{"gene":"PF3D7_1460600","run_date":"2026-09-08T16:56:21+00:00","timeline":{"discoveries":[{"year":2013,"finding":"ISP3 (PF3D7_1460600) localises to the anterior apical end of the parasite where IMC organisation is initiated, and is expressed at all developmental stages, especially invasive ones. ISP3 is N-myristoylated, phosphorylated, and membrane-bound. Gene disruption of ISP3 in P. berghei is not lethal but causes altered apical localisation of ISP1 in all invasive stages (ookinetes, sporozoites), indicating ISP3 is required for proper apical polarity organisation.","method":"Live cell imaging, reverse genetics (gene disruption), localization studies in P. berghei","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean gene disruption with defined phenotypic readout (mislocalization of ISP1) plus direct localization and biochemical characterization; single lab, multiple orthogonal methods","pmids":["24244852"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"finding":"PfISP3 (PF3D7_1460600) carries an N-terminal acylation motif (dual-acylated: myristoylated and palmitoylated) and co-distributes with the IMC-localised palmitoyl acyltransferase PfDHHC1 during schizogony; specific N-terminal sequence requirements govern its IMC membrane recruitment.","method":"Phylogenetic analysis, subcellular co-localization, mutagenesis of acylation motifs, IMC fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization with defined PAT plus sequence-requirement mutagenesis; single lab, two complementary approaches","pmids":["25425642"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"Palmitoylation of N-terminal cysteines of ISP3 (PF3D7_1460600) is catalysed by the IMC-resident palmitoyl-S-acyl-transferase DHHC2, and this palmitoylation is required for IMC localisation of ISP3 and for normal zygote-to-ookinete differentiation. IMC-anchored ISP3 physically interacts with the microtubule component β-tubulin, functioning as a tether to maintain proper subpellicular microtubule structure during zygote elongation.","method":"In vivo palmitoylation assay, co-immunoprecipitation (ISP3 with β-tubulin), gene disruption/conditional knockouts, live imaging of ookinete morphogenesis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (palmitoylation assay, Co-IP with β-tubulin, genetic disruption, live morphogenesis imaging) in a single rigorous study establishing mechanism","pmids":["32395856"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"finding":"ISP3 (PF3D7_1460600) is one of eight structurally distinct IMC proteins in Plasmodium; colocalization studies reveal differential spatial-temporal localisation patterns across motile life-cycle stages, and the IMC (including ISP3) plays an essential structural role during sexual-stage development and gametocyte morphogenesis.","method":"Fluorescence colocalization with centromere/spindle markers, live cell imaging across life-cycle stages","journal":"Molecular biology and evolution","confidence":"Low","confidence_rationale":"Tier 3 / Weak — colocalization-based localization without functional disruption specific to ISP3; single approach, ISP3-specific mechanistic detail not separated from other IMC proteins in abstract","pmids":["22389454"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"ISP3 (PF3D7_1460600) is a dual-acylated (N-myristoylated and palmitoylated) IMC sub-compartment protein whose palmitoylation on N-terminal cysteines is catalysed by the IMC-resident PAT DHHC2 (and in P. falciparum schizogony by PfDHHC1), targeting ISP3 to the anterior apical IMC where it tethers subpellicular microtubules via direct interaction with β-tubulin to maintain cytoskeletal integrity during ookinete morphogenesis, and where it cooperates with ISP1 to organise apical polarity across invasive stages."},"narrative":{"mechanistic_narrative":"ISP3 (PF3D7_1460600) is a dual-acylated inner membrane complex (IMC) sub-compartment protein that organizes apical polarity and tethers the subpellicular cytoskeleton during Plasmodium morphogenesis [PMID:24244852, PMID:32395856]. It localizes to the anterior apical end of the parasite where IMC assembly is initiated, is expressed across developmental stages with enrichment in invasive stages, and its disruption in P. berghei is non-lethal but mislocalizes ISP1 in ookinetes and sporozoites, establishing ISP3 as a determinant of proper apical polarity [PMID:24244852]. Membrane recruitment depends on an N-terminal acylation motif: ISP3 is both N-myristoylated and palmitoylated, and its N-terminal sequence requirements govern IMC targeting [PMID:24244852, PMID:25425642]. In P. berghei, palmitoylation of N-terminal cysteines by the IMC-resident palmitoyl-S-acyltransferase DHHC2 is required for IMC localization and for normal zygote-to-ookinete differentiation, while in P. falciparum schizogony PfISP3 co-distributes with the IMC-localized PAT PfDHHC1 [PMID:25425642, PMID:32395856]. Once anchored at the IMC, ISP3 physically interacts with β-tubulin, functioning as a tether that maintains subpellicular microtubule structure during zygote elongation [PMID:32395856]. Beyond these acylation, localization, and