{"gene":"TG_223940","gene_symbol":"GAP45","run_date":"2026-09-06T12:15:38+00:00","timeline":{"discoveries":[{"year":2004,"finding":"TgGAP45 was identified as a component of the myosin XIV motor complex in Toxoplasma gondii, together with TgMyoA, TgMLC1, and TgGAP50. The complex is localized to the inner membrane complex (IMC), where it is anchored by TgGAP50, an integral membrane glycoprotein. Assembly occurs in two spatially controlled stages.","method":"Co-immunoprecipitation, subcellular fractionation, immunofluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus localization experiments, foundational paper replicated across subsequent studies","pmids":["15123738"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"The myosin XIV motor complex (MyoA/MLC1/GAP45/GAP50) is firmly immobilized in the plane of the IMC. This immobilization requires cholesterol and is associated with detergent-resistant membrane (DRM) domains, but does not appear to depend on direct interactions with cytoskeletal elements.","method":"Fluorescence recovery after photobleaching (FRAP), detergent-resistant membrane fractionation, cholesterol depletion","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRAP and DRM fractionation in single lab, two orthogonal methods","pmids":["17538016"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"GAP45 is phosphorylated on multiple residues including Ser163 and Ser167. Phospho-mimetic mutation of both residues to Glu prevents association of the MyoA-MLC1-GAP45 soluble complex with the membrane anchor GAP50, demonstrating that phosphorylation of these sites controls the final step in myosin XIV motor complex assembly. Phospho-null mutations (Ala) do not block this association.","method":"Mass spectrometry phosphosite mapping, site-directed mutagenesis (Ser→Glu / Ser→Ala), co-immunoprecipitation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mass spectrometry site identification combined with mutagenesis and functional Co-IP readout, single lab but multiple orthogonal methods","pmids":["19047362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"GAP45 is anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminal extremity). The C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, while the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. Plasmodium falciparum GAP45 can functionally replace TgGAP45, demonstrated by transgenera complementation.","method":"Conditional/inducible knockout, domain truncation/mutagenesis, transgenera complementation, immunofluorescence localization, gliding/invasion/egress assays","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined phenotypic readouts, domain dissection, and cross-species complementation across multiple orthogonal assays","pmids":["20951968"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"The in vitro motility activity of the TgMyoA motor complex (containing TgMLC1, TgGAP45, and TgGAP50) is significantly reduced when TgMLC1 carries a compound-induced post-translational modification, providing evidence that the mechanical activity of the Class XIV myosin complex can be modulated by modifications to its associated light chain.","method":"In vitro motility assay comparing complexes from control vs. compound-treated parasites, electrophoretic mobility shift to detect TgMLC1 modification","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro motility assay with motor complex, single lab and modification not fully characterized","pmids":["20084115"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Calcium-dependent phosphorylation of six residues across GAP45, MLC1, and MyoA is correlated with invasion motor activity, as determined by SILAC-based quantitative LC-MS/MS analysis of Ca2+-stimulated tachyzoites.","method":"SILAC quantitative LC-MS/MS, 32P-labeling, 2-DE, Ca2+-mobilizing drug stimulation","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SILAC quantitative phosphoproteomics with orthogonal 32P labeling; correlation not proven causal but quantitative","pmids":["21980283"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"GAP45 is the acylated glideosome component that recruits the MyoA motor complex to the IMC pellicle and contributes to pellicle cohesion during motor traction. Conditional deletion of GAP45 reduces but does not fully abolish gliding or invasion, indicating that other mechanisms (e.g., MyoC-glideosome) can compensate. Loss of MyoA leads to relocalization of MyoC along the pellicle and apical cap, accounting for residual invasion.","method":"Inducible knockout (tetracycline-regulatable), gliding/invasion/egress assays, immunofluorescence","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — inducible KO with defined phenotypic readouts, replicated across two complementary studies in same period","pmids":["25393004","24632839"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Phospho-mimetic and phospho-null mutations of the twelve-residue