{"gene":"PF3D7_0302500","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2011,"finding":"CLAG3.1 (PF3D7_0302500) and its paralog clag3.2 are the parasite genes responsible for PSAC (plasmodial surface anion channel) activity at the host erythrocyte membrane; DNA transfection and in vitro selection experiments demonstrated that PSAC-inhibitor interactions are encoded by the clag3 gene family, and the encoded protein inserts as an integral protein in the host membrane, increasing host cell permeability to diverse solutes.","method":"High-throughput nutrient-uptake inhibitor screening, genetic cross linkage analysis (Dd2 × HB3), DNA transfection, in vitro selections, inhibitor specificity assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic cross linkage, transfection, in vitro selection, functional transport assays) converging on the same conclusion in a single rigorous study","pmids":["21620134"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2001,"finding":"CLAG3.1 (PF3D7_0302500; encoded by PFC0120w on chromosome 3) is identified as a component of the high-molecular-mass RhopH1 subunit of the merozoite rhoptry RhopH complex; peptide sequencing by mass spectrometry and Edman degradation of monoclonal antibody-purified RhopH complex identified chromosome-3 clag family members as encoding RhopH1 in P. falciparum.","method":"Monoclonal antibody purification of RhopH complex, peptide mass spectrometry, Edman degradation sequencing","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical identification of peptides from purified complex, but CLAG3.1 vs. CLAG3.2 not individually distinguished within chromosome-3 members","pmids":["11738712"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"CLAG3.1 protein localizes to the apical end of merozoites in segmented schizonts and is a component of the PfRhopH complex; immunoprecipitation with anti-Clag9 antibody did not co-precipitate CLAG3.1, indicating that individual PfRhopH complexes contain only one RhopH1/clag gene product at a time.","method":"Specific antisera localization (immunofluorescence), immunoprecipitation of PfRhopH complex with anti-Clag9, anti-Clag2, and anti-Clag3.1 antibodies","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal immunoprecipitation and localization experiments, single lab, two orthogonal methods","pmids":["15953647"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"HB3, 3D7, Dd2"}]},{"year":2013,"finding":"CLAG3.1 and CLAG3.2 undergo mutually exclusive, epigenetically regulated expression switching; selection with low-dose blasticidin S caused parasites to switch from clag3.2 to clag3.1 expression, demonstrating that the two clag3 isoforms confer different solute transport efficiencies to PSAC. Simultaneous silencing of both clag3 genes severely reduced PSAC-mediated uptake. Switches were not accompanied by genetic mutations at the clag3 loci.","method":"Drug selection (blasticidin S at varying concentrations), solute uptake assays, RT-PCR expression analysis, whole-genome analysis for mutations","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional transport readout combined with expression switching and gene-silencing analysis, single lab","pmids":["23819786"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"Silencing of clag3 paralogs (including clag3.1) is associated with enrichment of heterochromatin-associated histone modifications, as shown by chromatin immunoprecipitation. Parasites with both clag3.1 and clag3.2 silenced (clag3-null) exhibit significant growth inhibition, providing genetic evidence that clag3 gene products are required for efficient parasite proliferation.","method":"Chromatin immunoprecipitation (ChIP) for histone modifications, gene disruption of clag3.2 locus, drug-selection-mediated silencing of clag3.1, growth assays","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic disruption with defined growth phenotype and ChIP evidence for epigenetic mechanism, single lab","pmids":["21320181"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"finding":"CLAG3.1 (PF3D7_0302500) undergoes self-association (oligomerization) at the host erythrocyte membrane. Engineering parasites to express two CLAG3 isoforms simultaneously showed stoichiometric trafficking of both to the host membrane and yielded intermediate PSAC channel phenotypes. Co-immunoprecipitation and surface labeling confirmed CLAG3 oligomer formation. In vitro selections produced mutants with correlated changes in CLAG3 sequence and channel activity, supporting CLAG3 oligomers as the channel-forming unit.","method":"Transfection to co-express two CLAG3 isoforms, co-immunoprecipitation, surface biotin labeling, electrophysiological patch-clamp (PSAC phenotype), in vitro inhibitor selection","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, surface labeling, functional channel assays, in vitro selection with correlated sequence/phenotype changes) in a single rigorous study","pmids":["29739907"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"CLAG3 conditional knockdown in a genetic cross where one parent carries a single clag3 gene confirmed CLAG3's primary role in PSAC nutrient transport. However, complete CLAG3 knockout did not abolish transport activity under standard culture conditions (unlike RhopH2/RhopH3 knockouts), but the CLAG3 knockout parasite failed to propagate in medium resembling human plasma, indicating CLAG3 is required under physiological nutrient conditions. These results implicate oligomerization of CLAG paralogs from other chromosomes in channel formation.","method":"Conditional knockdown (TetR-DOZI aptamer system), complete knockout by genetic disruption, linkage analysis in P. falciparum genetic cross, nutrient/solute uptake assays, propagation