{"gene":"PF3D7_0629500","run_date":"2026-09-08T12:51:26","timeline":{"discoveries":[{"year":2018,"finding":"AAT1 (PF3D7_0629500) expressed heterologously in yeast functions as a drug transporter: expression produced chloroquine hypersensitivity coincident with increased CQ uptake (~4-fold increased quinine uptake), and sensitized cells to amodiaquine, mefloquine, and quinine. Drug sensitivity was reversed by introducing the S258L SNP linked to CQ resistance. Quinine hypersensitivity was suppressible with tryptophan, consistent with a shared amino acid-inhibitable transport mechanism with the yeast Tat2p transporter. The protein localised primarily to the yeast plasma membrane.","method":"Heterologous expression in yeast, drug sensitivity assays, radiolabelled drug uptake assays, site-directed mutagenesis (S258L SNP), tryptophan competition assay, fluorescence localization","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro functional reconstitution in heterologous system with direct drug uptake measurement, mutagenesis confirming SNP reversal, and competition assay establishing amino acid-inhibitable mechanism; single lab but multiple orthogonal methods","pmids":["29410428"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"A mutation in aat1 (the P. chabaudi orthologue of PF3D7_0629500) was identified as the critical mutation conferring the first level of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi, mapped by quantitative genome-wide linkage group selection and whole-genome resequencing of CQ-resistant mutants.","method":"Linkage group selection genetic cross, quantitative whole-genome resequencing, identification of point mutation at locus of selection valley","journal":"BMC genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via linkage group selection cross in P. chabaudi, replicated by quantitative genome resequencing; single lab but two orthogonal genomic approaches","pmids":["22435897"],"is_preprint":false,"attribution":[{"species":"Plasmodium chabaudi","strain":""}]},{"year":2023,"finding":"AAT1 S258L potentiates CQ resistance but at a fitness cost, while AAT1 F313S (a common Southeast Asian polymorphism) reduces CQ resistance while restoring fitness. A chromosome 6 quantitative trait locus containing pfaat1 was identified by parasite genetic crosses as being selected by CQ treatment. Population genomics showed temporal selection of pfaat1 S258L in parallel with pfcrt K76T in The Gambia (rising from 0% to 97% between 1984 and 2014). Gene editing confirmed differential effects of S258L and F313S on CQ resistance and parasite fitness.","method":"Population genomics (longitudinal temporal analysis), parasite genetic crosses (QTL mapping), CRISPR/gene editing of pfaat1 alleles, in vitro drug sensitivity assays, parasite growth/fitness assays","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic approaches (QTL cross + gene editing), multiple orthogonal methods, replicated across population genomics and experimental genetics in a single study","pmids":["37169919"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"finding":"PfAAT1 is essential in parasites harboring mutant PfCRT but largely dispensable in a wild-type PfCRT background; replacing mutant pfcrt with wild-type abolished the growth defect from pfaat1 knockdown, indicating functional overlap between PfCRT and PfAAT1. PfAAT1 knockdown causes accumulation of haemoglobin-derived peptides, establishing that PfAAT1 participates in export/utilisation of Hb-derived peptides from the digestive vacuole. Field-derived variants S258L and F313S confer a fitness advantage over wild-type PfAAT1 in the mutant PfCRT background, consistent with gain-of-function enhancement of peptide transport to compensate for impaired PfCRT. F313S imparts pronounced fitness advantage under cysteine- and methionine-limiting conditions. PfAAT1 mutations do not independently alter CQ sensitivity; selection of pfaat1 alleles in the field is driven primarily by epistatic interaction with pfcrt.","method":"Conditional knockdown of pfaat1, isogenic parasite line allele replacement (mutant vs wild-type pfcrt), peptide accumulation assays (mass spectrometry), comparative growth analyses under amino acid-limiting conditions, genetic epistasis analysis","journal":"Research square","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — knockdown with defined molecular phenotype (peptide accumulation), allele replacement epistasis, amino acid limitation experiments; multiple orthogonal methods in single study; preprint but mechanistically detailed","pmids":["42370240"],"is_preprint":true,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"Bioinformatic analysis of the P. falciparum genome identified PF3D7_0629500 (AAT1) as a putative amino acid transporter based on hydropathy plot analysis and sequence homology, assigning it to the amino acid transporter family.","method":"Computational genome-wide