cytoskeletal-tethering roles, no further mechanistic detail for ISP3 has been characterized in the available corpus.","teleology":[{"year":2012,"claim":"Before ISP3 was functionally distinguished, it was unknown how IMC sub-compartment proteins were spatially organized across motile stages; surveying the IMC family placed ISP3 among structurally distinct members with stage-specific localization patterns during sexual-stage development.","evidence":"Fluorescence colocalization with centromere/spindle markers and live imaging across life-cycle stages in P. falciparum","pmids":["22389454"],"confidence":"Low","gaps":["ISP3-specific function not separated from other IMC proteins","no functional disruption of ISP3 in this study","colocalization-only, no mechanism"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"It was unknown whether ISP3 contributes to apical organization; gene disruption showed ISP3 is dispensable for viability but required to correctly localize ISP1, establishing it as an organizer of apical polarity in invasive stages.","evidence":"Reverse genetics (gene disruption), live cell imaging and localization in P. berghei","pmids":["24244852"],"confidence":"Medium","gaps":["molecular basis of ISP1 mislocalization not defined","role of myristoylation vs phosphorylation in membrane binding not dissected","no binding partners identified"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"claim":"How ISP3 achieves IMC membrane association was unresolved; mapping its dual-acylation motif and co-distribution with the IMC PAT PfDHHC1 during schizogony showed that N-terminal acylation and specific sequence requirements drive IMC recruitment.","evidence":"Phylogenetic analysis, subcellular co-localization with PfDHHC1, acylation-motif mutagenesis and IMC fractionation in P. falciparum","pmids":["25425642"],"confidence":"Medium","gaps":["direct enzyme-substrate relationship with PfDHHC1 not demonstrated","functional consequence of acylation in P. falciparum not tested","co-localization does not prove catalysis"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"The enzyme responsible for ISP3 palmitoylation and the downstream cytoskeletal role were unknown; DHHC2 was identified as the PAT palmitoylating ISP3 N-terminal cysteines, and IMC-anchored ISP3 was shown to bind β-tubulin to maintain subpellicular microtubule integrity during ookinete morphogenesis.","evidence":"In vivo palmitoylation assay, ISP3–β-tubulin co-immunoprecipitation, conditional knockouts and live morphogenesis imaging in P. berghei","pmids":["32395856"],"confidence":"High","gaps":["structural basis of the ISP3–β-tubulin interaction not defined","whether the P. falciparum ortholog uses the same DHHC2/β-tubulin mechanism not tested","reciprocal validation across stages beyond ookinete not shown"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":null,"claim":"It remains unknown how ISP3 mechanistically coordinates ISP1 apical positioning with microtubule tethering, and whether its P. falciparum functions mirror the P. berghei cytoskeletal role.","evidence":"","pmids":[],"confidence":"Medium","gaps":["no structural model of ISP3 or its complexes","mechanism linking apical polarity organization to β-tubulin tethering unresolved","P. falciparum functional phenotype not established"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Inner membrane complex (IMC)"],"partners":["Β-TUBULIN (DIRECT INTERACTION)","DHHC2 (PALMITOYL-S-ACYLTRANSFERASE, P. BERGHEI)","PFDHHC1 (PALMITOYL ACYLTRANSFERASE, CO-DISTRIBUTION, P. FALCIPARUM)","ISP1 (APICAL POLARITY CO-ORGANIZATION)"],"other_free_text":["N-myristoylated and palmitoylated (dual-acylated); palmitoylation on N-terminal cysteines required for IMC localization","Gene disruption non-lethal in P. berghei but impairs ISP1 apical localization and zygote-to-ookinete differentiation","Functions as a tether maintaining subpellicular microtubule structure during ookinete morphogenesis"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"22389454","id":"PMC_22389454","title":"Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite.","date":"2012","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/22389454","citation_count":105,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24244852","id":"PMC_24244852","title":"Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite.","date":"2013","source":"Biology open","url":"https://pubmed.ncbi.nlm.nih.gov/24244852","citation_count":45,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["ISP3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32395856","id":"PMC_32395856","title":"A protein palmitoylation cascade regulates microtubule cytoskeleton integrity in Plasmodium.","date":"2020","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/32395856","citation_count":43,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["ISP3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25425642","id":"PMC_25425642","title":"The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25425642","citation_count":40,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfISP3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4765,"output_tokens":1370,"usd":0.017423,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6251,"output_tokens":2464,"usd":0.046427,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01BW7vvc7h5iV39oJ1MHMzXw","stage2_batch_id":"msgbatch_01Kh5HbgbPXepNfNCbktYkSD","note":"batch