in modified (physiological) medium","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic cross linkage, conditional knockdown, and complete knockout with multiple functional readouts in one rigorous study","pmids":["32069335"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"Live-cell FRET imaging showed that CLAG3 and RhopH2 remain physically associated from rhoptry organelles through merozoite egress, parasitophorous vacuole deposition, and export to the infected erythrocyte membrane; the two proteins traffic together without dissociation throughout this entire journey. Whole-cell patch-clamp demonstrated that doubly-infected erythrocytes have twice the PSAC functional copy number, establishing a quantitative (stoichiometric) relationship between RhopH complex copy number at the membrane and channel activity.","method":"Live-cell FRET (mCerulean/mVenus-tagged CLAG3 and RhopH2), fluorescence imaging of intracellular trafficking, whole-cell patch-clamp electrophysiology","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — FRET (direct protein-protein interaction measurement in living cells) combined with electrophysiology and quantitative imaging, multiple orthogonal methods in one rigorous study","pmids":["32900800"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"During merozoite invasion, CLAG3.1 (as the RhopH complex component Clag3) is released directly into the host cell cytoplasm, while RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Assembly of the Clag3/RhopH2/RhopH3 complex occurs only after export of RhopH2 and RhopH3 from the parasitophorous vacuole into the erythrocyte cytoplasm, followed by incorporation into the host cell membrane concomitant with nutrient uptake activation. This compartmentalization prevents premature channel assembly.","method":"Super-resolution microscopy, conditional knockdown, immunofluorescence localization, co-immunoprecipitation during defined invasion stages","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — super-resolution imaging, conditional KD, and co-IP at defined trafficking stages with multiple orthogonal methods establishing assembly mechanism","pmids":["35393572"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"Biochemical fractionation and native PAGE of CLAG3.1-expressing infected erythrocyte membranes identified at least three distinct high-molecular-weight complexes containing CLAG3: ~720 kDa (CLAG3 + RhopH2 + RhopH3), ~620 kDa (CLAG3 + RhopH2), and ~480 kDa (CLAG3 alone, likely a homo-oligomer). Extracellular biotin labeling and protease susceptibility studies localized the ~480 kDa CLAG3-only complex to the erythrocyte membrane surface, suggesting it may represent the functional pore unit of PSAC.","method":"Co-immunoprecipitation from membrane fractions, native PAGE, mass spectrometry, extracellular biotin labeling, protease protection/susceptibility assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods (native PAGE, MS, biotin surface labeling, protease assay), single lab, no functional reconstitution","pmids":["27299521"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"The N-terminal one-third of CLAG3.1, which contains a conserved domain shared with Toxoplasma gondii RON2, cannot associate with the RhopH complex when expressed as a GFP chimera; however, a c-Myc-CLAG3.1 chimera lacking the C-terminus successfully associates with the RhopH complex, indicating that the middle region of CLAG3.1 is required for RhopH complex association but the C-terminus is not necessary.","method":"Transgene expression constructs in P. falciparum, co-immunoprecipitation of chimeric proteins with RhopH complex","journal":"Parasitology international","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — domain deletion/chimera analysis with co-IP, single lab, limited follow-up","pmids":["17175193"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"Synthetic peptides spanning the CLAG3.1 sequence were tested in cell-binding assays; twelve peptides (HABPs) bound C32 cells (which express CD36) with high affinity (Kd 100–800 nM), and five of these also bound erythrocytes. Cross-linking and SDS-PAGE identified erythrocyte-binding proteins of ~26 kDa and ~59 kDa and a ~53 kDa protein as a possible receptor on C32 cells for CLAG3.1 peptides.","method":"Synthetic peptide binding assays (C32 cells and erythrocytes), affinity constant determination, cross-linking, SDS-PAGE","journal":"Protein science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — peptide binding assays with putative receptor identification, no in-parasite validation, single lab","pmids":["15659379"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"FCB2"}]}],"current_model":"CLAG3.1 (PF3D7_0302500) is an integral host erythrocyte membrane protein that functions as the primary determinant of the plasmodial surface anion channel (PSAC): it is synthesized in merozoite rhoptries as part of the RhopH complex (with RhopH2 and RhopH3), released directly into the host cell cytoplasm during invasion (while RhopH2/3 enter the parasitophorous vacuole), reassembles with exported RhopH2/RhopH3 in the erythrocyte cytoplasm, and inserts into the host membrane as a CLAG3.1 homo-oligomer (and higher-order complexes with RhopH2/RhopH3) that forms the broad-selectivity nutrient pore; expression of clag3.1 versus clag3.2 is mutually exclusive and epigenetically regulated, and the isoform expressed determines PSAC pharmacological and transport properties."