{"gene":"TG_219320","gene_symbol":"GAP50","run_date":"2026-09-06T08:22:39","timeline":{"discoveries":[{"year":2004,"finding":"TgGAP50 is an integral membrane glycoprotein of the inner membrane complex (IMC) that serves as the membrane anchor for the myosin XIV motor complex, binding TgMyoA, TgMLC1, and TgGAP45; it provides the first molecular description of an integral membrane protein as a specific receptor for a myosin motor.","method":"Co-immunoprecipitation, subcellular fractionation, localization studies","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and fractionation, widely replicated across multiple subsequent studies","pmids":["15123738"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"The myosin XIV motor complex, anchored to the IMC through GAP50, is firmly immobilized in the plane of the IMC in a cholesterol-dependent manner; both the motor complex and cholesterol reside in detergent-resistant membrane domains covering a large fraction of the IMC surface.","method":"FRAP (fluorescence recovery after photobleaching), detergent-resistant membrane fractionation, cholesterol depletion experiments","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRAP and biochemical fractionation in a single study, two orthogonal methods","pmids":["17538016"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"Phosphorylation of GAP45 at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Glu) mutations at these residues prevent association of the MyoA-MLC1-GAP45 sub-complex with GAP50, while phospho-null (Ala) mutations do not affect complex assembly.","method":"Mass spectrometry phosphosite identification, site-directed mutagenesis (Ser→Glu / Ser→Ala), co-immunoprecipitation","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with Co-IP in a single lab study; later work (PMID:24779470) partially contradicted the functional significance","pmids":["19047362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"GAP40, a polytopic IMC protein, is identified as an additional glideosome component; GAP50 (together with GAP40) anchors the glideosome motor complex to the IMC, while GAP45 bridges the plasma membrane and IMC via N- and C-terminal extremities respectively, with the C-terminal region recruiting MLC1-MyoA to the IMC.","method":"Co-immunoprecipitation, localization by IFA, genetic complementation (transgenera), conditional knockdown","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, IFA, complementation, KD), independently replicated","pmids":["20951968"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"In vitro motility assay demonstrated that the TgGAP50-containing myosin motor complex (TgMyoA, TgMLC1, TgGAP45, TgGAP50) exhibits motor activity that is inhibitable by posttranslational modification of TgMLC1; the assay directly measures mechanical output of the intact four-protein complex.","method":"In vitro motility assay using motor complexes purified from control and compound-treated parasites","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted motility assay, single lab study","pmids":["20084115"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"All three N-glycosylation sites of GAP50 are occupied by unusual N-glycan structures; site-directed mutagenesis of these sites shows that N-glycosylation is required for GAP50 trafficking from the ER to the Golgi and subsequently to the IMC, and for assembly with gliding partners (including GAP40) and parasite motility.","method":"Glycomic/proteomic analysis, site-directed mutagenesis of N-glycosylation sites, affinity purification, localization by IFA, motility assays","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis of specific glycosylation sites combined with multiple orthogonal readouts (trafficking, pulldown, motility) in a single rigorous study","pmids":["21610105"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"GAP50 (along with GAP40) also interacts with the MyoC-glideosome, anchoring myosin C at the basal polar ring; the MyoA- and MyoC-glideosomes share integral IMC proteins GAP50 and GAP40 but differ in their peripheral scaffolding proteins (GAP45 vs. GAP80) and localization determinants.","method":"Co-immunoprecipitation, conditional knockout/knockdown, IFA localization","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and genetic disruption with localization readouts, single lab","pmids":["25393004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Phospho-mimetic and phospho-null mutations in the GAP45 phosphorylation cluster (including the Ser163/Ser167 sites previously implicated) did not impair interaction with GAP50, glideosome assembly, or parasite motility in a clean complementation