phosphorylation cluster in TgGAP45, and of phosphorylation sites in TgMLC1, do not impair glideosome assembly or parasite motility when tested in a complementation strategy using an inducible GAP45 knockout background. The previously reported loss-of-interaction phenotype of a phospho-mimetic GAP45 mutant (Ser163/167Glu) with GAP50 was an artifact caused by competition with a co-existing wild-type copy. Thus, GAP45 phosphorylation is not critical for glideosome assembly or function.","method":"Allelic replacement, phospho-mimetic/phospho-null mutagenesis, inducible knockout complementation, co-immunoprecipitation, motility assays","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible KO complementation with phospho-mutants and Co-IP, single lab; contradicts prior Eukaryotic Cell paper but uses more rigorous genetic strategy","pmids":["24779470"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"TgCaMKrk (a calmodulin-independent CaMK homolog localizing to the apical end of tachyzoites) phosphorylates TgGAP45 in vitro, identifying it as a candidate kinase for GAP45 phosphorylation.","method":"In vitro kinase assay with purified proteins","journal":"Parasites & vectors","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single in vitro kinase assay, single lab, no in vivo validation of the phosphorylation event","pmids":["27444499"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"TgMLC1 palmitoylation on Cys8 and Cys11 is required for its binding to TgGAP45. Blocking palmitoylation completely abrogates TgMLC1–TgGAP45 interaction. Surprisingly, loss of this interaction has little effect on parasite motility, challenging the necessity of MLC1-GAP45 binding for force transduction.","method":"Site-directed mutagenesis of palmitoylation sites, co-immunoprecipitation, gliding motility assays","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus Co-IP plus functional motility assay, single lab with multiple orthogonal methods","pmids":["34011689"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgGAP45 is a dually acylated glideosome scaffolding protein anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminus), where it recruits the MyoA–MLC1 motor complex to the pellicle, maintains pellicle cohesion during invasion, and is immobilized within cholesterol-rich detergent-resistant membrane domains; its assembly into the motor complex can be regulated by phosphorylation (with TgCaMKrk as a candidate kinase), though the phosphorylation cluster itself is not strictly required for glideosome assembly or motility, and while MLC1 palmitoylation is needed for MLC1–GAP45 binding, this interaction is surprisingly dispensable for parasite movement, indicating that additional force-transduction mechanisms exist."},"narrative":{"mechanistic_narrative":"TgGAP45 is a scaffolding component of the myosin XIV glideosome that powers gliding motility, host-cell invasion, and egress in Toxoplasma gondii [PMID:15123738, PMID:20951968]. It bridges two membranes of the parasite pellicle: N-terminal acylation anchors it to the plasma membrane while its C-terminal extremity attaches to the inner membrane complex (IMC), where it recruits the MyoA–MLC1 motor to the pellicle, and its N-terminal acylation together with a coiled-coil domain preserves pellicle integrity during the mechanical stress of invasion [PMID:20951968]. Within the IMC the assembled MyoA/MLC1/GAP45/GAP50 complex is immobilized in cholesterol-dependent, detergent-resistant membrane domains rather than by direct cytoskeletal tethering [PMID:17538016]. Conditional deletion of GAP45 reduces but does not abolish gliding and invasion, because a MyoC-based glideosome can partially compensate [PMID:25393004, PMID:24632839]. MLC1 palmitoylation on Cys8/Cys11 is strictly required for MLC1–GAP45 binding, yet loss of this interaction has little effect on motility, indicating that additional force-transduction routes exist [PMID:34011689]. GAP45 phosphorylation, initially implicated in the final GAP50 assembly step, was shown by rigorous inducible-knockout complementation to be dispensable for glideosome assembly and motility [PMID:19047362, PMID:24779470].","teleology":[{"year":2004,"claim":"Establishing which proteins constitute the invasion motor was the first requirement; identifying GAP45 as part of the myosin XIV complex anchored at the IMC defined the molecular machine driving parasite motility.","evidence":"Reciprocal co-immunoprecipitation, subcellular fractionation and immunofluorescence in T. gondii","pmids":["15123738"],"confidence":"High","gaps":["Role of GAP45 within the complex not resolved","Membrane anchoring of GAP45 itself not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"claim":"How the motor complex is held in place at the pellicle was unknown; FRAP and DRM analysis showed it is immobilized in cholesterol-rich detergent-resistant domains, not by cytoskeletal contacts, defining the