},"narrative":{"mechanistic_narrative":"CLAG3.1 (PF3D7_0302500) is a parasite-encoded determinant of the plasmodial surface anion channel (PSAC), the broad-selectivity nutrient conduit that plasmodial infection installs in the host erythrocyte membrane [PMID:21620134]. Genetic cross linkage, DNA transfection, and in vitro inhibitor selections establish that the clag3 gene family encodes PSAC activity and that the CLAG3.1 protein inserts as an integral component of the host membrane to raise permeability to diverse solutes [PMID:21620134], with in-parasite mutants showing correlated changes in CLAG3 sequence and channel pharmacology [PMID:29739907]. CLAG3.1 is synthesized as the RhopH1 subunit of the merozoite rhoptry RhopH complex together with RhopH2 and RhopH3 [PMID:11738712, PMID:15953647], and during invasion it is released directly into the host cytoplasm while RhopH2/RhopH3 enter the parasitophorous vacuole; only after those partners are exported to the erythrocyte cytoplasm does the Clag3/RhopH2/RhopH3 complex assemble and insert into the host membrane, a compartmentalization that prevents premature channel assembly [PMID:35393572]. CLAG3.1 and RhopH2 remain physically associated throughout this trafficking route, and channel activity scales stoichiometrically with RhopH complex copy number at the membrane [PMID:32900800]. At the surface, CLAG3.1 self-associates into oligomers, and biochemical fractionation resolves distinct complexes including a surface-exposed CLAG3-only assembly implicated as the pore-forming unit [PMID:29739907, PMID:27299521]. Expression of clag3.1 versus its paralog clag3.2 is mutually exclusive and epigenetically controlled through heterochromatin-associated histone modifications, and the isoform expressed dictates PSAC solute-transport and pharmacological properties [PMID:23819786, PMID:21320181]; clag3-null parasites are severely growth-impaired, and CLAG3 is required for propagation under physiological plasma-like nutrient conditions [PMID:21320181, PMID:32069335]. The middle region of CLAG3.1 is required for RhopH complex association while its C-terminus is dispensable [PMID:17175193].","teleology":[{"year":2001,"claim":"Establishing the molecular identity of the RhopH1 subunit answered which gene product occupies the high-molecular-mass position of the merozoite rhoptry RhopH complex.","evidence":"monoclonal-antibody purification of the RhopH complex followed by peptide mass spectrometry and Edman sequencing in P. falciparum","pmids":["11738712"],"confidence":"Medium","gaps":["CLAG3.1 not distinguished from CLAG3.2 within chromosome-3 members","no functional role assigned"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"Localization and reciprocal immunoprecipitation established that CLAG3.1 sits at the apical merozoite end and that each RhopH complex contains only a single clag/RhopH1 gene product.","evidence":"specific antisera immunofluorescence and immunoprecipitation with anti-Clag9/Clag2/Clag3.1 antibodies in HB3, 3D7, Dd2","pmids":["15953647"],"confidence":"Medium","gaps":["did not test whether one-clag-per-complex rule applies at the host membrane","no channel-function link"],"attribution":[{"species":"Plasmodium falciparum","strain":"HB3, 3D7, Dd2"}]},{"year":2005,"claim":"Peptide-binding surveys attempted to identify host-cell receptors for CLAG3.1 sequences, addressing whether the protein has adhesive/receptor-binding activity.","evidence":"synthetic peptide (HABP) binding to CD36-expressing C32 cells and erythrocytes with cross-linking and SDS-PAGE in strain FCB2","pmids":["15659379"],"confidence":"Low","gaps":["not validated in intact parasites","putative receptors of ~26/53/59 kDa not molecularly identified","relevance to PSAC function unestablished"],"attribution":[{"species":"Plasmodium falciparum","strain":"FCB2"}]},{"year":2006,"claim":"Domain-mapping defined which region of CLAG3.1 mediates incorporation into the RhopH complex.","evidence":"GFP/c-Myc chimera deletion constructs with co-immunoprecipitation in P. falciparum","pmids":["17175193"],"confidence":"Medium","gaps":["precise interacting residues not mapped","single lab, limited follow-up","no structural basis for association"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"Genetic mapping resolved the long-standing question of what parasite gene encodes PSAC, assigning channel activity and inhibitor specificity to the clag3 family.","evidence":"high-throughput nutrient-uptake inhibitor screening, Dd2 x HB3 cross linkage, DNA transfection, and in vitro selections","pmids":["21620134"],"confidence":"High","gaps":["did not resolve whether CLAG3 alone forms the pore or requires partners","structure of the channel unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2011,"claim":"Silencing and disruption studies established that clag3 expression is epigenetically controlled by heterochromatin and that clag3 gene products are needed for efficient proliferation.","evidence":"ChIP for histone modifications, clag3.2 disruption plus drug-selection silencing of clag3.1, and growth assays in P. falciparum","pmids":["21320181"],"confidence":"Medium","gaps":["mechanism linking heterochromatin to mutually exclusive choice not defined","growth requirement not tied to a specific transport substrate"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Expression switching between clag3.1 and clag3.2 was shown to be mutually exclusive, epigenetic, and functionally consequential for solute transport.","evidence":"blasticidin S selection, solute uptake assays, RT-PCR, and whole-genome mutation analysis in P. falciparum","pmids":["23819786"],"confidence":"Medium","gaps":["molecular trigger of switching not identified","structural basis for isoform-specific transport differences unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"claim":"Native biochemistry resolved distinct CLAG3-containing membrane complexes and localized a surface-exposed CLAG3-only assembly as a candidate functional pore unit.","evidence":"membrane co-IP, native