hydropathy plot analysis and sequence homology assignment","journal":"Genome biology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational prediction only, no experimental validation in this paper","pmids":["15774027"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PfAAT1 (PF3D7_0629500) is a digestive vacuole-associated amino acid transporter in P. falciparum that mediates export of haemoglobin-derived peptides from the digestive vacuole; it is largely dispensable when PfCRT is functional but becomes essential when PfCRT is mutated (K76T), because mutant PfCRT loses its native peptide export activity, and field-selected PfAAT1 variants (S258L in Africa; F313S in Southeast Asia) represent gain-of-function mutations that compensate for impaired PfCRT peptide transport, thereby restoring parasite fitness at the cost of (S258L) or with restoration of (F313S) CQ sensitivity, with allele selection driven primarily by epistatic interaction with pfcrt rather than by direct effects on CQ transport."},"narrative":{"mechanistic_narrative":"AAT1 (PF3D7_0629500) is a digestive vacuole-associated amino acid transporter of Plasmodium falciparum that participates in the export and utilisation of haemoglobin-derived peptides from the digestive vacuole [PMID:42370240]. Conditional knockdown of PfAAT1 causes accumulation of Hb-derived peptides and reveals a functional overlap with PfCRT: AAT1 is largely dispensable when PfCRT is wild-type but becomes essential in a mutant PfCRT background, and restoring wild-type pfcrt abolishes the growth defect of pfaat1 knockdown [PMID:42370240]. Field-selected variants act as gain-of-function alleles that compensate for impaired PfCRT peptide transport — S258L and F313S confer a fitness advantage in the mutant PfCRT background, with F313S particularly favoured under cysteine- and methionine-limiting conditions [PMID:42370240], and their selection in the field is driven by epistatic interaction with pfcrt rather than by direct effects on chloroquine transport [PMID:42370240]. Consistent with a role in drug handling, heterologous expression of AAT1 in yeast produces chloroquine hypersensitivity and increased CQ and quinine uptake in an amino acid-inhibitable manner reversed by the S258L SNP [PMID:29410428], and population genomics documents temporal co-selection of pfaat1 S258L alongside pfcrt K76T [PMID:37169919].","teleology":[{"year":2005,"claim":"Before any functional data, it was unknown whether PF3D7_0629500 encoded a transporter at all; genome annotation placed it within a candidate gene family to be tested.","evidence":"Genome-wide computational hydropathy and sequence-homology analysis of the P. falciparum genome","pmids":["15774027"],"confidence":"Low","gaps":["Computational prediction only, no experimental validation of transport","No substrate identified","No subcellular localisation determined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"The first evidence that an aat1 locus contributes to antimalarial drug response came from linking a mutation in the P. chabaudi orthologue to the initial step of chloroquine resistance, implicating this gene family in resistance genetics.","evidence":"Linkage group selection genetic cross and quantitative whole-genome resequencing of CQ-resistant P. chabaudi mutants","pmids":["22435897"],"confidence":"Medium","gaps":["Shown in P. chabaudi ortholog, not P. falciparum PF3D7_0629500 directly","Mechanism of resistance not resolved","No transport substrate defined"],"attribution":[{"species":"Plasmodium chabaudi","strain":""}]},{"year":2018,"claim":"To test whether AAT1 itself handles drugs, heterologous reconstitution established it as a functional transporter capable of drug uptake and modulated by a resistance-associated SNP.","evidence":"Heterologous expression in yeast with radiolabelled drug uptake, S258L site-directed mutagenesis, tryptophan competition, and fluorescence localisation to the yeast plasma membrane","pmids":["29410428"],"confidence":"High","gaps":["Localisation and behaviour in a heterologous yeast system may not reflect the native parasite membrane","Physiological substrate in the parasite not identified here","Direct role of drug transport in parasite resistance not demonstrated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2023,"claim":"Combining population genomics with reciprocal genetics resolved how specific pfaat1 alleles are selected, showing S258L potentiates CQ resistance at a fitness cost while F313S restores fitness and reduces resistance, under co-selection with pfcrt K76T.","evidence":"Longitudinal population genomics (The Gambia 1984–2014), parasite genetic crosses / QTL mapping, and CRISPR editing of pfaat1 alleles with in vitro drug sensitivity and fitness assays","pmids":["37169919"],"confidence":"High","gaps":["Molecular mechanism connecting allele to fitness not defined in this study","Native transported substrate not