{"gene":"TG_248480","gene_symbol":"RPS9","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2001,"finding":"The first 55 amino acids of the RPS9 leader sequence are sufficient for apicoplast targeting in T. gondii, and transit peptide processing is a multi-step event occurring both outside of and at/within the apicoplast.","method":"Deletional and insertional mutagenesis with HA tagging, fluorescence localization, and Western blot analysis in transfected T. gondii","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct mutagenesis with two orthogonal methods (localization + Western blot), single lab","pmids":["11704269"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Two cis-acting regions within the RPS9 transit domain (amino acids 38–49 and 79–86), together with the signal sequence, are necessary and sufficient for apicoplast targeting; affinity purification identified proteins of 92, 90, 86, and 160 kDa that associate with the RPS9 leader, with the 92- and 90-kDa species being subtilisin-like proteins and the 86-kDa species identified as the T. gondii molecular chaperone BiP.","method":"Cis-element mutagenesis, GST-fusion affinity chromatography, and mass spectrometry in T. gondii transfectants","journal":"The Journal of parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity pulldown with mass spectrometry identification and functional mutagenesis, single lab","pmids":["14533689"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"T. gondii RPS9 is a nuclear-encoded apicoplast ribosomal protein whose bipartite leader directs it to the apicoplast via a multi-step processing pathway: the first 55 amino acids are sufficient for targeting, two specific transit-domain regions (aa 38–49 and 79–86) act as cis elements, and the leader interacts with subtilisin-like proteases and the chaperone BiP during transit to and/or import into the apicoplast."},"narrative":{"mechanistic_narrative":"In Toxoplasma gondii, RPS9 is a nuclear-encoded apicoplast ribosomal protein that carries a bipartite N-terminal leader directing it into the apicoplast through a multi-step processing pathway [PMID:11704269]. The first 55 amino acids of this leader are sufficient for apicoplast targeting, and processing of the transit peptide occurs as a multi-step event both outside of and at/within the organelle [PMID:11704269]. Two discrete cis-acting regions within the transit domain (amino acids 38–49 and 79–86), acting together with the signal sequence, are necessary and sufficient for targeting [PMID:14533689]. During transit, the leader physically associates with subtilisin-like proteases (92- and 90-kDa species) and the T. gondii molecular chaperone BiP (86-kDa species), implicating these factors in leader processing and/or import [PMID:14533689]. Beyond this targeting and processing pathway, the biochemical function of RPS9 within the assembled apicoplast ribosome has not been characterized in the available corpus.","teleology":[{"year":2001,"claim":"Whether the RPS9 leader alone could route a nuclear-encoded protein to the apicoplast, and whether transit peptide removal was a single or multi-step event, was unknown; defining the minimal targeting determinant established the logic of apicoplast protein import.","evidence":"Deletional/insertional mutagenesis with HA tagging, fluorescence localization, and Western blotting in transfected T. gondii","pmids":["11704269"],"confidence":"Medium","gaps":["The specific proteases or chaperones mediating each processing step were not identified","The precise cleavage sites within the leader were not mapped","Function of mature RPS9 in the apicoplast ribosome was not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Having localized targeting to the first 55 residues, the next question was which specific sequence elements act as cis determinants and which trans factors engage the leader; mapping two transit-domain regions and identifying associated proteins built the molecular basis of leader recognition and processing.","evidence":"Cis-element mutagenesis, GST-fusion affinity chromatography, and mass spectrometry of leader-associated proteins in T. gondii transfectants","pmids":["14533689"],"confidence":"Medium","gaps":["Direct enzymatic activity of the subtilisin-like proteases on the RPS9 leader was not demonstrated","The functional role of BiP association during import was not tested by loss-of-function","The 160-kDa associated protein was not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The function of mature RPS9 within the apicoplast ribosome and the mechanistic role of the associated proteases and BiP in import remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural or functional characterization of RPS9 in the assembled ribosome","No loss-of-function or reconstitution of the protease/chaperone-mediated import step"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["SUBTILISIN-LIKE PROTEASES (92 AND 90 KDA)","BIP (86 KDA MOLECULAR CHAPERONE)"],"other_free_text":["Bipartite N-terminal leader: first 55 aa sufficient for apicoplast targeting","Two cis-acting transit-domain regions (aa 38-49 and 79-86) necessary and sufficient with the signal sequence","Transit peptide processing is multi-step, occurring both outside of and at/within the apicoplast","Unidentified 160-kDa leader-associated protein"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"11704269","id":"PMC_11704269","title":"Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis.","date":"2001","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/11704269","citation_count":28,"is_preprint":false},{"pmid":"14533689","id":"PMC_14533689","title":"Cis and trans factors involved in apicoplast targeting in Toxoplasma gondii.","date":"2003","source":"The Journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/14533689","citation_count":13,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3138,"output_tokens":654,"usd":0.009612,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5363,"output_tokens":1797,"usd":0.03587,"stage2_stop_reason":"end_turn"},"total_usd":0.045482,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"The first 55 amino acids of the RPS9 leader sequence are sufficient for apicoplast targeting in T. gondii, and transit peptide processing is a multi-step event occurring both outside of and at/within the apicoplast.