{"gene":"TG_306060","gene_symbol":"RON8","run_date":"2026-09-08T15:15:13","timeline":{"discoveries":[{"year":2008,"finding":"RON8 co-immunoprecipitates RON5 and other known moving junction proteins (RON2, RON4) from extracellular parasites, demonstrating that a preformed RON complex exists within the parasite prior to secretion.","method":"Co-immunoprecipitation from extracellular parasites","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP method, but finding replicated independently in a concurrent paper (PMID:19247437)","pmids":["19134112"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"finding":"Upon secretion during invasion, RON8 localizes to the cytoplasmic face of the host plasma membrane at the moving junction, as shown by differential permeabilization and expression of RON8 in mammalian cells where C-terminal sequences are required for peripheral targeting.","method":"Differential permeabilization, heterologous expression in mammalian cells with C-terminal deletion analysis","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — localization established by differential permeabilization and heterologous expression with domain dissection, single lab","pmids":["19134112"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"RON8 is an additional member of the AMA1/RON2/RON4/RON5 moving junction complex, identified biochemically; all RON proteins (including RON8) are present at the moving junction during invasion, and RON4/5/8 associate with the cytoplasmic face of the host cell membrane.","method":"Biochemical fractionation, immunoprecipitation, differential permeabilization, loading of RON-specific antibodies into the host cell","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (biochemical fractionation, Co-IP, differential permeabilization, antibody loading), replicated across two independent labs","pmids":["19247437"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"RON proteins, including RON8, undergo proteolytic maturation during trafficking to their respective organelles (rhoptry neck) and can associate as non-mature forms in vitro.","method":"Metabolic labelling, immunoprecipitation, in vitro association assay","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — metabolic labelling plus immunoprecipitation in a single lab, two orthogonal methods","pmids":["19247437"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Genetic disruption of RON8 (Δron8) severely impairs both parasite attachment to and invasion of host cells, demonstrating that RON8 is required to establish firm attachment and commit the parasite to invasion; remaining MJ components trail behind invading knockout parasites, indicating a malformed complex without RON8.","method":"Genetic knockout (Δron8), invasion and attachment assays, complementation","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with complementation rescue, multiple functional readouts (attachment, invasion, MJ morphology), in vitro and in vivo validation","pmids":["21423671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Complementation of Δron8 parasites reveals a proteolytic processing event at the RON8 C-terminus, and replacement of the RON8 N-terminal region with mCherry separates the region required for rhoptry neck targeting and MJ complex formation from the region required for function during invasion.","method":"Complementation, domain-swap reporter construct (mCherry), subcellular localization and invasion assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain dissection with reporter fusion, complementation controls, and multiple functional readouts in a single rigorous study","pmids":["21423671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"RON8 is restricted to the coccidia (no orthologue detected in Plasmodium), indicating it is a coccidian-specific component of the moving junction complex.","method":"Comparative genomic/bioinformatic analysis supported by experimental identification","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 3 / Strong — phylogenetic inference confirmed by two independent experimental studies identifying RON8 only in coccidians","pmids":["21423671","19134112"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Conditional knockdown of RON5 causes degradation of RON2 and mistargeting of RON4, but RON8 levels and localization are unaffected by loss of RON5, indicating RON8 trafficking to the rhoptry neck is independent of RON5.","method":"Conditional knockdown (tetracycline-regulatable), immunofluorescence, Western blot","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic system with multiple readouts; RON8 independence from RON5 directly demonstrated","pmids":["24651769"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Traffic of RON8 to the moving junction is not affected by absence of RON2, RON4, or RON5, suggesting RON8 (together with RON4L1) may constitute an independent MJ sub-complex distinct from the RON2/RON4/RON5 complex.","method":"Knockout cell lines for RON2, RON4, and RON5 with immunofluorescence localization of RON8","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockouts with direct localization readout, single lab","pmids":["29263399"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"RON8 is a coccidian-specific rhoptry neck protein that forms a preformed complex with RON2, RON4, and RON5 inside the parasite; upon secretion during invasion it is injected into the host cell and anchors to the cytoplasmic face of the host plasma membrane at the moving junction, where its C-terminal region mediates rhoptry neck targeting and complex association while a separate N-terminal region is essential for establishing firm parasite attachment and committing the parasite to host cell entry, independently of RON5-mediated complex organization."