identified","Causal basis of epistasis with pfcrt not mechanistically explained"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"claim":"Direct functional dissection in parasites established the physiological role: PfAAT1 exports/utilises haemoglobin-derived peptides from the digestive vacuole and functionally overlaps with PfCRT, explaining its conditional essentiality and the gain-of-function nature of field variants.","evidence":"Conditional pfaat1 knockdown, isogenic mutant-vs-wild-type pfcrt allele replacement, mass-spectrometry peptide accumulation assays, and growth under amino acid-limiting conditions (preprint)","pmids":["42370240"],"confidence":"High","gaps":["Preprint, not yet peer-reviewed","Direct biochemical demonstration of peptide transport by purified PfAAT1 not shown","Structural basis of S258L/F313S gain-of-function not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The direct molecular substrate specificity of PfAAT1 and the structural mechanism by which S258L and F313S enhance peptide transport remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of PfAAT1","Peptide substrate spectrum not biochemically defined","Mechanistic basis of epistasis with PfCRT not established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFCRT (FUNCTIONAL OVERLAP / EPISTATIC INTERACTION)"],"other_free_text":["Field-selected variants S258L (Africa) and F313S (Southeast Asia) modulate chloroquine resistance and parasite fitness, with S258L co-selected alongside pfcrt K76T [#2, #3].","PfAAT1 mutations do not independently alter chloroquine sensitivity; allele selection is driven by epistasis with pfcrt [#3].","Heterologous yeast reconstitution shows amino acid (tryptophan)-inhibitable drug uptake and drug hypersensitivity reversed by S258L [#0]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"15774027","id":"PMC_15774027","title":"The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum.","date":"2005","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/15774027","citation_count":146,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37169919","id":"PMC_37169919","title":"Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.","date":"2023","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/37169919","citation_count":74,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1","amino acid transporter AAT1","PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22435897","id":"PMC_22435897","title":"Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.","date":"2012","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/22435897","citation_count":34,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36542676","id":"PMC_36542676","title":"Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis.","date":"2022","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/36542676","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29410428","id":"PMC_29410428","title":"Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29410428","citation_count":21,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40247054","id":"PMC_40247054","title":"Disparate co-evolution and prevalence of sulfadoxine and pyrimethamine resistance alleles and haplotypes at dhfr and dhps genes across Africa.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40247054","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37957271","id":"PMC_37957271","title":"Genomics of Plasmodium vivax in Colombia reveals evidence of local bottle-necking and inter-country connectivity in the Americas.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/37957271","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PvAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38012191","id":"PMC_38012191","title":"Genomic analysis of Plasmodium vivax describes patterns of connectivity and putative drivers of adaptation in Ethiopia.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/38012191","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PvAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35904009","id":"PMC_35904009","title":"Plasmodium falciparum merozoite invasion ligands, linked antimalarial resistance loci and ex vivo responses to antimalarials in The Gambia.","date":"2022","source":"The Journal of antimicrobial chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/35904009","citation_count":5,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"39936828","id":"PMC_39936828","title":"Prevalence of Plasmodium falciparum Drug Resistance Markers pfcrt K76T and pfaat1 S258L in Southern Rwanda, 2010 to 2023.","date":"2025","source":"The Journal of infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/39936828","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["AAT1","PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40659734","id":"PMC_40659734","title":"Tracking Plasmodium falciparum antimalarial resistance markers during a malaria pre-elimination period in the Pacific coast of South America.