physical platform for force transduction.","evidence":"FRAP, detergent-resistant membrane fractionation, and cholesterol depletion in T. gondii","pmids":["17538016"],"confidence":"Medium","gaps":["Does not resolve which complex component confers immobilization","Mechanism linking DRM residence to motility not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Whether motor assembly is regulated was untested; phosphosite mapping plus phospho-mimetic mutagenesis indicated that phosphorylation of GAP45 Ser163/Ser167 controls the final docking of the soluble MyoA-MLC1-GAP45 complex onto GAP50.","evidence":"Mass spectrometry phosphosite mapping, Ser→Glu/Ser→Ala mutagenesis, and co-immunoprecipitation in T. gondii","pmids":["19047362"],"confidence":"High","gaps":["Physiological kinase not identified","Phospho-mimetic readout later shown to be confounded by a co-existing wild-type copy"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"The topology and functional necessity of GAP45 were unresolved; conditional knockout, domain dissection and cross-genus complementation showed GAP45 spans plasma membrane and IMC, recruits MLC1-MyoA via its C-terminus, preserves pellicle integrity via its N-terminus, and is essential for gliding, invasion and egress.","evidence":"Inducible knockout, domain truncation/mutagenesis, Plasmodium falciparum GAP45 transgenera complementation and motility assays in T. gondii","pmids":["20951968"],"confidence":"High","gaps":["Extent of functional redundancy not quantified here","Regulation of the two membrane attachments not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Linking motor activity to signalling, calcium-stimulated quantitative phosphoproteomics correlated phosphorylation of six residues across GAP45, MLC1 and MyoA with invasion motor activity.","evidence":"SILAC LC-MS/MS, 32P-labeling and 2-DE of Ca2+-stimulated tachyzoites in T. gondii","pmids":["21980283"],"confidence":"Medium","gaps":["Correlation not shown to be causal","Responsible kinases not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The strict essentiality of GAP45 was reassessed; inducible knockouts showed deletion reduces but does not abolish gliding/invasion because a MyoC-based glideosome can compensate, redefining GAP45 as important but partially redundant.","evidence":"Tetracycline-regulatable inducible knockout, gliding/invasion/egress assays and immunofluorescence in T. gondii","pmids":["25393004","24632839"],"confidence":"High","gaps":["Extent to which MyoC glideosome uses GAP45 not defined","Molecular basis of residual motility not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"A rigorous genetic test overturned the earlier phosphoregulation model, showing phospho-mimetic/null mutation of the GAP45 phosphorylation cluster (and of MLC1 sites) does not impair assembly or motility, and that the prior Ser163/167Glu phenotype was an artifact of competition with wild-type GAP45.","evidence":"Allelic replacement, phospho-mutagenesis, inducible knockout complementation, Co-IP and motility assays in T. gondii","pmids":["24779470"],"confidence":"Medium","gaps":["Does not exclude a subtle regulatory role in vivo","Physiological function of the phosphosites left unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Seeking the kinase acting on GAP45, an in vitro assay identified TgCaMKrk, an apically localized calmodulin-independent CaMK homolog, as a candidate that phosphorylates GAP45.","evidence":"In vitro kinase assay with purified proteins in T. gondii","pmids":["27444499"],"confidence":"Low","gaps":["No in vivo validation of the phosphorylation event","Physiological consequence untested given phospho-cluster dispensability"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"The requirement of the MLC1-GAP45 linkage for force transduction was tested; palmitoylation of MLC1 Cys8/Cys11 was found necessary for MLC1-GAP45 binding, yet abolishing the interaction barely affected motility, implying additional force-transduction mechanisms.","evidence":"Palmitoylation-site mutagenesis, co-immunoprecipitation and gliding motility assays in T. gondii","pmids":["34011689"],"confidence":"Medium","gaps":["Alternative force-transduction route not identified","Structural basis of palmitoylation-dependent binding not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The mechanism by which GAP45 transmits motor force to the pellicle despite dispensability of both its phosphorylation cluster and the MLC1-GAP45 interaction remains unresolved, as does the in vivo kinase and the molecular basis of MyoC-glideosome compensation.","evidence":"","pmids":[],"confidence":"Low","gaps":["No defined alternative force-transduction pathway","In vivo kinase for GAP45 unconfirmed","Structural model of GAP45 dual-membrane bridging absent"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Myosin XIV glideosome motor complex (TgMyoA-TgMLC1-TgGAP45-TgGAP50)"],"partners":["TGMYOA","TGMLC1","TGGAP50","TGCAMKRK (CANDIDATE KINASE, IN VITRO)"],"other_free_text":["N-terminal acylation anchors GAP45 to the plasma membrane; C-terminal extremity attaches to the IMC and recruits MLC1-MyoA [#3].","Motor complex immobilized in cholesterol-dependent detergent-resistant membrane domains [#1].","MLC1 palmitoylation (Cys8/Cys11) required for MLC1-GAP45 binding, but the interaction is dispensable for motility [#9].","GAP45 phosphorylation cluster is not required for glideosome assembly or motility; prior loss-of-interaction phenotype was an artifact [#7].","MyoC-based glideosome partially compensates for GAP45 loss [#6]."]