},"narrative":{"mechanistic_narrative":"RON8 is a coccidian-specific rhoptry neck protein that functions as a core component of the moving junction (MJ) machinery driving Toxoplasma gondii invasion of host cells [PMID:19247437, PMID:21423671, PMID:19134112]. Within extracellular parasites it exists in a preformed complex with the other RON proteins RON2, RON4, and RON5 [PMID:19134112], and, like its partners, undergoes proteolytic maturation during trafficking to the rhoptry neck [PMID:19247437]. During invasion RON8 is secreted and delivered across the host plasma membrane, where it anchors to the cytoplasmic face of the membrane at the moving junction [PMID:19134112, PMID:19247437]; its C-terminal region mediates peripheral targeting and rhoptry neck/MJ complex association [PMID:19134112, PMID:21423671]. Genetic disruption of RON8 severely impairs both attachment and invasion, with the residual MJ components trailing behind the parasite, establishing that RON8 is required to build a functional junction and commit the parasite to entry [PMID:21423671]. Domain-swap analysis separates the C-terminal targeting region from an N-terminal region essential for function during invasion [PMID:21423671]. RON8 traffics to the rhoptry neck and MJ independently of RON2, RON4, and RON5, indicating it constitutes a distinct MJ sub-complex rather than depending on the RON2/RON4/RON5 organization for its localization [PMID:24651769, PMID:29263399].","teleology":[{"year":2008,"claim":"Establishing that RON8 is physically part of the moving junction machinery answered whether it acts alone or as part of an organized complex assembled before secretion.","evidence":"Co-immunoprecipitation of RON5, RON2, and RON4 by RON8 from extracellular parasites","pmids":["19134112"],"confidence":"Medium","gaps":["Single Co-IP method; stoichiometry and direct binding partners within the complex not resolved","Whether all interactions are direct or bridged is unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Defining where RON8 goes upon secretion answered whether it acts on the parasite or host side of the junction.","evidence":"Differential permeabilization and heterologous expression in mammalian cells with C-terminal deletion analysis","pmids":["19134112"],"confidence":"Medium","gaps":["Membrane-anchoring mechanism on the host cytoplasmic face not defined","How C-terminal sequences mediate peripheral targeting mechanistically unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Multiple orthogonal methods across independent labs confirmed RON8 as a bona fide MJ complex member present at the junction during invasion, consolidating the biochemical picture.","evidence":"Biochemical fractionation, immunoprecipitation, differential permeabilization, and antibody loading into host cells","pmids":["19247437"],"confidence":"High","gaps":["Functional contribution of RON8 to invasion not yet tested by genetics"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Showing that RON proteins including RON8 are proteolytically matured during trafficking clarified how the complex is prepared en route to the rhoptry neck.","evidence":"Metabolic labelling, immunoprecipitation, and in vitro association assay","pmids":["19247437"],"confidence":"Medium","gaps":["Protease responsible for maturation not identified","Functional consequence of maturation vs non-mature association not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Genetic knockout defined RON8's causal role, answering whether it is required for invasion rather than merely present.","evidence":"Genetic knockout (Δron8) with complementation rescue and attachment/invasion assays","pmids":["21423671"],"confidence":"High","gaps":["Molecular basis of how RON8 establishes firm attachment not defined","Host-side binding partner of RON8 not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Domain dissection separated the region controlling targeting/complex formation from the region required for invasion, defining a modular architecture and a distinct functional N-terminus.","evidence":"Complementation with mCherry domain-swap reporter constructs plus localization and invasion assays","pmids":["21423671"],"confidence":"High","gaps":["Molecular function of the N-terminal invasion-essential region unknown","C-terminal processing event's role not fully defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Comparative genomics placed RON8 as coccidian-specific, framing it as a lineage-restricted invasion determinant absent in Plasmodium.","evidence":"Comparative genomic/bioinformatic analysis with experimental identification","pmids":["21423671","19134112"],"confidence":"Medium","gaps":["Whether a functional analogue substitutes for RON8 in Plasmodium unaddressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Conditional loss of RON5 tested RON8's dependence on the rest of the complex, showing RON8 trafficking is RON5-independent.","evidence":"Tetracycline-regulatable RON5 knockdown with immunofluorescence and Western blot","pmids":["24651769"],"confidence":"High","gaps":["What does drive RON8 rhoptry neck targeting remains unidentified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Systematic RON2/RON4/RON5 knockouts refined the model, indicating RON8 forms an independent MJ sub-complex distinct from the RON2/RON4/RON5 module.","evidence":"Knockout cell lines for RON2, RON4, and RON5 with immunofluorescence localization of RON8","pmids":["29263399"],"confidence":"Medium","gaps":["The composition and binding partners of the proposed RON8/RON4L1 sub-complex not biochemically defined","How two sub-complexes coordinate at the junction unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The host-cell receptor or membrane feature that RON8 engages to establish firm attachment, and the biochemical function of its invasion-essential N-terminus, remain unidentified.