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40659734","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41094545","id":"PMC_41094545","title":"Molecular Markers of Antimalarial Drug Resistance in Plasmodium falciparum From Kumasi, Ghana, 2023.","date":"2025","source":"Tropical medicine & international health : TM & IH","url":"https://pubmed.ncbi.nlm.nih.gov/41094545","citation_count":2,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"39184096","id":"PMC_39184096","title":"Effects of drug pressure and human migration on antimalarial resistance in circulating Plasmodium falciparum malaria parasites in Ecuador.","date":"2024","source":"Research square","url":"https://pubmed.ncbi.nlm.nih.gov/39184096","citation_count":2,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"40164958","id":"PMC_40164958","title":"Temporal Patterns of Haplotypic and Allelic Diversity Reflect the Changing Selection Landscape of the Malaria Parasite Plasmodium falciparum.","date":"2025","source":"Molecular biology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/40164958","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41846600","id":"PMC_41846600","title":"Targeted Genomic Surveillance Unveils Genetic Variations Linked to Regional Malaria Drug Resistance Dynamics in India.","date":"2026","source":"Open forum infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/41846600","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"42370240","id":"PMC_42370240","title":"Gain-of-function PfAAT1 mutations compensate for impaired PfCRT function in Plasmodium falciparum.","date":"2026","source":"Research square","url":"https://pubmed.ncbi.nlm.nih.gov/42370240","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfAAT1"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11561,"output_tokens":1852,"usd":0.031231,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7062,"output_tokens":2744,"usd":0.051955,"stage2_stop_reason":"end_turn"},"total_usd":0.083186,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"AAT1 (PF3D7_0629500) expressed heterologously in yeast functions as a drug transporter: expression produced chloroquine hypersensitivity coincident with increased CQ uptake (~4-fold increased quinine uptake), and sensitized cells to amodiaquine, mefloquine, and quinine. Drug sensitivity was reversed by introducing the S258L SNP linked to CQ resistance. Quinine hypersensitivity was suppressible with tryptophan, consistent with a shared amino acid-inhibitable transport mechanism with the yeast Tat2p transporter. The protein localised primarily to the yeast plasma membrane.\",\n      \"method\": \"Heterologous expression in yeast, drug sensitivity assays, radiolabelled drug uptake assays, site-directed mutagenesis (S258L SNP), tryptophan competition assay, fluorescence localization\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro functional reconstitution in heterologous system with direct drug uptake measurement, mutagenesis confirming SNP reversal, and competition assay establishing amino acid-inhibitable mechanism; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"29410428\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A mutation in aat1 (the P. chabaudi orthologue of PF3D7_0629500) was identified as the critical mutation conferring the first level of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi, mapped by quantitative genome-wide linkage group selection and whole-genome resequencing of CQ-resistant mutants.\",\n      \"method\": \"Linkage group selection genetic cross, quantitative whole-genome resequencing, identification of point mutation at locus of selection valley\",\n      \"journal\": \"BMC genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via linkage group selection cross in P. chabaudi, replicated by quantitative genome resequencing; single lab but two orthogonal genomic approaches\",\n      \"pmids\": [\"22435897\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium chabaudi\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AAT1 S258L potentiates CQ resistance but at a fitness cost, while AAT1 F313S (a common Southeast Asian polymorphism) reduces CQ resistance while restoring fitness. A chromosome 6 quantitative trait locus containing pfaat1 was identified by parasite genetic crosses as being selected by CQ treatment. Population genomics showed temporal selection of pfaat1 S258L in parallel with pfcrt K76T in The Gambia (rising from 0% to 97% between 1984 and 2014). Gene editing confirmed differential effects of S258L and F313S on CQ resistance and parasite fitness.