},"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":"24632839","id":"PMC_24632839","title":"The toxoplasma Acto-MyoA motor complex is important but not essential for gliding motility and host cell invasion.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24632839","citation_count":91,"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":"21980283","id":"PMC_21980283","title":"Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.","date":"2011","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/21980283","citation_count":73,"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":"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":"18315523","id":"PMC_18315523","title":"Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex.","date":"2008","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/18315523","citation_count":34,"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":"34011689","id":"PMC_34011689","title":"Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility.","date":"2021","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/34011689","citation_count":11,"is_preprint":false},{"pmid":"40625742","id":"PMC_40625742","title":"PLGA nanoparticles as an efficient carrier in Toxoplasma GAP45: a more effective vaccine against acute toxoplasmosis than traditional ones.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40625742","citation_count":4,"is_preprint":false},{"pmid":"27444499","id":"PMC_27444499","title":"Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog.","date":"2016","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/27444499","citation_count":4,"is_preprint":false},{"pmid":"40936913","id":"PMC_40936913","title":"Commentary: PLGA nanoparticles as an efficient carrier in Toxoplasma GAP45: a more effective vaccine against acute toxoplasmosis than traditional ones.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40936913","citation_count":1,"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":9674,"output_tokens":2883,"usd":0.036133,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8592,"output_tokens":4104,"usd":0.07278,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"TgGAP45 was identified as a component of the myosin XIV motor complex in Toxoplasma gondii, together with TgMyoA, TgMLC1, and TgGAP50. The complex is localized to the inner membrane complex (IMC), where it is anchored by TgGAP50, an integral membrane glycoprotein. Assembly occurs in two spatially controlled stages.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, immunofluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus localization experiments, foundational paper replicated across 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 (MyoA/MLC1/GAP45/GAP50) is firmly immobilized in the plane of the IMC. This immobilization requires cholesterol and is associated with detergent-resistant membrane (DRM) domains, but does not appear to depend on direct interactions with cytoskeletal elements.\",\n      \"method\": \"Fluorescence recovery after photobleaching (FRAP), detergent-resistant membrane fractionation, cholesterol depletion\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRAP and DRM fractionation in single lab, two orthogonal methods\",\n      \"pmids\": [\"17538016\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GAP45 is phosphorylated on multiple residues including Ser163 and Ser167. Phospho-mimetic mutation of both residues to Glu prevents association of the MyoA-MLC1-GAP45 soluble complex with the membrane anchor GAP50, demonstrating that phosphorylation of these sites controls the final step in myosin XIV motor complex assembly. Phospho-null mutations (Ala) do not block this association.\",\n      \"method\": \"Mass spectrometry phosphosite mapping, site-directed mutagenesis (Ser→Glu / Ser→Ala), co-immunoprecipitation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mass spectrometry site identification combined with mutagenesis and functional Co-IP readout, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"19047362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GAP45 is anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminal extremity). The C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, while the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. Plasmodium falciparum GAP45 can functionally replace TgGAP45, demonstrated by transgenera complementation.