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No host-side binding partner identified","Molecular activity of the N-terminal region undefined","Composition of the RON8-independent sub-complex not resolved biochemically"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Moving junction RON complex (RON2/RON4/RON5/RON8)","Proposed RON8/RON4L1 sub-complex"],"partners":["RON2","RON4","RON5","RON4L1","AMA1"],"other_free_text":["Coccidian-specific rhoptry neck protein; no orthologue in Plasmodium [#6]","Secreted and delivered to the cytoplasmic face of the host plasma membrane at the moving junction [#1, #2]","C-terminal region mediates rhoptry neck targeting and MJ complex association; N-terminal region essential for function during invasion [#5]","Undergoes proteolytic maturation during trafficking and C-terminal processing [#3, #5]","Required for firm host-cell attachment and commitment to invasion [#4]","Traffics to the rhoptry neck/MJ independently of RON2, RON4, and RON5 [#7, #8]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"19247437","id":"PMC_19247437","title":"Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.","date":"2009","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/19247437","citation_count":238,"is_preprint":false},{"pmid":"19134112","id":"PMC_19134112","title":"Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements.","date":"2008","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/19134112","citation_count":92,"is_preprint":false},{"pmid":"21423671","id":"PMC_21423671","title":"The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii.","date":"2011","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/21423671","citation_count":52,"is_preprint":false},{"pmid":"24651769","id":"PMC_24651769","title":"RON5 is critical for organization and function of the Toxoplasma moving junction complex.","date":"2014","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/24651769","citation_count":47,"is_preprint":false},{"pmid":"29263399","id":"PMC_29263399","title":"RON4L1 is a new member of the moving junction complex in Toxoplasma gondii.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29263399","citation_count":16,"is_preprint":false},{"pmid":"40881926","id":"PMC_40881926","title":"In silico evaluation of Toxoplasma gondii rhoptry neck proteins (TgRONs) for potential immunogenic epitopes.","date":"2025","source":"EXCLI journal","url":"https://pubmed.ncbi.nlm.nih.gov/40881926","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5325,"output_tokens":2107,"usd":0.02379,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7504,"output_tokens":3307,"usd":0.060097,"stage2_stop_reason":"end_turn"},"total_usd":0.083887,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"RON8 co-immunoprecipitates RON5 and other known moving junction proteins (RON2, RON4) from extracellular parasites, demonstrating that a preformed RON complex exists within the parasite prior to secretion.\",\n      \"method\": \"Co-immunoprecipitation from extracellular parasites\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP method, but finding replicated independently in a concurrent paper (PMID:19247437)\",\n      \"pmids\": [\"19134112\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Upon secretion during invasion, RON8 localizes to the cytoplasmic face of the host plasma membrane at the moving junction, as shown by differential permeabilization and expression of RON8 in mammalian cells where C-terminal sequences are required for peripheral targeting.\",\n      \"method\": \"Differential permeabilization, heterologous expression in mammalian cells with C-terminal deletion analysis\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — localization established by differential permeabilization and heterologous expression with domain dissection, single lab\",\n      \"pmids\": [\"19134112\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RON8 is an additional member of the AMA1/RON2/RON4/RON5 moving junction complex, identified biochemically; all RON proteins (including RON8) are present at the moving junction during invasion, and RON4/5/8 associate with the cytoplasmic face of the host cell membrane.\",\n      \"method\": \"Biochemical fractionation, immunoprecipitation, differential permeabilization, loading of RON-specific antibodies into the host cell\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (biochemical fractionation, Co-IP, differential permeabilization, antibody loading), replicated across two independent labs\",\n      \"pmids\": [\"19247437\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RON proteins, including RON8, undergo proteolytic maturation during trafficking to their respective organelles (rhoptry neck) and can associate as non-mature forms in vitro.\",\n      \"method\": \"Metabolic labelling, immunoprecipitation, in vitro association assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — metabolic labelling plus immunoprecipitation in a single lab, two orthogonal methods\",\n      \"pmids\": [\"19247437\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Genetic disruption of RON8 (Δron8) severely impairs both parasite attachment to and invasion of host cells, demonstrating that RON8 is required to establish firm attachment and commit the parasite to invasion; remaining MJ components trail behind invading knockout parasites, indicating a malformed complex without RON8.