PAGE, mass spectrometry, extracellular biotin labeling, and protease-protection assays in P. falciparum","pmids":["27299521"],"confidence":"Medium","gaps":["no functional reconstitution of the ~480 kDa complex","stoichiometry of the oligomer not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"claim":"Self-association of CLAG3 was demonstrated and linked to channel formation, addressing whether CLAG3 oligomers constitute the transport unit.","evidence":"co-expression of two CLAG3 isoforms with co-IP, surface biotin labeling, patch-clamp, and in vitro selection yielding correlated sequence/phenotype changes","pmids":["29739907"],"confidence":"High","gaps":["atomic structure of the oligomer not determined","exact number of subunits per pore unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Conditional knockdown and complete knockout dissected CLAG3's necessity, showing it is the primary PSAC determinant yet dispensable for residual transport in standard culture but essential under physiological plasma-like conditions.","evidence":"TetR-DOZI conditional knockdown, complete knockout, genetic-cross linkage, and uptake assays in modified medium in P. falciparum","pmids":["32069335"],"confidence":"High","gaps":["identity of paralogs compensating in knockout not resolved","molecular basis of the physiological-medium requirement unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Live-cell FRET established that CLAG3 and RhopH2 traffic together without dissociation and that channel activity scales quantitatively with membrane complex copy number.","evidence":"FRET imaging of tagged CLAG3/RhopH2 and whole-cell patch-clamp of doubly-infected erythrocytes in P. falciparum","pmids":["32900800"],"confidence":"High","gaps":["timing of oligomerization relative to trafficking not fully resolved","does not define membrane-insertion mechanism"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"Compartmentalized release and staged assembly were defined, explaining how premature channel formation is avoided during invasion.","evidence":"super-resolution microscopy, conditional knockdown, immunofluorescence, and stage-defined co-IP in P. falciparum","pmids":["35393572"],"confidence":"High","gaps":["export machinery moving RhopH2/RhopH3 across the PV membrane not identified","signal controlling final membrane insertion unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The atomic structure of the CLAG3.1 oligomeric pore and the mechanism of its insertion into the host erythrocyte membrane remain undefined.","evidence":"","pmids":[],"confidence":"High","gaps":["no high-resolution structure of the channel-forming CLAG3 oligomer","membrane-insertion mechanism and topology not resolved","how isoform sequence dictates transport selectivity structurally unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["RhopH complex (CLAG3.1/RhopH1 + RhopH2 + RhopH3), ~720 kDa","CLAG3 + RhopH2 subcomplex, ~620 kDa","CLAG3-only homo-oligomer, ~480 kDa (candidate PSAC pore unit)"],"partners":["RHOPH2","RHOPH3"],"other_free_text":["CLAG3.1 is the RhopH1 subunit of the merozoite rhoptry RhopH complex [#1, #2].","clag3.1 and clag3.2 are expressed in a mutually exclusive, epigenetically switched manner; the expressed isoform sets PSAC transport and pharmacology [#3, #4].","The middle region of CLAG3.1 is required for RhopH complex association; the C-terminus is dispensable; the N-terminal third contains a domain shared with T. gondii RON2 [#10].","clag3-null parasites show severe growth inhibition, and CLAG3 is required for propagation under physiological plasma-like nutrient conditions [#4, #6].","Complete CLAG3 knockout did not abolish residual transport in standard culture, implicating other-chromosome CLAG paralogs in compensatory channel formation [#6]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"21620134","id":"PMC_21620134","title":"Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells.","date":"2011","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21620134","citation_count":205,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"11738712","id":"PMC_11738712","title":"The high molecular mass rhoptry protein, RhopH1, is encoded by members of the clag multigene family in Plasmodium falciparum and Plasmodium yoelii.","date":"2001","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/11738712","citation_count":72,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PyRhopH1A"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15953647","id":"PMC_15953647","title":"Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15953647","citation_count":68,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PFC0120w"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23819786","id":"PMC_23819786","title":"Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites.","date":"2013","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23819786","citation_count":63,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21320181","id":"PMC_21320181","title":"Functional analysis of epigenetic regulation of tandem RhopH1/clag genes reveals a role in Plasmodium falciparum growth.","date":"2011","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21320181","citation_count":46,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18155305","id":"PMC_18155305","title":"Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum.","date":"2007","source":"Molecular and biochemical 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research","url":"https://pubmed.ncbi.nlm.nih.gov/22782471","citation_count":2,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PFC0120w"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10165,"output_tokens":3951,"usd":0.04488,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10002,"output_tokens":4798,"usd":0.08498,"stage2_stop_reason":"end_turn"},"total_usd":0.12986,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"CLAG3.1 (PF3D7_0302500) and its paralog clag3.2 are the parasite genes responsible for PSAC (plasmodial surface anion channel) activity at the host erythrocyte membrane; DNA transfection and in vitro selection experiments demonstrated that PSAC-inhibitor interactions are encoded by the clag3 gene family, and the encoded protein inserts as an integral protein in the host membrane, increasing host cell permeability to diverse solutes.