pricing = 50% of standard","skipped":true},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"ISP3 (PF3D7_1460600) localises to the anterior apical end of the parasite where IMC organisation is initiated, and is expressed at all developmental stages, especially invasive ones. ISP3 is N-myristoylated, phosphorylated, and membrane-bound. Gene disruption of ISP3 in P. berghei is not lethal but causes altered apical localisation of ISP1 in all invasive stages (ookinetes, sporozoites), indicating ISP3 is required for proper apical polarity organisation.\",\n      \"method\": \"Live cell imaging, reverse genetics (gene disruption), localization studies in P. berghei\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean gene disruption with defined phenotypic readout (mislocalization of ISP1) plus direct localization and biochemical characterization; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"24244852\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PfISP3 (PF3D7_1460600) carries an N-terminal acylation motif (dual-acylated: myristoylated and palmitoylated) and co-distributes with the IMC-localised palmitoyl acyltransferase PfDHHC1 during schizogony; specific N-terminal sequence requirements govern its IMC membrane recruitment.\",\n      \"method\": \"Phylogenetic analysis, subcellular co-localization, mutagenesis of acylation motifs, IMC fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization with defined PAT plus sequence-requirement mutagenesis; single lab, two complementary approaches\",\n      \"pmids\": [\"25425642\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Palmitoylation of N-terminal cysteines of ISP3 (PF3D7_1460600) is catalysed by the IMC-resident palmitoyl-S-acyl-transferase DHHC2, and this palmitoylation is required for IMC localisation of ISP3 and for normal zygote-to-ookinete differentiation. IMC-anchored ISP3 physically interacts with the microtubule component β-tubulin, functioning as a tether to maintain proper subpellicular microtubule structure during zygote elongation.\",\n      \"method\": \"In vivo palmitoylation assay, co-immunoprecipitation (ISP3 with β-tubulin), gene disruption/conditional knockouts, live imaging of ookinete morphogenesis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (palmitoylation assay, Co-IP with β-tubulin, genetic disruption, live morphogenesis imaging) in a single rigorous study establishing mechanism\",\n      \"pmids\": [\"32395856\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ISP3 (PF3D7_1460600) is one of eight structurally distinct IMC proteins in Plasmodium; colocalization studies reveal differential spatial-temporal localisation patterns across motile life-cycle stages, and the IMC (including ISP3) plays an essential structural role during sexual-stage development and gametocyte morphogenesis.\",\n      \"method\": \"Fluorescence colocalization with centromere/spindle markers, live cell imaging across life-cycle stages\",\n      \"journal\": \"Molecular biology and evolution\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — colocalization-based localization without functional disruption specific to ISP3; single approach, ISP3-specific mechanistic detail not separated from other IMC proteins in abstract\",\n      \"pmids\": [\"22389454\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"ISP3 (PF3D7_1460600) is a dual-acylated (N-myristoylated and palmitoylated) IMC sub-compartment protein whose palmitoylation on N-terminal cysteines is catalysed by the IMC-resident PAT DHHC2 (and in P. falciparum schizogony by PfDHHC1), targeting ISP3 to the anterior apical IMC where it tethers subpellicular microtubules via direct interaction with β-tubulin to maintain cytoskeletal integrity during ookinete morphogenesis, and where it cooperates with ISP1 to organise apical polarity across invasive stages.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ISP3 (PF3D7_1460600) is a dual-acylated inner membrane complex (IMC) sub-compartment protein that organizes apical polarity and tethers the subpellicular cytoskeleton during Plasmodium morphogenesis [#0, #2]. It localizes to the anterior apical end of the parasite where IMC assembly is initiated, is expressed across developmental stages with enrichment in invasive stages, and its disruption in P. berghei is non-lethal but mislocalizes ISP1 in ookinetes and sporozoites, establishing ISP3 as a determinant of proper apical polarity [#0]. Membrane recruitment depends on an N-terminal acylation motif: ISP3 is both N-myristoylated and palmitoylated, and its N-terminal sequence requirements govern IMC targeting [#0, #1]. In P. berghei, palmitoylation of N-terminal cysteines by the IMC-resident palmitoyl-S-acyltransferase DHHC2 is required for IMC localization and for normal zygote-to-ookinete differentiation, while in P. falciparum schizogony PfISP3 co-distributes with the IMC-localized PAT PfDHHC1 [#1, #2]. Once anchored at the IMC, ISP3 physically interacts with \\u03b2-tubulin, functioning as a tether that maintains subpellicular microtubule structure during zygote elongation [#2]. Beyond these acylation, localization, and cytoskeletal-tethering roles, no further mechanistic detail for ISP3 has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Before ISP3 was functionally distinguished, it was unknown how IMC sub-compartment proteins were spatially organized across motile stages; surveying the IMC family placed ISP3 among structurally distinct members with stage-specific localization patterns during sexual-stage development.