\",\n      \"method\": \"Conditional/inducible knockout, domain truncation/mutagenesis, transgenera complementation, immunofluorescence localization, gliding/invasion/egress assays\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined phenotypic readouts, domain dissection, and cross-species complementation across multiple orthogonal assays\",\n      \"pmids\": [\"20951968\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The in vitro motility activity of the TgMyoA motor complex (containing TgMLC1, TgGAP45, and TgGAP50) is significantly reduced when TgMLC1 carries a compound-induced post-translational modification, providing evidence that the mechanical activity of the Class XIV myosin complex can be modulated by modifications to its associated light chain.\",\n      \"method\": \"In vitro motility assay comparing complexes from control vs. compound-treated parasites, electrophoretic mobility shift to detect TgMLC1 modification\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro motility assay with motor complex, single lab and modification not fully characterized\",\n      \"pmids\": [\"20084115\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Calcium-dependent phosphorylation of six residues across GAP45, MLC1, and MyoA is correlated with invasion motor activity, as determined by SILAC-based quantitative LC-MS/MS analysis of Ca2+-stimulated tachyzoites.\",\n      \"method\": \"SILAC quantitative LC-MS/MS, 32P-labeling, 2-DE, Ca2+-mobilizing drug stimulation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SILAC quantitative phosphoproteomics with orthogonal 32P labeling; correlation not proven causal but quantitative\",\n      \"pmids\": [\"21980283\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GAP45 is the acylated glideosome component that recruits the MyoA motor complex to the IMC pellicle and contributes to pellicle cohesion during motor traction. Conditional deletion of GAP45 reduces but does not fully abolish gliding or invasion, indicating that other mechanisms (e.g., MyoC-glideosome) can compensate. Loss of MyoA leads to relocalization of MyoC along the pellicle and apical cap, accounting for residual invasion.\",\n      \"method\": \"Inducible knockout (tetracycline-regulatable), gliding/invasion/egress assays, immunofluorescence\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — inducible KO with defined phenotypic readouts, replicated across two complementary studies in same period\",\n      \"pmids\": [\"25393004\", \"24632839\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phospho-mimetic and phospho-null mutations of the twelve-residue phosphorylation cluster in TgGAP45, and of phosphorylation sites in TgMLC1, do not impair glideosome assembly or parasite motility when tested in a complementation strategy using an inducible GAP45 knockout background. The previously reported loss-of-interaction phenotype of a phospho-mimetic GAP45 mutant (Ser163/167Glu) with GAP50 was an artifact caused by competition with a co-existing wild-type copy. Thus, GAP45 phosphorylation is not critical for glideosome assembly or function.\",\n      \"method\": \"Allelic replacement, phospho-mimetic/phospho-null mutagenesis, inducible knockout complementation, co-immunoprecipitation, motility assays\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible KO complementation with phospho-mutants and Co-IP, single lab; contradicts prior Eukaryotic Cell paper but uses more rigorous genetic strategy\",\n      \"pmids\": [\"24779470\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TgCaMKrk (a calmodulin-independent CaMK homolog localizing to the apical end of tachyzoites) phosphorylates TgGAP45 in vitro, identifying it as a candidate kinase for GAP45 phosphorylation.\",\n      \"method\": \"In vitro kinase assay with purified proteins\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single in vitro kinase assay, single lab, no in vivo validation of the phosphorylation event\",\n      \"pmids\": [\"27444499\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TgMLC1 palmitoylation on Cys8 and Cys11 is required for its binding to TgGAP45. Blocking palmitoylation completely abrogates TgMLC1–TgGAP45 interaction. Surprisingly, loss of this interaction has little effect on parasite motility, challenging the necessity of MLC1-GAP45 binding for force transduction.