\",\n      \"method\": \"Genetic knockout (Δron8), invasion and attachment assays, complementation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with complementation rescue, multiple functional readouts (attachment, invasion, MJ morphology), in vitro and in vivo validation\",\n      \"pmids\": [\"21423671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Complementation of Δron8 parasites reveals a proteolytic processing event at the RON8 C-terminus, and replacement of the RON8 N-terminal region with mCherry separates the region required for rhoptry neck targeting and MJ complex formation from the region required for function during invasion.\",\n      \"method\": \"Complementation, domain-swap reporter construct (mCherry), subcellular localization and invasion assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain dissection with reporter fusion, complementation controls, and multiple functional readouts in a single rigorous study\",\n      \"pmids\": [\"21423671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RON8 is restricted to the coccidia (no orthologue detected in Plasmodium), indicating it is a coccidian-specific component of the moving junction complex.\",\n      \"method\": \"Comparative genomic/bioinformatic analysis supported by experimental identification\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Strong — phylogenetic inference confirmed by two independent experimental studies identifying RON8 only in coccidians\",\n      \"pmids\": [\"21423671\", \"19134112\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Conditional knockdown of RON5 causes degradation of RON2 and mistargeting of RON4, but RON8 levels and localization are unaffected by loss of RON5, indicating RON8 trafficking to the rhoptry neck is independent of RON5.\",\n      \"method\": \"Conditional knockdown (tetracycline-regulatable), immunofluorescence, Western blot\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic system with multiple readouts; RON8 independence from RON5 directly demonstrated\",\n      \"pmids\": [\"24651769\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Traffic of RON8 to the moving junction is not affected by absence of RON2, RON4, or RON5, suggesting RON8 (together with RON4L1) may constitute an independent MJ sub-complex distinct from the RON2/RON4/RON5 complex.\",\n      \"method\": \"Knockout cell lines for RON2, RON4, and RON5 with immunofluorescence localization of RON8\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockouts with direct localization readout, single lab\",\n      \"pmids\": [\"29263399\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"RON8 is a coccidian-specific rhoptry neck protein that forms a preformed complex with RON2, RON4, and RON5 inside the parasite; upon secretion during invasion it is injected into the host cell and anchors to the cytoplasmic face of the host plasma membrane at the moving junction, where its C-terminal region mediates rhoptry neck targeting and complex association while a separate N-terminal region is essential for establishing firm parasite attachment and committing the parasite to host cell entry, independently of RON5-mediated complex organization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RON8 is a coccidian-specific rhoptry neck protein that functions as a core component of the moving junction (MJ) machinery driving Toxoplasma gondii invasion of host cells [#2, #6]. Within extracellular parasites it exists in a preformed complex with the other RON proteins RON2, RON4, and RON5 [#0], and, like its partners, undergoes proteolytic maturation during trafficking to the rhoptry neck [#3]. During invasion RON8 is secreted and delivered across the host plasma membrane, where it anchors to the cytoplasmic face of the membrane at the moving junction [#1, #2]; its C-terminal region mediates peripheral targeting and rhoptry neck/MJ complex association [#1, #5]. Genetic disruption of RON8 severely impairs both attachment and invasion, with the residual MJ components trailing behind the parasite, establishing that RON8 is required to build a functional junction and commit the parasite to entry [#4]. Domain-swap analysis separates the C-terminal targeting region from an N-terminal region essential for function during invasion [#5]. RON8 traffics to the rhoptry neck and MJ independently of RON2, RON4, and RON5, indicating it constitutes a distinct MJ sub-complex rather than depending on the RON2/RON4/RON5 organization for its localization [#7, #8].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing that RON8 is physically part of the moving junction machinery answered whether it acts alone or as part of an organized complex assembled before secretion.\",\n      \"evidence\": \"Co-immunoprecipitation of RON5, RON2, and RON4 by RON8 from extracellular parasites\",\n      \"pmids\": [\"19134112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP method; stoichiometry and direct binding partners within the complex not resolved\", \"Whether all interactions are direct or bridged is unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defining where RON8 goes upon secretion answered whether it acts on the parasite or host side of the junction.\",\n      \"evidence\": \"Differential permeabilization and heterologous expression in mammalian cells with C-terminal deletion analysis\",\n      \"pmids\": [\"19134112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Membrane-anchoring mechanism on the host cytoplasmic face not defined\", \"How C-terminal sequences mediate peripheral targeting mechanistically unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Multiple orthogonal methods across independent labs confirmed RON8 as a bona fide MJ complex member present at the junction during invasion, consolidating the biochemical picture.