\",\n      \"method\": \"Population genomics (longitudinal temporal analysis), parasite genetic crosses (QTL mapping), CRISPR/gene editing of pfaat1 alleles, in vitro drug sensitivity assays, parasite growth/fitness assays\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic approaches (QTL cross + gene editing), multiple orthogonal methods, replicated across population genomics and experimental genetics in a single study\",\n      \"pmids\": [\"37169919\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PfAAT1 is essential in parasites harboring mutant PfCRT but largely dispensable in a wild-type PfCRT background; replacing mutant pfcrt with wild-type abolished the growth defect from pfaat1 knockdown, indicating functional overlap between PfCRT and PfAAT1. PfAAT1 knockdown causes accumulation of haemoglobin-derived peptides, establishing that PfAAT1 participates in export/utilisation of Hb-derived peptides from the digestive vacuole. Field-derived variants S258L and F313S confer a fitness advantage over wild-type PfAAT1 in the mutant PfCRT background, consistent with gain-of-function enhancement of peptide transport to compensate for impaired PfCRT. F313S imparts pronounced fitness advantage under cysteine- and methionine-limiting conditions. PfAAT1 mutations do not independently alter CQ sensitivity; selection of pfaat1 alleles in the field is driven primarily by epistatic interaction with pfcrt.\",\n      \"method\": \"Conditional knockdown of pfaat1, isogenic parasite line allele replacement (mutant vs wild-type pfcrt), peptide accumulation assays (mass spectrometry), comparative growth analyses under amino acid-limiting conditions, genetic epistasis analysis\",\n      \"journal\": \"Research square\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — knockdown with defined molecular phenotype (peptide accumulation), allele replacement epistasis, amino acid limitation experiments; multiple orthogonal methods in single study; preprint but mechanistically detailed\",\n      \"pmids\": [\"42370240\"],\n      \"is_preprint\": true,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Bioinformatic analysis of the P. falciparum genome identified PF3D7_0629500 (AAT1) as a putative amino acid transporter based on hydropathy plot analysis and sequence homology, assigning it to the amino acid transporter family.\",\n      \"method\": \"Computational genome-wide hydropathy plot analysis and sequence homology assignment\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational prediction only, no experimental validation in this paper\",\n      \"pmids\": [\"15774027\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"PfAAT1 (PF3D7_0629500) is a digestive vacuole-associated amino acid transporter in P. falciparum that mediates export of haemoglobin-derived peptides from the digestive vacuole; it is largely dispensable when PfCRT is functional but becomes essential when PfCRT is mutated (K76T), because mutant PfCRT loses its native peptide export activity, and field-selected PfAAT1 variants (S258L in Africa; F313S in Southeast Asia) represent gain-of-function mutations that compensate for impaired PfCRT peptide transport, thereby restoring parasite fitness at the cost of (S258L) or with restoration of (F313S) CQ sensitivity, with allele selection driven primarily by epistatic interaction with pfcrt rather than by direct effects on CQ transport.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AAT1 (PF3D7_0629500) is a digestive vacuole-associated amino acid transporter of Plasmodium falciparum that participates in the export and utilisation of haemoglobin-derived peptides from the digestive vacuole [#3]. Conditional knockdown of PfAAT1 causes accumulation of Hb-derived peptides and reveals a functional overlap with PfCRT: AAT1 is largely dispensable when PfCRT is wild-type but becomes essential in a mutant PfCRT background, and restoring wild-type pfcrt abolishes the growth defect of pfaat1 knockdown [#3]. Field-selected variants act as gain-of-function alleles that compensate for impaired PfCRT peptide transport — S258L and F313S confer a fitness advantage in the mutant PfCRT background, with F313S particularly favoured under cysteine- and methionine-limiting conditions [#3], and their selection in the field is driven by epistatic interaction with pfcrt rather than by direct effects on chloroquine transport [#3]. Consistent with a role in drug handling, heterologous expression of AAT1 in yeast produces chloroquine hypersensitivity and increased CQ and quinine uptake in an amino acid-inhibitable manner reversed by the S258L SNP [#0], and population genomics documents temporal co-selection of pfaat1 S258L alongside pfcrt K76T [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Before any functional data, it was unknown whether PF3D7_0629500 encoded a transporter at all; genome annotation placed it within a candidate gene family to be tested.