\",\n      \"method\": \"High-throughput nutrient-uptake inhibitor screening, genetic cross linkage analysis (Dd2 × HB3), DNA transfection, in vitro selections, inhibitor specificity assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic cross linkage, transfection, in vitro selection, functional transport assays) converging on the same conclusion in a single rigorous study\",\n      \"pmids\": [\"21620134\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CLAG3.1 (PF3D7_0302500; encoded by PFC0120w on chromosome 3) is identified as a component of the high-molecular-mass RhopH1 subunit of the merozoite rhoptry RhopH complex; peptide sequencing by mass spectrometry and Edman degradation of monoclonal antibody-purified RhopH complex identified chromosome-3 clag family members as encoding RhopH1 in P. falciparum.\",\n      \"method\": \"Monoclonal antibody purification of RhopH complex, peptide mass spectrometry, Edman degradation sequencing\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical identification of peptides from purified complex, but CLAG3.1 vs. CLAG3.2 not individually distinguished within chromosome-3 members\",\n      \"pmids\": [\"11738712\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CLAG3.1 protein localizes to the apical end of merozoites in segmented schizonts and is a component of the PfRhopH complex; immunoprecipitation with anti-Clag9 antibody did not co-precipitate CLAG3.1, indicating that individual PfRhopH complexes contain only one RhopH1/clag gene product at a time.\",\n      \"method\": \"Specific antisera localization (immunofluorescence), immunoprecipitation of PfRhopH complex with anti-Clag9, anti-Clag2, and anti-Clag3.1 antibodies\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal immunoprecipitation and localization experiments, single lab, two orthogonal methods\",\n      \"pmids\": [\"15953647\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"HB3, 3D7, Dd2\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CLAG3.1 and CLAG3.2 undergo mutually exclusive, epigenetically regulated expression switching; selection with low-dose blasticidin S caused parasites to switch from clag3.2 to clag3.1 expression, demonstrating that the two clag3 isoforms confer different solute transport efficiencies to PSAC. Simultaneous silencing of both clag3 genes severely reduced PSAC-mediated uptake. Switches were not accompanied by genetic mutations at the clag3 loci.\",\n      \"method\": \"Drug selection (blasticidin S at varying concentrations), solute uptake assays, RT-PCR expression analysis, whole-genome analysis for mutations\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional transport readout combined with expression switching and gene-silencing analysis, single lab\",\n      \"pmids\": [\"23819786\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Silencing of clag3 paralogs (including clag3.1) is associated with enrichment of heterochromatin-associated histone modifications, as shown by chromatin immunoprecipitation. Parasites with both clag3.1 and clag3.2 silenced (clag3-null) exhibit significant growth inhibition, providing genetic evidence that clag3 gene products are required for efficient parasite proliferation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for histone modifications, gene disruption of clag3.2 locus, drug-selection-mediated silencing of clag3.1, growth assays\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic disruption with defined growth phenotype and ChIP evidence for epigenetic mechanism, single lab\",\n      \"pmids\": [\"21320181\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CLAG3.1 (PF3D7_0302500) undergoes self-association (oligomerization) at the host erythrocyte membrane. Engineering parasites to express two CLAG3 isoforms simultaneously showed stoichiometric trafficking of both to the host membrane and yielded intermediate PSAC channel phenotypes. Co-immunoprecipitation and surface labeling confirmed CLAG3 oligomer formation. In vitro selections produced mutants with correlated changes in CLAG3 sequence and channel activity, supporting CLAG3 oligomers as the channel-forming unit.\",\n      \"method\": \"Transfection to co-express two CLAG3 isoforms, co-immunoprecipitation, surface biotin labeling, electrophysiological patch-clamp (PSAC phenotype), in vitro inhibitor selection\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, surface labeling, functional channel assays, in vitro selection with correlated sequence/phenotype changes) in a single rigorous study\",\n      \"pmids\": [\"29739907\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLAG3 conditional knockdown in a genetic cross where one parent carries a single clag3 gene confirmed CLAG3's primary role in PSAC nutrient transport. However, complete CLAG3 knockout did not abolish transport activity under standard culture conditions (unlike RhopH2/RhopH3 knockouts), but the CLAG3 knockout parasite failed to propagate in medium resembling human plasma, indicating CLAG3 is required under physiological nutrient conditions. These results implicate oligomerization of CLAG paralogs from other chromosomes in channel formation.