strategy; the previously reported loss of GAP45–GAP50 interaction by the phospho-mimetic GAP45 mutant depended on competition from co-expressed wild-type GAP45.","method":"Phospho-mutant generation, inducible knockout complementation, co-immunoprecipitation, motility assays","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous complementation in inducible KO background; contradicts PMID:19047362 regarding GAP45 phosphorylation controlling GAP50 binding","pmids":["24779470"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"GAP50 (and GAP40) are essential for the biogenesis of the IMC during intracellular replication; deletion or disruption of GAP50 causes rapid collapse of developing daughter parasites after cell cycle initiation and redistribution of other glideosome components, revealing a structural role in IMC formation beyond motility.","method":"Conditional/inducible gene deletion, IFA, electron microscopy, phenotypic analysis of dividing parasites","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular phenotype (IMC collapse), single lab","pmids":["26845335"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgGAP50 is an N-glycosylated integral membrane glycoprotein of the Toxoplasma gondii inner membrane complex (IMC) that functions as the obligate membrane anchor for both the MyoA-glideosome and the MyoC-glideosome motor complexes; N-glycosylation is required for its ER-to-IMC trafficking and for assembly with gliding partners, it resides in cholesterol-rich detergent-resistant membrane domains that immobilize the motor, and it plays an additional structural role in IMC biogenesis during intracellular replication independent of its role in motility."},"narrative":{"mechanistic_narrative":"TgGAP50 is an N-glycosylated integral membrane glycoprotein of the Toxoplasma gondii inner membrane complex (IMC) that serves as the membrane anchor tethering the parasite's actomyosin gliding motor to the IMC, thereby coupling motor force to the cellular functions of gliding motility and invasion [PMID:15123738]. Together with the polytopic IMC protein GAP40, it anchors the MyoA-glideosome — comprising the myosin TgMyoA, its light chain TgMLC1, and the peripheral scaffold GAP45 — and the assembled four-protein complex exhibits measurable motor activity in vitro [PMID:20951968, PMID:20084115]. The same GAP50/GAP40 integral core also anchors a distinct MyoC-glideosome at the basal polar ring, with the two motors differing in their peripheral scaffolding and localization determinants (GAP45 versus GAP80) [PMID:25393004]. All three of GAP50's N-glycosylation sites carry unusual N-glycans that are required for its trafficking from the ER through the Golgi to the IMC and for its assembly with gliding partners and parasite motility [PMID:21610105]. Within the IMC, the anchored motor is immobilized in the membrane plane within cholesterol-rich, detergent-resistant membrane domains [PMID:17538016]. Beyond its role in motility, GAP50 is essential for IMC biogenesis during intracellular replication, its disruption causing collapse of developing daughter parasites and redistribution of other glideosome components [PMID:26845335].","teleology":[{"year":2004,"claim":"Establishing how a myosin motor is fixed to the parasite periphery, GAP50 was identified as an integral IMC membrane protein serving as the specific receptor anchoring the myosin XIV motor complex, providing the first molecular description of a membrane anchor for a myosin motor.","evidence":"Co-immunoprecipitation, subcellular fractionation and localization in Toxoplasma gondii","pmids":["15123738"],"confidence":"High","gaps":["Did not define which motor partners contact GAP50 directly versus indirectly","No structural basis for the anchoring interaction"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"claim":"Addressing how the anchored motor is stabilized to transmit force, the GAP50-anchored motor was shown to be immobilized in the plane of the IMC within cholesterol-dependent detergent-resistant membrane domains.","evidence":"FRAP, detergent-resistant membrane fractionation and cholesterol depletion in Toxoplasma gondii","pmids":["17538016"],"confidence":"Medium","gaps":["Mechanism by which cholesterol domains organize GAP50 not defined","Single-study observation of immobilization"],"attribution":[]},{"year":2008,"claim":"Testing whether phosphorylation gates motor assembly, GAP45 Ser163/Ser167 phospho-mimetic mutations were reported to prevent the MyoA-MLC1-GAP45 sub-complex from associating with GAP50.","evidence":"Mass spectrometry phosphosite mapping, Ser→Glu/Ala mutagenesis and Co-IP in Toxoplasma