\",\n      \"method\": \"Site-directed mutagenesis of palmitoylation sites, co-immunoprecipitation, gliding motility assays\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus Co-IP plus functional motility assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34011689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgGAP45 is a dually acylated glideosome scaffolding protein anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminus), where it recruits the MyoA–MLC1 motor complex to the pellicle, maintains pellicle cohesion during invasion, and is immobilized within cholesterol-rich detergent-resistant membrane domains; its assembly into the motor complex can be regulated by phosphorylation (with TgCaMKrk as a candidate kinase), though the phosphorylation cluster itself is not strictly required for glideosome assembly or motility, and while MLC1 palmitoylation is needed for MLC1–GAP45 binding, this interaction is surprisingly dispensable for parasite movement, indicating that additional force-transduction mechanisms exist.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgGAP45 is a scaffolding component of the myosin XIV glideosome that powers gliding motility, host-cell invasion, and egress in Toxoplasma gondii [#0, #3]. It bridges two membranes of the parasite pellicle: N-terminal acylation anchors it to the plasma membrane while its C-terminal extremity attaches to the inner membrane complex (IMC), where it recruits the MyoA–MLC1 motor to the pellicle, and its N-terminal acylation together with a coiled-coil domain preserves pellicle integrity during the mechanical stress of invasion [#3]. Within the IMC the assembled MyoA/MLC1/GAP45/GAP50 complex is immobilized in cholesterol-dependent, detergent-resistant membrane domains rather than by direct cytoskeletal tethering [#1]. Conditional deletion of GAP45 reduces but does not abolish gliding and invasion, because a MyoC-based glideosome can partially compensate [#6]. MLC1 palmitoylation on Cys8/Cys11 is strictly required for MLC1–GAP45 binding, yet loss of this interaction has little effect on motility, indicating that additional force-transduction routes exist [#9]. GAP45 phosphorylation, initially implicated in the final GAP50 assembly step, was shown by rigorous inducible-knockout complementation to be dispensable for glideosome assembly and motility [#2, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing which proteins constitute the invasion motor was the first requirement; identifying GAP45 as part of the myosin XIV complex anchored at the IMC defined the molecular machine driving parasite motility.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, subcellular fractionation and immunofluorescence in T. gondii\",\n      \"pmids\": [\"15123738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of GAP45 within the complex not resolved\", \"Membrane anchoring of GAP45 itself not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"How the motor complex is held in place at the pellicle was unknown; FRAP and DRM analysis showed it is immobilized in cholesterol-rich detergent-resistant domains, not by cytoskeletal contacts, defining the physical platform for force transduction.\",\n      \"evidence\": \"FRAP, detergent-resistant membrane fractionation, and cholesterol depletion in T. gondii\",\n      \"pmids\": [\"17538016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not resolve which complex component confers immobilization\", \"Mechanism linking DRM residence to motility not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Whether motor assembly is regulated was untested; phosphosite mapping plus phospho-mimetic mutagenesis indicated that phosphorylation of GAP45 Ser163/Ser167 controls the final docking of the soluble MyoA-MLC1-GAP45 complex onto GAP50.\",\n      \"evidence\": \"Mass spectrometry phosphosite mapping, Ser\\u2192Glu/Ser\\u2192Ala mutagenesis, and co-immunoprecipitation in T. gondii\",\n      \"pmids\": [\"19047362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological kinase not identified\", \"Phospho-mimetic readout later shown to be confounded by a co-existing wild-type copy\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The topology and functional necessity of GAP45 were unresolved; conditional knockout, domain dissection and cross-genus complementation showed GAP45 spans plasma membrane and IMC, recruits MLC1-MyoA via its C-terminus, preserves pellicle integrity via its N-terminus, and is essential for gliding, invasion and egress.\",\n      \"evidence\": \"Inducible knockout, domain truncation/mutagenesis, Plasmodium falciparum GAP45 transgenera complementation and motility assays in T. gondii\",\n      \"pmids\": [\"20951968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Extent of functional redundancy not quantified here\", \"Regulation of the two membrane attachments not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linking motor activity to signalling, calcium-stimulated quantitative phosphoproteomics correlated phosphorylation of six residues across GAP45, MLC1 and MyoA with invasion motor activity.\",\n      \"evidence\": \"SILAC LC-MS/MS, 32P-labeling and 2-DE of Ca2+-stimulated tachyzoites in T. gondii\",\n      \"pmids\": [\"21980283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlation not shown to be causal\", \"Responsible kinases not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The strict essentiality of GAP45 was reassessed; inducible knockouts showed deletion reduces but does not abolish gliding/invasion because a MyoC-based glideosome can compensate, redefining GAP45 as important but partially redundant.