\",\n      \"evidence\": \"Fluorescence colocalization with centromere/spindle markers and live imaging across life-cycle stages in P. falciparum\",\n      \"pmids\": [\"22389454\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"ISP3-specific function not separated from other IMC proteins\", \"no functional disruption of ISP3 in this study\", \"colocalization-only, no mechanism\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"It was unknown whether ISP3 contributes to apical organization; gene disruption showed ISP3 is dispensable for viability but required to correctly localize ISP1, establishing it as an organizer of apical polarity in invasive stages.\",\n      \"evidence\": \"Reverse genetics (gene disruption), live cell imaging and localization in P. berghei\",\n      \"pmids\": [\"24244852\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"molecular basis of ISP1 mislocalization not defined\", \"role of myristoylation vs phosphorylation in membrane binding not dissected\", \"no binding partners identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"How ISP3 achieves IMC membrane association was unresolved; mapping its dual-acylation motif and co-distribution with the IMC PAT PfDHHC1 during schizogony showed that N-terminal acylation and specific sequence requirements drive IMC recruitment.\",\n      \"evidence\": \"Phylogenetic analysis, subcellular co-localization with PfDHHC1, acylation-motif mutagenesis and IMC fractionation in P. falciparum\",\n      \"pmids\": [\"25425642\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct enzyme-substrate relationship with PfDHHC1 not demonstrated\", \"functional consequence of acylation in P. falciparum not tested\", \"co-localization does not prove catalysis\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The enzyme responsible for ISP3 palmitoylation and the downstream cytoskeletal role were unknown; DHHC2 was identified as the PAT palmitoylating ISP3 N-terminal cysteines, and IMC-anchored ISP3 was shown to bind \\u03b2-tubulin to maintain subpellicular microtubule integrity during ookinete morphogenesis.\",\n      \"evidence\": \"In vivo palmitoylation assay, ISP3\\u2013\\u03b2-tubulin co-immunoprecipitation, conditional knockouts and live morphogenesis imaging in P. berghei\",\n      \"pmids\": [\"32395856\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of the ISP3\\u2013\\u03b2-tubulin interaction not defined\", \"whether the P. falciparum ortholog uses the same DHHC2/\\u03b2-tubulin mechanism not tested\", \"reciprocal validation across stages beyond ookinete not shown\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how ISP3 mechanistically coordinates ISP1 apical positioning with microtubule tethering, and whether its P. falciparum functions mirror the P. berghei cytoskeletal role.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"no structural model of ISP3 or its complexes\", \"mechanism linking apical polarity organization to \\u03b2-tubulin tethering unresolved\", \"P. falciparum functional phenotype not established\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008017\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0070258\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007163\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\n      \"Inner membrane complex (IMC)\"\n    ],\n    \"partners\": [\n      \"\\u03b2-tubulin (direct interaction)\",\n      \"DHHC2 (palmitoyl-S-acyltransferase, P. berghei)\",\n      \"PfDHHC1 (palmitoyl acyltransferase, co-distribution, P. falciparum)\",\n      \"ISP1 (apical polarity co-organization)\"\n    ],\n    \"other_free_text\": [\n      \"N-myristoylated and palmitoylated (dual-acylated); palmitoylation on N-terminal cysteines required for IMC localization\",\n      \"Gene disruption non-lethal in P. berghei but impairs ISP1 apical localization and zygote-to-ookinete differentiation\",\n      \"Functions as a tether maintaining subpellicular microtubule structure during ookinete morphogenesis\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1460600","name":null,"product":"inner membrane complex sub-compartment protein 3","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1324300","name":null,"product":"inner membrane complex sub-compartment protein 3","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1221100","name":null,"product":"inner membrane complex sub-compartment protein 3, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"ISP3","evaluation":null}