gondii","pmids":["19047362"],"confidence":"Medium","gaps":["Functional significance later contradicted (see 2014)","Effect measured in presence of co-expressed wild-type GAP45"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Resolving the composition of the anchoring module, GAP40 was identified as a second integral IMC anchor acting with GAP50, while GAP45 was shown to bridge plasma membrane and IMC and recruit MLC1-MyoA, defining the architecture of glideosome attachment.","evidence":"Co-IP, IFA, transgenera complementation and conditional knockdown in Toxoplasma gondii","pmids":["20951968"],"confidence":"High","gaps":["Stoichiometry of the GAP50/GAP40 core unknown","Direct binding interface between GAP50 and partners not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Demonstrating that the anchored complex is a functional motor, the intact TgMyoA-MLC1-GAP45-GAP50 complex produced mechanical output in vitro that was inhibitable by post-translational modification of MLC1.","evidence":"In vitro motility assay using motor complexes purified from control and compound-treated Toxoplasma gondii","pmids":["20084115"],"confidence":"Medium","gaps":["Did not isolate GAP50's specific contribution to mechanical output","Single-lab reconstitution"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Explaining how GAP50 reaches the IMC and assembles a functional glideosome, all three N-glycosylation sites were shown to be occupied and required for ER-to-Golgi-to-IMC trafficking, partner assembly and motility.","evidence":"Glycomic/proteomic analysis, N-glycosylation site mutagenesis, affinity purification, IFA and motility assays in Toxoplasma gondii","pmids":["21610105"],"confidence":"High","gaps":["Whether glycans act on folding, trafficking receptors, or partner recognition not resolved","Identity of trafficking machinery reading the glycans unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Broadening GAP50's role beyond a single motor, GAP50/GAP40 were shown to also anchor the MyoC-glideosome at the basal polar ring, revealing a shared integral core serving distinct motors defined by their peripheral scaffolds.","evidence":"Co-IP, conditional knockout/knockdown and IFA localization in Toxoplasma gondii","pmids":["25393004"],"confidence":"Medium","gaps":["How GAP50 discriminates or partitions between MyoA and MyoC glideosomes unknown","Localization determinants for each motor not fully mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Revising the phosphorylation model, clean inducible-knockout complementation showed GAP45 phospho-mutants did not impair GAP50 binding, glideosome assembly, or motility, attributing the earlier phenotype to competition from wild-type GAP45.","evidence":"Phospho-mutant generation, inducible KO complementation, Co-IP and motility assays in Toxoplasma gondii","pmids":["24779470"],"confidence":"Medium","gaps":["Whether any physiological phospho-regulation of GAP50 assembly exists remains open","Directly contradicts 2008 phosphorylation model"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Uncovering a motility-independent function, conditional deletion of GAP50 caused collapse of developing daughter parasites and glideosome redistribution, establishing an essential structural role in IMC biogenesis during replication.","evidence":"Conditional/inducible gene deletion, IFA, electron microscopy and phenotypic analysis in dividing Toxoplasma gondii","pmids":["26845335"],"confidence":"Medium","gaps":["Molecular mechanism linking GAP50 to IMC membrane formation unknown","Whether the structural and motor roles use the same molecular interactions unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The structural interface by which GAP50 binds its glideosome partners, the mechanism linking it to IMC membrane biogenesis, and how it partitions between the MyoA and MyoC motors remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No high-resolution structure of GAP50 or its complexes","Molecular basis of the IMC-biogenesis function unresolved","Determinants of motor-specific anchoring unmapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MyoA-glideosome (TgMyoA-TgMLC1-TgGAP45-GAP50-GAP40)","MyoC-glideosome (TgMyoC-GAP80-GAP50-GAP40, basal polar ring)"],"partners":["TGMYOA","TGMLC1","TGGAP45","GAP40","TGMYOC","GAP80"],"other_free_text":["Integral IMC glycoprotein carrying unusual N-glycans at three sites required for ER-to-Golgi-to-IMC trafficking, partner assembly and motility [#5]","Motor complex immobilized in cholesterol-dependent detergent-resistant membrane domains of the IMC [#1]","Essential structural role in IMC biogenesis during intracellular replication, independent of motility [#8]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"20951968","id":"PMC_20951968","title":"Functional dissection of the apicomplexan glideosome molecular architecture.","date":"2010","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/20951968","citation_count":234,"is_preprint":false},{"pmid":"15123738","id":"PMC_15123738","title":"Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii.","date":"2004","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15123738","citation_count":214,"is_preprint":false},{"pmid":"25393004","id":"PMC_25393004","title":"Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion.","date":"2014","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/25393004","citation_count":77,"is_preprint":false},{"pmid":"19047362","id":"PMC_19047362","title":"GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex.","date":"2008","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/19047362","citation_count":65,"is_preprint":false},{"pmid":"17538016","id":"PMC_17538016","title":"Immobilization of the type XIV myosin complex in Toxoplasma gondii.","date":"2007","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/17538016","citation_count":64,"is_preprint":false},{"pmid":"26845335","id":"PMC_26845335","title":"Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii.","date":"2016","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26845335","citation_count":52,"is_preprint":false},{"pmid":"20084115","id":"PMC_20084115","title":"A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.","date":"2010","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/20084115","citation_count":39,"is_preprint":false},{"pmid":"24779470","id":"PMC_24779470","title":"Assessment of phosphorylation in Toxoplasma glideosome assembly and function.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24779470","citation_count":25,"is_preprint":false},{"pmid":"21610105","id":"PMC_21610105","title":"Unusual N-glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility.","date":"2011","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/21610105","citation_count":23,"is_preprint":false},{"pmid":"38894968","id":"PMC_38894968","title":"Identification of novel biomarkers for anti-Toxoplasma gondii IgM detection and the potential application in rapid diagnostic fluorescent tests.","date":"2024","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/38894968","citation_count":3,"is_preprint":false},{"pmid":"30130043","id":"PMC_30130043","title":"[Research Advances on Gliding-associated Proteins of Toxoplasma gondii].","date":"2016","source":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/30130043","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7188,"output_tokens":2411,"usd":0.028865,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7924,"output_tokens":4004,"usd":0.06986,"stage2_stop_reason":"end_turn"},"total_usd":0.098725,"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\": 2004,\n      \"finding\": \"TgGAP50 is an integral membrane glycoprotein of the inner membrane complex (IMC) that serves as the membrane anchor for the myosin XIV motor complex, binding TgMyoA, TgMLC1, and TgGAP45; it provides the first molecular description of an integral membrane protein as a specific receptor for a myosin motor.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, localization studies\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and fractionation, widely replicated across multiple subsequent studies\",\n      \"pmids\": [\"15123738\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The myosin XIV motor complex, anchored to the IMC through GAP50, is firmly immobilized in the plane of the IMC in a cholesterol-dependent manner; both the motor complex and cholesterol reside in detergent-resistant membrane domains covering a large fraction of the IMC surface.\",\n      \"method\": \"FRAP (fluorescence recovery after photobleaching), detergent-resistant membrane fractionation, cholesterol depletion experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRAP and biochemical fractionation in a single study, two orthogonal methods\",\n      \"pmids\": [\"17538016\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Phosphorylation of GAP45 at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Glu) mutations at these residues prevent association of the MyoA-MLC1-GAP45 sub-complex with GAP50, while phospho-null (Ala) mutations do not affect complex assembly.