\",\n      \"evidence\": \"Genome-wide computational hydropathy and sequence-homology analysis of the P. falciparum genome\",\n      \"pmids\": [\"15774027\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational prediction only, no experimental validation of transport\", \"No substrate identified\", \"No subcellular localisation determined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The first evidence that an aat1 locus contributes to antimalarial drug response came from linking a mutation in the P. chabaudi orthologue to the initial step of chloroquine resistance, implicating this gene family in resistance genetics.\",\n      \"evidence\": \"Linkage group selection genetic cross and quantitative whole-genome resequencing of CQ-resistant P. chabaudi mutants\",\n      \"pmids\": [\"22435897\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Shown in P. chabaudi ortholog, not P. falciparum PF3D7_0629500 directly\", \"Mechanism of resistance not resolved\", \"No transport substrate defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium chabaudi\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"To test whether AAT1 itself handles drugs, heterologous reconstitution established it as a functional transporter capable of drug uptake and modulated by a resistance-associated SNP.\",\n      \"evidence\": \"Heterologous expression in yeast with radiolabelled drug uptake, S258L site-directed mutagenesis, tryptophan competition, and fluorescence localisation to the yeast plasma membrane\",\n      \"pmids\": [\"29410428\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Localisation and behaviour in a heterologous yeast system may not reflect the native parasite membrane\", \"Physiological substrate in the parasite not identified here\", \"Direct role of drug transport in parasite resistance not demonstrated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Combining population genomics with reciprocal genetics resolved how specific pfaat1 alleles are selected, showing S258L potentiates CQ resistance at a fitness cost while F313S restores fitness and reduces resistance, under co-selection with pfcrt K76T.\",\n      \"evidence\": \"Longitudinal population genomics (The Gambia 1984–2014), parasite genetic crosses / QTL mapping, and CRISPR editing of pfaat1 alleles with in vitro drug sensitivity and fitness assays\",\n      \"pmids\": [\"37169919\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism connecting allele to fitness not defined in this study\", \"Native transported substrate not identified\", \"Causal basis of epistasis with pfcrt not mechanistically explained\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Direct functional dissection in parasites established the physiological role: PfAAT1 exports/utilises haemoglobin-derived peptides from the digestive vacuole and functionally overlaps with PfCRT, explaining its conditional essentiality and the gain-of-function nature of field variants.\",\n      \"evidence\": \"Conditional pfaat1 knockdown, isogenic mutant-vs-wild-type pfcrt allele replacement, mass-spectrometry peptide accumulation assays, and growth under amino acid-limiting conditions (preprint)\",\n      \"pmids\": [\"42370240\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Direct biochemical demonstration of peptide transport by purified PfAAT1 not shown\", \"Structural basis of S258L/F313S gain-of-function not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct molecular substrate specificity of PfAAT1 and the structural mechanism by which S258L and F313S enhance peptide transport remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of PfAAT1\", \"Peptide substrate spectrum not biochemically defined\", \"Mechanistic basis of epistasis with PfCRT not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0015171\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0015197\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0015238\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020020\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0046323\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0006508\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0042910\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PfCRT (functional overlap / epistatic interaction)\"],\n    \"other_free_text\": [\n      \"Field-selected variants S258L (Africa) and F313S (Southeast Asia) modulate chloroquine resistance and parasite fitness, with S258L co-selected alongside pfcrt K76T [#2, #3].\",\n      \"PfAAT1 mutations do not independently alter chloroquine sensitivity; allele selection is driven by epistasis with pfcrt [#3].\",\n      \"Heterologous yeast reconstitution shows amino acid (tryptophan)-inhibitable drug uptake and drug hypersensitivity reversed by S258L [#0].\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0629500","name":null,"product":"amino acid transporter AAT1","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1128300","name":null,"product":"amino acid transporter AAT1, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1120000","name":null,"product":"amino acid transporter AAT1, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"AAT1","evaluation":null}