\",\n      \"method\": \"Deletional and insertional mutagenesis with HA tagging, fluorescence localization, and Western blot analysis in transfected T. gondii\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct mutagenesis with two orthogonal methods (localization + Western blot), single lab\",\n      \"pmids\": [\"11704269\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two cis-acting regions within the RPS9 transit domain (amino acids 38–49 and 79–86), together with the signal sequence, are necessary and sufficient for apicoplast targeting; affinity purification identified proteins of 92, 90, 86, and 160 kDa that associate with the RPS9 leader, with the 92- and 90-kDa species being subtilisin-like proteins and the 86-kDa species identified as the T. gondii molecular chaperone BiP.\",\n      \"method\": \"Cis-element mutagenesis, GST-fusion affinity chromatography, and mass spectrometry in T. gondii transfectants\",\n      \"journal\": \"The Journal of parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity pulldown with mass spectrometry identification and functional mutagenesis, single lab\",\n      \"pmids\": [\"14533689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"T. gondii RPS9 is a nuclear-encoded apicoplast ribosomal protein whose bipartite leader directs it to the apicoplast via a multi-step processing pathway: the first 55 amino acids are sufficient for targeting, two specific transit-domain regions (aa 38–49 and 79–86) act as cis elements, and the leader interacts with subtilisin-like proteases and the chaperone BiP during transit to and/or import into the apicoplast.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"In Toxoplasma gondii, RPS9 is a nuclear-encoded apicoplast ribosomal protein that carries a bipartite N-terminal leader directing it into the apicoplast through a multi-step processing pathway [#0]. The first 55 amino acids of this leader are sufficient for apicoplast targeting, and processing of the transit peptide occurs as a multi-step event both outside of and at/within the organelle [#0]. Two discrete cis-acting regions within the transit domain (amino acids 38\\u201349 and 79\\u201386), acting together with the signal sequence, are necessary and sufficient for targeting [#1]. During transit, the leader physically associates with subtilisin-like proteases (92- and 90-kDa species) and the T. gondii molecular chaperone BiP (86-kDa species), implicating these factors in leader processing and/or import [#1]. Beyond this targeting and processing pathway, the biochemical function of RPS9 within the assembled apicoplast ribosome has not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Whether the RPS9 leader alone could route a nuclear-encoded protein to the apicoplast, and whether transit peptide removal was a single or multi-step event, was unknown; defining the minimal targeting determinant established the logic of apicoplast protein import.\",\n      \"evidence\": \"Deletional/insertional mutagenesis with HA tagging, fluorescence localization, and Western blotting in transfected T. gondii\",\n      \"pmids\": [\n        \"11704269\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The specific proteases or chaperones mediating each processing step were not identified\",\n        \"The precise cleavage sites within the leader were not mapped\",\n        \"Function of mature RPS9 in the apicoplast ribosome was not addressed\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Having localized targeting to the first 55 residues, the next question was which specific sequence elements act as cis determinants and which trans factors engage the leader; mapping two transit-domain regions and identifying associated proteins built the molecular basis of leader recognition and processing.\",\n      \"evidence\": \"Cis-element mutagenesis, GST-fusion affinity chromatography, and mass spectrometry of leader-associated proteins in T. gondii transfectants\",\n      \"pmids\": [\n        \"14533689\"\n      ],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct enzymatic activity of the subtilisin-like proteases on the RPS9 leader was not demonstrated\",\n        \"The functional role of BiP association during import was not tested by loss-of-function\",\n        \"The 160-kDa associated protein was not identified\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The function of mature RPS9 within the apicoplast ribosome and the mechanistic role of the associated proteases and BiP in import remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural or functional characterization of RPS9 in the assembled ribosome\",\n        \"No loss-of-function or reconstitution of the protease/chaperone-mediated import step\"\n      ],\n      \"attribution\": [\n        {\n          \"species\": \"Toxoplasma gondii\",\n          \"strain\": \"\"\n        }\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0003735\",\n        \"supporting_discovery_ids\": [0, 1],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0009507\",\n        \"supporting_discovery_ids\": [0, 1],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"GO:0006605\",\n        \"supporting_discovery_ids\": [0, 1],\n        \"attribution\": [\n          {\n            \"species\": \"Toxoplasma gondii\",\n            \"strain\": \"\"\n          }\n        ]\n      }\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"Subtilisin-like proteases (92 and 90 kDa)\",\n      \"BiP (86 kDa molecular chaperone)\"\n    ],\n    \"other_free_text\": [\n      \"Bipartite N-terminal leader: first 55 aa sufficient for apicoplast targeting\",\n      \"Two cis-acting transit-domain regions (aa 38-49 and 79-86) necessary and sufficient with the signal sequence\",\n      \"Transit peptide processing is multi-step, occurring both outside of and at/within the apicoplast\",\n      \"Unidentified 160-kDa leader-associated protein\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_248480","name":"RPS9","product":"ribosomal protein RPS9","uniprot_acc":"A0A125YWF6","chromosome":"TGME49_chrXII","start_pos":3884269},{"strain":"GT1","gene_id":"TGGT1_248480","name":null,"product":"ribosomal protein RPS9","uniprot_acc":null,"chromosome":"TGGT1_chrXII","start_pos":3816916},{"strain":"RH88","gene_id":"TGRH88_062360","name":null,"product":"ribosomal protein RPS9","uniprot_acc":null,"chromosome":"CM023093","start_pos":3937496},{"strain":"VEG","gene_id":"TGVEG_248480","name":null,"product":"ribosomal protein RPS9","uniprot_acc":"B9PQU8","chromosome":"TGVEG_chrXII","start_pos":3855189}]}