\",\n      \"method\": \"Mass spectrometry phosphosite identification, site-directed mutagenesis (Ser→Glu / Ser→Ala), co-immunoprecipitation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with Co-IP in a single lab study; later work (PMID:24779470) partially contradicted the functional significance\",\n      \"pmids\": [\"19047362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GAP40, a polytopic IMC protein, is identified as an additional glideosome component; GAP50 (together with GAP40) anchors the glideosome motor complex to the IMC, while GAP45 bridges the plasma membrane and IMC via N- and C-terminal extremities respectively, with the C-terminal region recruiting MLC1-MyoA to the IMC.\",\n      \"method\": \"Co-immunoprecipitation, localization by IFA, genetic complementation (transgenera), conditional knockdown\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, IFA, complementation, KD), independently replicated\",\n      \"pmids\": [\"20951968\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In vitro motility assay demonstrated that the TgGAP50-containing myosin motor complex (TgMyoA, TgMLC1, TgGAP45, TgGAP50) exhibits motor activity that is inhibitable by posttranslational modification of TgMLC1; the assay directly measures mechanical output of the intact four-protein complex.\",\n      \"method\": \"In vitro motility assay using motor complexes purified from control and compound-treated parasites\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted motility assay, single lab study\",\n      \"pmids\": [\"20084115\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"All three N-glycosylation sites of GAP50 are occupied by unusual N-glycan structures; site-directed mutagenesis of these sites shows that N-glycosylation is required for GAP50 trafficking from the ER to the Golgi and subsequently to the IMC, and for assembly with gliding partners (including GAP40) and parasite motility.\",\n      \"method\": \"Glycomic/proteomic analysis, site-directed mutagenesis of N-glycosylation sites, affinity purification, localization by IFA, motility assays\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis of specific glycosylation sites combined with multiple orthogonal readouts (trafficking, pulldown, motility) in a single rigorous study\",\n      \"pmids\": [\"21610105\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GAP50 (along with GAP40) also interacts with the MyoC-glideosome, anchoring myosin C at the basal polar ring; the MyoA- and MyoC-glideosomes share integral IMC proteins GAP50 and GAP40 but differ in their peripheral scaffolding proteins (GAP45 vs. GAP80) and localization determinants.\",\n      \"method\": \"Co-immunoprecipitation, conditional knockout/knockdown, IFA localization\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and genetic disruption with localization readouts, single lab\",\n      \"pmids\": [\"25393004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phospho-mimetic and phospho-null mutations in the GAP45 phosphorylation cluster (including the Ser163/Ser167 sites previously implicated) did not impair interaction with GAP50, glideosome assembly, or parasite motility in a clean complementation strategy; the previously reported loss of GAP45–GAP50 interaction by the phospho-mimetic GAP45 mutant depended on competition from co-expressed wild-type GAP45.\",\n      \"method\": \"Phospho-mutant generation, inducible knockout complementation, co-immunoprecipitation, motility assays\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous complementation in inducible KO background; contradicts PMID:19047362 regarding GAP45 phosphorylation controlling GAP50 binding\",\n      \"pmids\": [\"24779470\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GAP50 (and GAP40) are essential for the biogenesis of the IMC during intracellular replication; deletion or disruption of GAP50 causes rapid collapse of developing daughter parasites after cell cycle initiation and redistribution of other glideosome components, revealing a structural role in IMC formation beyond motility.\",\n      \"method\": \"Conditional/inducible gene deletion, IFA, electron microscopy, phenotypic analysis of dividing parasites\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular phenotype (IMC collapse), single lab\",\n      \"pmids\": [\"26845335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgGAP50 is an N-glycosylated integral membrane glycoprotein of the Toxoplasma gondii inner membrane complex (IMC) that functions as the obligate membrane anchor for both the MyoA-glideosome and the MyoC-glideosome motor complexes; N-glycosylation is required for its ER-to-IMC trafficking and for assembly with gliding partners, it resides in cholesterol-rich detergent-resistant membrane domains that immobilize the motor, and it plays an additional structural role in IMC biogenesis during intracellular replication