\",\n      \"method\": \"Conditional knockdown (TetR-DOZI aptamer system), complete knockout by genetic disruption, linkage analysis in P. falciparum genetic cross, nutrient/solute uptake assays, propagation in modified (physiological) medium\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic cross linkage, conditional knockdown, and complete knockout with multiple functional readouts in one rigorous study\",\n      \"pmids\": [\"32069335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Live-cell FRET imaging showed that CLAG3 and RhopH2 remain physically associated from rhoptry organelles through merozoite egress, parasitophorous vacuole deposition, and export to the infected erythrocyte membrane; the two proteins traffic together without dissociation throughout this entire journey. Whole-cell patch-clamp demonstrated that doubly-infected erythrocytes have twice the PSAC functional copy number, establishing a quantitative (stoichiometric) relationship between RhopH complex copy number at the membrane and channel activity.\",\n      \"method\": \"Live-cell FRET (mCerulean/mVenus-tagged CLAG3 and RhopH2), fluorescence imaging of intracellular trafficking, whole-cell patch-clamp electrophysiology\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — FRET (direct protein-protein interaction measurement in living cells) combined with electrophysiology and quantitative imaging, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"32900800\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"During merozoite invasion, CLAG3.1 (as the RhopH complex component Clag3) is released directly into the host cell cytoplasm, while RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Assembly of the Clag3/RhopH2/RhopH3 complex occurs only after export of RhopH2 and RhopH3 from the parasitophorous vacuole into the erythrocyte cytoplasm, followed by incorporation into the host cell membrane concomitant with nutrient uptake activation. This compartmentalization prevents premature channel assembly.\",\n      \"method\": \"Super-resolution microscopy, conditional knockdown, immunofluorescence localization, co-immunoprecipitation during defined invasion stages\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — super-resolution imaging, conditional KD, and co-IP at defined trafficking stages with multiple orthogonal methods establishing assembly mechanism\",\n      \"pmids\": [\"35393572\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Biochemical fractionation and native PAGE of CLAG3.1-expressing infected erythrocyte membranes identified at least three distinct high-molecular-weight complexes containing CLAG3: ~720 kDa (CLAG3 + RhopH2 + RhopH3), ~620 kDa (CLAG3 + RhopH2), and ~480 kDa (CLAG3 alone, likely a homo-oligomer). Extracellular biotin labeling and protease susceptibility studies localized the ~480 kDa CLAG3-only complex to the erythrocyte membrane surface, suggesting it may represent the functional pore unit of PSAC.\",\n      \"method\": \"Co-immunoprecipitation from membrane fractions, native PAGE, mass spectrometry, extracellular biotin labeling, protease protection/susceptibility assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods (native PAGE, MS, biotin surface labeling, protease assay), single lab, no functional reconstitution\",\n      \"pmids\": [\"27299521\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal one-third of CLAG3.1, which contains a conserved domain shared with Toxoplasma gondii RON2, cannot associate with the RhopH complex when expressed as a GFP chimera; however, a c-Myc-CLAG3.1 chimera lacking the C-terminus successfully associates with the RhopH complex, indicating that the middle region of CLAG3.1 is required for RhopH complex association but the C-terminus is not necessary.\",\n      \"method\": \"Transgene expression constructs in P. falciparum, co-immunoprecipitation of chimeric proteins with RhopH complex\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — domain deletion/chimera analysis with co-IP, single lab, limited follow-up\",\n      \"pmids\": [\"17175193\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Synthetic peptides spanning the CLAG3.1 sequence were tested in cell-binding assays; twelve peptides (HABPs) bound C32 cells (which express CD36) with high affinity (Kd 100–800 nM), and five of these also bound erythrocytes. Cross-linking and SDS-PAGE identified erythrocyte-binding proteins of ~26 kDa and ~59 kDa and a ~53 kDa protein as a possible receptor on C32 cells for CLAG3.1 peptides.\",\n      \"method\": \"Synthetic peptide binding assays (C32 cells and erythrocytes), affinity constant determination, cross-linking, SDS-PAGE\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — peptide binding assays with putative receptor identification, no in-parasite validation, single lab\",\n      \"pmids\": [\"15659379\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"FCB2\"}]\n    }\n  ],\n  \"current_model\": \"CLAG3.1 (PF3D7_0302500) is an integral host erythrocyte membrane protein that functions as the primary determinant of the plasmodial surface anion channel (PSAC): it is synthesized in merozoite rhoptries as part of the RhopH complex (with RhopH2 and RhopH3), released directly into the host cell cytoplasm during invasion (while RhopH2/3 enter the parasitophorous vacuole), reassembles with exported RhopH2/RhopH3 in the erythrocyte cytoplasm, and inserts into the host membrane as a CLAG3.1 homo-oligomer (and higher-order complexes with RhopH2/RhopH3) that forms the broad-selectivity nutrient pore; expression of clag3.1 versus clag3.2 is mutually exclusive and epigenetically regulated, and the isoform expressed determines PSAC pharmacological and transport properties.