\",\n      \"evidence\": \"Tetracycline-regulatable inducible knockout, gliding/invasion/egress assays and immunofluorescence in T. gondii\",\n      \"pmids\": [\"25393004\", \"24632839\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Extent to which MyoC glideosome uses GAP45 not defined\", \"Molecular basis of residual motility not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A rigorous genetic test overturned the earlier phosphoregulation model, showing phospho-mimetic/null mutation of the GAP45 phosphorylation cluster (and of MLC1 sites) does not impair assembly or motility, and that the prior Ser163/167Glu phenotype was an artifact of competition with wild-type GAP45.\",\n      \"evidence\": \"Allelic replacement, phospho-mutagenesis, inducible knockout complementation, Co-IP and motility assays in T. gondii\",\n      \"pmids\": [\"24779470\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not exclude a subtle regulatory role in vivo\", \"Physiological function of the phosphosites left unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Seeking the kinase acting on GAP45, an in vitro assay identified TgCaMKrk, an apically localized calmodulin-independent CaMK homolog, as a candidate that phosphorylates GAP45.\",\n      \"evidence\": \"In vitro kinase assay with purified proteins in T. gondii\",\n      \"pmids\": [\"27444499\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vivo validation of the phosphorylation event\", \"Physiological consequence untested given phospho-cluster dispensability\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The requirement of the MLC1-GAP45 linkage for force transduction was tested; palmitoylation of MLC1 Cys8/Cys11 was found necessary for MLC1-GAP45 binding, yet abolishing the interaction barely affected motility, implying additional force-transduction mechanisms.\",\n      \"evidence\": \"Palmitoylation-site mutagenesis, co-immunoprecipitation and gliding motility assays in T. gondii\",\n      \"pmids\": [\"34011689\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Alternative force-transduction route not identified\", \"Structural basis of palmitoylation-dependent binding not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which GAP45 transmits motor force to the pellicle despite dispensability of both its phosphorylation cluster and the MLC1-GAP45 interaction remains unresolved, as does the in vivo kinase and the molecular basis of MyoC-glideosome compensation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No defined alternative force-transduction pathway\", \"In vivo kinase for GAP45 unconfirmed\", \"Structural model of GAP45 dual-membrane bridging absent\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 3, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0032403\", \"supporting_discovery_ids\": [0, 3, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0016020\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0045178\", \"supporting_discovery_ids\": [0, 1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0071702\", \"supporting_discovery_ids\": [3, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006928\", \"supporting_discovery_ids\": [3, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Myosin XIV glideosome motor complex (TgMyoA-TgMLC1-TgGAP45-TgGAP50)\"\n    ],\n    \"partners\": [\n      \"TgMyoA\",\n      \"TgMLC1\",\n      \"TgGAP50\",\n      \"TgCaMKrk (candidate kinase, in vitro)\"\n    ],\n    \"other_free_text\": [\n      \"N-terminal acylation anchors GAP45 to the plasma membrane; C-terminal extremity attaches to the IMC and recruits MLC1-MyoA [#3].\",\n      \"Motor complex immobilized in cholesterol-dependent detergent-resistant membrane domains [#1].\",\n      \"MLC1 palmitoylation (Cys8/Cys11) required for MLC1-GAP45 binding, but the interaction is dispensable for motility [#9].\",\n      \"GAP45 phosphorylation cluster is not required for glideosome assembly or motility; prior loss-of-interaction phenotype was an artifact [#7].\",\n      \"MyoC-based glideosome partially compensates for GAP45 loss [#6].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_223940","name":"GAP45","product":"gliding-associated protein GAP45","uniprot_acc":"S8GEQ1","chromosome":"TGME49_chrX","start_pos":3440383},{"strain":"GT1","gene_id":"TGGT1_223940","name":"GAP45","product":"GAP45 protein","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":3344621},{"strain":"RH88","gene_id":"TGRH88_043730","name":null,"product":"putative GAP45","uniprot_acc":null,"chromosome":"CM023091","start_pos":4123295},{"strain":"VEG","gene_id":"TGVEG_223940","name":null,"product":"GAP45","uniprot_acc":"A0A0F7V9A2","chromosome":"TGVEG_chrX","start_pos":3344273}]}