independent of its role in motility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgGAP50 is an N-glycosylated integral membrane glycoprotein of the Toxoplasma gondii inner membrane complex (IMC) that serves as the membrane anchor tethering the parasite's actomyosin gliding motor to the IMC, thereby coupling motor force to the cellular functions of gliding motility and invasion [#0]. Together with the polytopic IMC protein GAP40, it anchors the MyoA-glideosome — comprising the myosin TgMyoA, its light chain TgMLC1, and the peripheral scaffold GAP45 — and the assembled four-protein complex exhibits measurable motor activity in vitro [#3, #4]. The same GAP50/GAP40 integral core also anchors a distinct MyoC-glideosome at the basal polar ring, with the two motors differing in their peripheral scaffolding and localization determinants (GAP45 versus GAP80) [#6]. All three of GAP50's N-glycosylation sites carry unusual N-glycans that are required for its trafficking from the ER through the Golgi to the IMC and for its assembly with gliding partners and parasite motility [#5]. Within the IMC, the anchored motor is immobilized in the membrane plane within cholesterol-rich, detergent-resistant membrane domains [#1]. Beyond its role in motility, GAP50 is essential for IMC biogenesis during intracellular replication, its disruption causing collapse of developing daughter parasites and redistribution of other glideosome components [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing how a myosin motor is fixed to the parasite periphery, GAP50 was identified as an integral IMC membrane protein serving as the specific receptor anchoring the myosin XIV motor complex, providing the first molecular description of a membrane anchor for a myosin motor.\",\n      \"evidence\": \"Co-immunoprecipitation, subcellular fractionation and localization in Toxoplasma gondii\",\n      \"pmids\": [\"15123738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define which motor partners contact GAP50 directly versus indirectly\", \"No structural basis for the anchoring interaction\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Addressing how the anchored motor is stabilized to transmit force, the GAP50-anchored motor was shown to be immobilized in the plane of the IMC within cholesterol-dependent detergent-resistant membrane domains.\",\n      \"evidence\": \"FRAP, detergent-resistant membrane fractionation and cholesterol depletion in Toxoplasma gondii\",\n      \"pmids\": [\"17538016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which cholesterol domains organize GAP50 not defined\", \"Single-study observation of immobilization\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Testing whether phosphorylation gates motor assembly, GAP45 Ser163/Ser167 phospho-mimetic mutations were reported to prevent the MyoA-MLC1-GAP45 sub-complex from associating with GAP50.\",\n      \"evidence\": \"Mass spectrometry phosphosite mapping, Ser\\u2192Glu/Ala mutagenesis and Co-IP in Toxoplasma gondii\",\n      \"pmids\": [\"19047362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance later contradicted (see 2014)\", \"Effect measured in presence of co-expressed wild-type GAP45\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolving the composition of the anchoring module, GAP40 was identified as a second integral IMC anchor acting with GAP50, while GAP45 was shown to bridge plasma membrane and IMC and recruit MLC1-MyoA, defining the architecture of glideosome attachment.\",\n      \"evidence\": \"Co-IP, IFA, transgenera complementation and conditional knockdown in Toxoplasma gondii\",\n      \"pmids\": [\"20951968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the GAP50/GAP40 core unknown\", \"Direct binding interface between GAP50 and partners not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that the anchored complex is a functional motor, the intact TgMyoA-MLC1-GAP45-GAP50 complex produced mechanical output in vitro that was inhibitable by post-translational modification of MLC1.\",\n      \"evidence\": \"In vitro motility assay using motor complexes purified from control and compound-treated Toxoplasma gondii\",\n      \"pmids\": [\"20084115\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not isolate GAP50's specific contribution to mechanical output\", \"Single-lab reconstitution\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Explaining how GAP50 reaches the IMC and assembles a functional glideosome, all three N-glycosylation sites were shown to be occupied and required for ER-to-Golgi-to-IMC trafficking, partner assembly and motility.