\",\n      \"evidence\": \"Biochemical fractionation, immunoprecipitation, differential permeabilization, and antibody loading into host cells\",\n      \"pmids\": [\"19247437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional contribution of RON8 to invasion not yet tested by genetics\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showing that RON proteins including RON8 are proteolytically matured during trafficking clarified how the complex is prepared en route to the rhoptry neck.\",\n      \"evidence\": \"Metabolic labelling, immunoprecipitation, and in vitro association assay\",\n      \"pmids\": [\"19247437\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Protease responsible for maturation not identified\", \"Functional consequence of maturation vs non-mature association not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic knockout defined RON8's causal role, answering whether it is required for invasion rather than merely present.\",\n      \"evidence\": \"Genetic knockout (\\u0394ron8) with complementation rescue and attachment/invasion assays\",\n      \"pmids\": [\"21423671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of how RON8 establishes firm attachment not defined\", \"Host-side binding partner of RON8 not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Domain dissection separated the region controlling targeting/complex formation from the region required for invasion, defining a modular architecture and a distinct functional N-terminus.\",\n      \"evidence\": \"Complementation with mCherry domain-swap reporter constructs plus localization and invasion assays\",\n      \"pmids\": [\"21423671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function of the N-terminal invasion-essential region unknown\", \"C-terminal processing event's role not fully defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Comparative genomics placed RON8 as coccidian-specific, framing it as a lineage-restricted invasion determinant absent in Plasmodium.\",\n      \"evidence\": \"Comparative genomic/bioinformatic analysis with experimental identification\",\n      \"pmids\": [\"21423671\", \"19134112\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether a functional analogue substitutes for RON8 in Plasmodium unaddressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Conditional loss of RON5 tested RON8's dependence on the rest of the complex, showing RON8 trafficking is RON5-independent.\",\n      \"evidence\": \"Tetracycline-regulatable RON5 knockdown with immunofluorescence and Western blot\",\n      \"pmids\": [\"24651769\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What does drive RON8 rhoptry neck targeting remains unidentified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Systematic RON2/RON4/RON5 knockouts refined the model, indicating RON8 forms an independent MJ sub-complex distinct from the RON2/RON4/RON5 module.\",\n      \"evidence\": \"Knockout cell lines for RON2, RON4, and RON5 with immunofluorescence localization of RON8\",\n      \"pmids\": [\"29263399\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The composition and binding partners of the proposed RON8/RON4L1 sub-complex not biochemically defined\", \"How two sub-complexes coordinate at the junction unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The host-cell receptor or membrane feature that RON8 engages to establish firm attachment, and the biochemical function of its invasion-essential N-terminus, remain unidentified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No host-side binding partner identified\", \"Molecular activity of the N-terminal region undefined\", \"Composition of the RON8-independent sub-complex not resolved biochemically\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\"term_id\": \"GO:0035838\", \"supporting_discovery_ids\": [1, 2, 3, 5, 7, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [2, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\"Moving junction RON complex (RON2/RON4/RON5/RON8)\", \"Proposed RON8/RON4L1 sub-complex\"],\n    \"partners\": [\"RON2\", \"RON4\", \"RON5\", \"RON4L1\", \"AMA1\"],\n    \"other_free_text\": [\n      \"Coccidian-specific rhoptry neck protein; no orthologue in Plasmodium [#6]\",\n      \"Secreted and delivered to the cytoplasmic face of the host plasma membrane at the moving junction [#1, #2]\",\n      \"C-terminal region mediates rhoptry neck targeting and MJ complex association; N-terminal region essential for function during invasion [#5]\",\n      \"Undergoes proteolytic maturation during trafficking and C-terminal processing [#3, #5]\",\n      \"Required for firm host-cell attachment and commitment to invasion [#4]\",\n      \"Traffics to the rhoptry neck/MJ independently of RON2, RON4, and RON5 [#7, #8]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_306060","name":"RON8","product":"rhoptry neck protein RON8","uniprot_acc":"S8F1T4","chromosome":"TGME49_chrIX","start_pos":5779179},{"strain":"GT1","gene_id":"TGGT1_306060","name":null,"product":"rhoptry neck protein RON8","uniprot_acc":null,"chromosome":"TGGT1_chrIX","start_pos":5560917},{"strain":"RH88","gene_id":"TGRH88_017600","name":null,"product":"rhoptry neck protein RON8","uniprot_acc":null,"chromosome":"CM023090","start_pos":5902524},{"strain":"VEG","gene_id":"TGVEG_306060","name":null,"product":"rhoptry neck protein RON8","uniprot_acc":"A0A125YRV5","chromosome":"TGVEG_chrIX","start_pos":5817900}]}