss of ISP2 causes severe daughter-cell formation defects during endodyogeny, indicating it plays an essential scaffolding role in parasite replication.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ISP2 is an inner membrane complex (IMC) sub-compartment protein of Toxoplasma gondii that acts as a scaffolding factor required for daughter-cell formation during endodyogeny [#0, #2]. It localizes specifically to the central sub-compartment of the IMC through an N-terminal targeting signal that depends on coordinated myristoylation and palmitoylation for membrane anchoring [#0]. Its sub-compartment identity is enforced hierarchically: the related protein ISP1 gates ISP2 away from the apical cap, and disruption of ISP1 relocalizes ISP2 to the apical cap through a mechanism requiring the ISP1 C-terminal domain [#1]. Structurally, ISP family members adopt a pleckstrin homology (PH) fold that has been repurposed for protein\\u2013protein interactions rather than phospholipid binding, with conserved surface residues overlaying the canonical lipid-binding site [#3, #4]. Loss of ISP2 produces severe defects in daughter-cell formation, establishing its essential role in coordinating parasite replication [#2]. The palmitoyl acyltransferase TgDHHC2 is implicated in the membrane anchoring of ISP family proteins [#5].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing where ISP2 resides and how it is anchored addressed how the IMC is partitioned into sub-compartments and defined a lipid-dependent targeting mechanism.\",\n      \"evidence\": \"Fluorescence microscopy with N-terminal targeting analysis and site-directed mutagenesis of predicted myristoylation/palmitoylation sites in T. gondii\",\n      \"pmids\": [\"20844581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific enzymes executing the lipid modifications were not identified in this study\", \"Direct binding partners in the central sub-compartment were not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Testing whether sub-compartment identity is imposed by other ISP proteins revealed a hierarchical gatekeeping system in which ISP1 restricts ISP2 to the central region.\",\n      \"evidence\": \"Genetic knockout of ISP1 with fluorescence readout of ISP2 localization plus ISP1 domain-deletion analysis in T. gondii\",\n      \"pmids\": [\"20844581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of ISP1-mediated exclusion (direct interaction vs indirect crowding) not resolved\", \"Which ISP1 C-terminal residues mediate ISP2 exclusion not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Determining the consequence of ISP2 loss assigned a functional role in replication, showing ISP2 is required for proper daughter-cell formation during endodyogeny.\",\n      \"evidence\": \"Gene disruption of ISP2 with phenotypic analysis by fluorescence and electron microscopy in T. gondii\",\n      \"pmids\": [\"20844581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The molecular scaffolding activity underlying the defect was not identified\", \"Interacting components of the daughter-cell machinery not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Solving ISP family structures addressed the biochemical nature of ISP proteins, showing they adopt a PH fold repurposed away from lipid binding toward protein\\u2013protein interactions.\",\n      \"evidence\": \"X-ray crystallography of TgISP1 and TgISP3 with phospholipid-binding assays and conserved surface residue mapping\",\n      \"pmids\": [\"24675080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ISP2 itself was not crystallized; inference relies on family membership\", \"The actual protein interaction partners engaged by the repurposed surface were not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Direct biochemical testing confirmed ISP family proteins do not bind phospholipids, ruling out a lipid-sensing role and reinforcing a protein-interaction function.\",\n      \"evidence\": \"In vitro phospholipid-binding assays with purified recombinant ISP1 and ISP3\",\n      \"pmids\": [\"24675080\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative binding result was not tested directly on ISP2\", \"Conditions tested may not cover all relevant phospholipid species\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying the palmitoyl acyltransferases addressed which enzyme catalyzes ISP membrane anchoring, implicating TgDHHC2 in maintaining ISP family membrane association.\",\n      \"evidence\": \"Conditional knockdown of TgDHHC2/TgDHHC14 with fluorescence microscopy of ISP1/ISP3 localization in T. gondii\",\n      \"pmids\": [\"32329212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ISP2 was not directly tested as a TgDHHC2 substrate; inferred by family membership\", \"Direct enzyme\\u2013substrate acylation of ISP2 not demonstrated biochemically\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct protein-interaction partners engaged by the repurposed PH surface of ISP2 and the molecular basis of its scaffolding role in endodyogeny remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No identified ISP2 binding partners\", \"No direct structural data on ISP2 itself\", \"Mechanistic link between ISP2 scaffolding and daughter-cell assembly unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005515\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0070258\", \"supporting_discovery_ids\": [0, 1, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0000281\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ISP1\", \"ISP3\", \"TgDHHC2\"],\n    \"other_free_text\": [\n      \"N-terminally myristoylated and palmitoylated for IMC membrane anchoring [#0]\",\n      \"Targeted to the central IMC sub-compartment; excluded from the apical cap by ISP1 gatekeeping [#0, #1]\",\n      \"Adopts a pleckstrin homology fold repurposed for protein\\u2013protein interactions rather than phospholipid binding [#3, #4]\",\n      \"Required for daughter-cell formation during endodyogeny [#2]\",\n      \"Membrane association depends on the palmitoyl acyltransferase TgDHHC2 (inferred by family membership) [#5]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_237820","name":"ISP2","product":"IMC sub-compartment protein ISP2","uniprot_acc":"A0A125YYN0","chromosome":"TGME49_chrIV","start_pos":2559233},{"strain":"GT1","gene_id":"TGGT1_237820","name":null,"product":"IMC sub-compartment protein ISP2","uniprot_acc":null,"chromosome":"TGGT1_chrIV","start_pos":2412658},{"strain":"RH88","gene_id":"TGRH88_009360","name":null,"product":"IMC sub-compartment protein ISP2","uniprot_acc":null,"chromosome":"CM023085","start_pos":2334825},{"strain":"VEG","gene_id":"TGVEG_237820","name":null,"product":"IMC sub-compartment protein ISP2","uniprot_acc":"B9Q3R2","chromosome":"TGVEG_chrIV","start_pos":2582120}]}