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLAG3.1 (PF3D7_0302500) is a parasite-encoded determinant of the plasmodial surface anion channel (PSAC), the broad-selectivity nutrient conduit that plasmodial infection installs in the host erythrocyte membrane [#0]. Genetic cross linkage, DNA transfection, and in vitro inhibitor selections establish that the clag3 gene family encodes PSAC activity and that the CLAG3.1 protein inserts as an integral component of the host membrane to raise permeability to diverse solutes [#0], with in-parasite mutants showing correlated changes in CLAG3 sequence and channel pharmacology [#5]. CLAG3.1 is synthesized as the RhopH1 subunit of the merozoite rhoptry RhopH complex together with RhopH2 and RhopH3 [#1, #2], and during invasion it is released directly into the host cytoplasm while RhopH2/RhopH3 enter the parasitophorous vacuole; only after those partners are exported to the erythrocyte cytoplasm does the Clag3/RhopH2/RhopH3 complex assemble and insert into the host membrane, a compartmentalization that prevents premature channel assembly [#8]. CLAG3.1 and RhopH2 remain physically associated throughout this trafficking route, and channel activity scales stoichiometrically with RhopH complex copy number at the membrane [#7]. At the surface, CLAG3.1 self-associates into oligomers, and biochemical fractionation resolves distinct complexes including a surface-exposed CLAG3-only assembly implicated as the pore-forming unit [#5, #9]. Expression of clag3.1 versus its paralog clag3.2 is mutually exclusive and epigenetically controlled through heterochromatin-associated histone modifications, and the isoform expressed dictates PSAC solute-transport and pharmacological properties [#3, #4]; clag3-null parasites are severely growth-impaired, and CLAG3 is required for propagation under physiological plasma-like nutrient conditions [#4, #6]. The middle region of CLAG3.1 is required for RhopH complex association while its C-terminus is dispensable [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing the molecular identity of the RhopH1 subunit answered which gene product occupies the high-molecular-mass position of the merozoite rhoptry RhopH complex.\",\n      \"evidence\": \"monoclonal-antibody purification of the RhopH complex followed by peptide mass spectrometry and Edman sequencing in P. falciparum\",\n      \"pmids\": [\"11738712\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CLAG3.1 not distinguished from CLAG3.2 within chromosome-3 members\", \"no functional role assigned\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Localization and reciprocal immunoprecipitation established that CLAG3.1 sits at the apical merozoite end and that each RhopH complex contains only a single clag/RhopH1 gene product.\",\n      \"evidence\": \"specific antisera immunofluorescence and immunoprecipitation with anti-Clag9/Clag2/Clag3.1 antibodies in HB3, 3D7, Dd2\",\n      \"pmids\": [\"15953647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"did not test whether one-clag-per-complex rule applies at the host membrane\", \"no channel-function link\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"HB3, 3D7, Dd2\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Peptide-binding surveys attempted to identify host-cell receptors for CLAG3.1 sequences, addressing whether the protein has adhesive/receptor-binding activity.\",\n      \"evidence\": \"synthetic peptide (HABP) binding to CD36-expressing C32 cells and erythrocytes with cross-linking and SDS-PAGE in strain FCB2\",\n      \"pmids\": [\"15659379\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"not validated in intact parasites\", \"putative receptors of ~26/53/59 kDa not molecularly identified\", \"relevance to PSAC function unestablished\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"FCB2\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Domain-mapping defined which region of CLAG3.1 mediates incorporation into the RhopH complex.\",\n      \"evidence\": \"GFP/c-Myc chimera deletion constructs with co-immunoprecipitation in P. falciparum\",\n      \"pmids\": [\"17175193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"precise interacting residues not mapped\", \"single lab, limited follow-up\", \"no structural basis for association\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic mapping resolved the long-standing question of what parasite gene encodes PSAC, assigning channel activity and inhibitor specificity to the clag3 family.\",\n      \"evidence\": \"high-throughput nutrient-uptake inhibitor screening, Dd2 x HB3 cross linkage, DNA transfection, and in vitro selections\",\n      \"pmids\": [\"21620134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"did not resolve whether CLAG3 alone forms the pore or requires partners\", \"structure of the channel unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Silencing and disruption studies established that clag3 expression is epigenetically controlled by heterochromatin and that clag3 gene products are needed for efficient proliferation.\",\n      \"evidence\": \"ChIP for histone modifications, clag3.2 disruption plus drug-selection silencing of clag3.1, and growth assays in P. falciparum\",\n      \"pmids\": [\"21320181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism linking heterochromatin to mutually exclusive choice not defined\", \"growth requirement not tied to a specific transport substrate\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expression switching between clag3.1 and clag3.2 was shown to be mutually exclusive, epigenetic, and functionally consequential for solute transport.