\",\n      \"evidence\": \"Glycomic/proteomic analysis, N-glycosylation site mutagenesis, affinity purification, IFA and motility assays in Toxoplasma gondii\",\n      \"pmids\": [\"21610105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether glycans act on folding, trafficking receptors, or partner recognition not resolved\", \"Identity of trafficking machinery reading the glycans unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Broadening GAP50's role beyond a single motor, GAP50/GAP40 were shown to also anchor the MyoC-glideosome at the basal polar ring, revealing a shared integral core serving distinct motors defined by their peripheral scaffolds.\",\n      \"evidence\": \"Co-IP, conditional knockout/knockdown and IFA localization in Toxoplasma gondii\",\n      \"pmids\": [\"25393004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How GAP50 discriminates or partitions between MyoA and MyoC glideosomes unknown\", \"Localization determinants for each motor not fully mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revising the phosphorylation model, clean inducible-knockout complementation showed GAP45 phospho-mutants did not impair GAP50 binding, glideosome assembly, or motility, attributing the earlier phenotype to competition from wild-type GAP45.\",\n      \"evidence\": \"Phospho-mutant generation, inducible KO complementation, Co-IP and motility assays in Toxoplasma gondii\",\n      \"pmids\": [\"24779470\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether any physiological phospho-regulation of GAP50 assembly exists remains open\", \"Directly contradicts 2008 phosphorylation model\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Uncovering a motility-independent function, conditional deletion of GAP50 caused collapse of developing daughter parasites and glideosome redistribution, establishing an essential structural role in IMC biogenesis during replication.\",\n      \"evidence\": \"Conditional/inducible gene deletion, IFA, electron microscopy and phenotypic analysis in dividing Toxoplasma gondii\",\n      \"pmids\": [\"26845335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking GAP50 to IMC membrane formation unknown\", \"Whether the structural and motor roles use the same molecular interactions unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural interface by which GAP50 binds its glideosome partners, the mechanism linking it to IMC membrane biogenesis, and how it partitions between the MyoA and MyoC motors remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of GAP50 or its complexes\", \"Molecular basis of the IMC-biogenesis function unresolved\", \"Determinants of motor-specific anchoring unmapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0017022\", \"supporting_discovery_ids\": [0, 3, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005178\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0070258\", \"supporting_discovery_ids\": [0, 1, 3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016021\", \"supporting_discovery_ids\": [0, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0045121\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0071973\", \"supporting_discovery_ids\": [0, 3, 4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006486\", \"supporting_discovery_ids\": [5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"MyoA-glideosome (TgMyoA-TgMLC1-TgGAP45-GAP50-GAP40)\",\n      \"MyoC-glideosome (TgMyoC-GAP80-GAP50-GAP40, basal polar ring)\"\n    ],\n    \"partners\": [\n      \"TgMyoA\",\n      \"TgMLC1\",\n      \"TgGAP45\",\n      \"GAP40\",\n      \"TgMyoC\",\n      \"GAP80\"\n    ],\n    \"other_free_text\": [\n      \"Integral IMC glycoprotein carrying unusual N-glycans at three sites required for ER-to-Golgi-to-IMC trafficking, partner assembly and motility [#5]\",\n      \"Motor complex immobilized in cholesterol-dependent detergent-resistant membrane domains of the IMC [#1]\",\n      \"Essential structural role in IMC biogenesis during intracellular replication, independent of motility [#8]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_219320","name":"GAP50","product":"gliding-associated protein GAP50","uniprot_acc":"A0A125YT84","chromosome":"TGME49_chrXII","start_pos":968519},{"strain":"GT1","gene_id":"TGGT1_219320","name":null,"product":"acid phosphatase GAP50","uniprot_acc":null,"chromosome":"TGGT1_chrXII","start_pos":907511},{"strain":"RH88","gene_id":"TGRH88_058240","name":null,"product":"acid phosphatase GAP50","uniprot_acc":null,"chromosome":"CM023093","start_pos":980277},{"strain":"VEG","gene_id":"TGVEG_219320","name":null,"product":"acid phosphatase GAP50","uniprot_acc":"A0A0F7VDV9","chromosome":"TGVEG_chrXII","start_pos":941844}]}