\",\n      \"evidence\": \"blasticidin S selection, solute uptake assays, RT-PCR, and whole-genome mutation analysis in P. falciparum\",\n      \"pmids\": [\"23819786\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"molecular trigger of switching not identified\", \"structural basis for isoform-specific transport differences unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Native biochemistry resolved distinct CLAG3-containing membrane complexes and localized a surface-exposed CLAG3-only assembly as a candidate functional pore unit.\",\n      \"evidence\": \"membrane co-IP, native PAGE, mass spectrometry, extracellular biotin labeling, and protease-protection assays in P. falciparum\",\n      \"pmids\": [\"27299521\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"no functional reconstitution of the ~480 kDa complex\", \"stoichiometry of the oligomer not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Self-association of CLAG3 was demonstrated and linked to channel formation, addressing whether CLAG3 oligomers constitute the transport unit.\",\n      \"evidence\": \"co-expression of two CLAG3 isoforms with co-IP, surface biotin labeling, patch-clamp, and in vitro selection yielding correlated sequence/phenotype changes\",\n      \"pmids\": [\"29739907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"atomic structure of the oligomer not determined\", \"exact number of subunits per pore unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Conditional knockdown and complete knockout dissected CLAG3's necessity, showing it is the primary PSAC determinant yet dispensable for residual transport in standard culture but essential under physiological plasma-like conditions.\",\n      \"evidence\": \"TetR-DOZI conditional knockdown, complete knockout, genetic-cross linkage, and uptake assays in modified medium in P. falciparum\",\n      \"pmids\": [\"32069335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"identity of paralogs compensating in knockout not resolved\", \"molecular basis of the physiological-medium requirement unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Live-cell FRET established that CLAG3 and RhopH2 traffic together without dissociation and that channel activity scales quantitatively with membrane complex copy number.\",\n      \"evidence\": \"FRET imaging of tagged CLAG3/RhopH2 and whole-cell patch-clamp of doubly-infected erythrocytes in P. falciparum\",\n      \"pmids\": [\"32900800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"timing of oligomerization relative to trafficking not fully resolved\", \"does not define membrane-insertion mechanism\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Compartmentalized release and staged assembly were defined, explaining how premature channel formation is avoided during invasion.\",\n      \"evidence\": \"super-resolution microscopy, conditional knockdown, immunofluorescence, and stage-defined co-IP in P. falciparum\",\n      \"pmids\": [\"35393572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"export machinery moving RhopH2/RhopH3 across the PV membrane not identified\", \"signal controlling final membrane insertion unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The atomic structure of the CLAG3.1 oligomeric pore and the mechanism of its insertion into the host erythrocyte membrane remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"no high-resolution structure of the channel-forming CLAG3 oligomer\", \"membrane-insertion mechanism and topology not resolved\", \"how isoform sequence dictates transport selectivity structurally unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008509\", \"supporting_discovery_ids\": [0, 3, 5, 6]},\n      {\"term_id\": \"GO:0042802\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"GO:0015267\", \"supporting_discovery_ids\": [0, 5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020008\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0020030\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0020002\", \"supporting_discovery_ids\": [0, 5, 9]},\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0015772\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"GO:0006811\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0006338\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [\n      \"RhopH complex (CLAG3.1/RhopH1 + RhopH2 + RhopH3), ~720 kDa\",\n      \"CLAG3 + RhopH2 subcomplex, ~620 kDa\",\n      \"CLAG3-only homo-oligomer, ~480 kDa (candidate PSAC pore unit)\"\n    ],\n    \"partners\": [\n      \"RhopH2\",\n      \"RhopH3\"\n    ],\n    \"other_free_text\": [\n      \"CLAG3.1 is the RhopH1 subunit of the merozoite rhoptry RhopH complex [#1, #2].\",\n      \"clag3.1 and clag3.2 are expressed in a mutually exclusive, epigenetically switched manner; the expressed isoform sets PSAC transport and pharmacology [#3, #4].\",\n      \"The middle region of CLAG3.1 is required for RhopH complex association; the C-terminus is dispensable; the N-terminal third contains a domain shared with T. gondii RON2 [#10].\",\n      \"clag3-null parasites show severe growth inhibition, and CLAG3 is required for propagation under physiological plasma-like nutrient conditions [#4, #6].\",\n      \"Complete CLAG3 knockout did not abolish residual transport in standard culture, implicating other-chromosome CLAG paralogs in compensatory channel formation [#6].\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0302500","name":null,"product":"cytoadherence linked asexual protein 3.1","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1400600","name":null,"product":"cytoadherence linked asexual protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1401300","name":null,"product":"cytoadherence linked asexual protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"RhopH1A","evaluation":null}