{"gene":"PF3D7_0929400","run_date":"2026-09-08T12:51:27","timeline":{"discoveries":[{"year":1985,"finding":"RhopH2 is a ~140 kDa protein synthesized during schizogony that localizes to the rhoptries (apical organelles) of P. falciparum merozoites and is not expressed on the merozoite surface, as determined by monoclonal antibody affinity purification and immunolocalization.","method":"Monoclonal antibody affinity chromatography, immunofluorescence, immunoelectron microscopy","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct protein purification and immunolocalization, replicated in multiple subsequent studies","pmids":["3887156"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1993,"finding":"During rhoptry biogenesis, RhopH2 (Rhop-H2) is detected in a low-density vesicle (pre-rhoptry compartment, peak density ~1.12 g/ml) at the four-nucleus schizont stage, before assembly into mature rhoptry vesicles of higher density (~1.16 g/ml) at the 16-nucleus stage, indicating a staggered assembly pathway.","method":"Subcellular fractionation by sucrose gradient, immunoelectron microscopy","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation with direct localization, single lab, two orthogonal methods","pmids":["8433718"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2001,"finding":"RhopH2 is a stoichiometric component of the high molecular mass RhopH complex (together with RhopH1/CLAG and RhopH3) in both P. falciparum and P. yoelii, as shown by monoclonal antibody purification of the intact complex.","method":"Monoclonal antibody purification, mass spectrometry, Edman degradation peptide sequencing","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical purification of complex replicated across two species and multiple labs","pmids":["11738712"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium yoelii","strain":""}]},{"year":2003,"finding":"RhopH2 is a single-copy gene product located on chromosome 9 of P. falciparum, transcribed during schizogony, with nine introns, and the encoded protein shares high amino acid sequence similarity with its P. yoelii ortholog; the intact RhopH complex (including RhopH2) is synthesized during schizogony and transferred to ring-stage parasites after erythrocyte invasion.","method":"Gene identification by peptide sequencing, oligonucleotide primer-based cloning, cDNA library screening with mAb 4E10, sequence analysis","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct gene characterization with protein-level validation, replicated across species","pmids":["12615335"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium yoelii","strain":""}]},{"year":2003,"finding":"RhopH2 resides in detergent-resistant membrane (DRM) rafts within the rhoptry organelles, and upon erythrocyte invasion it is inserted into the newly formed parasitophorous vacuole membrane alongside rhoptry stomatin.","method":"Detergent-resistant membrane fractionation, immunofluorescence co-localization, immunoelectron microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DRM fractionation combined with immunolocalization, single lab, two orthogonal methods","pmids":["12968029"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"RhopH2 co-localizes with Clag9 exclusively to the basal bulb region of rhoptries (not the apical ducts) in merozoites, and Clag9 is detected in the RhopH complex purified with anti-RhopH2 antibodies, confirming RhopH2 co-complex membership; after invasion, RhopH2 and Clag9 share identical ring-stage localization.","method":"Immunofluorescence co-localization, immunoelectron microscopy, immunoprecipitation with anti-RhopH2 antibody","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal immunoprecipitation and co-localization, single lab, two orthogonal methods","pmids":["15049814"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"The RhopH complex immunoprecipitated with anti-Clag9 antibody does not contain Clag2 or Clag3.1, indicating that individual RhopH complexes contain only single RhopH1/Clag gene products, while RhopH2 and RhopH3 (encoded by single-copy genes) are shared across all complex variants.","method":"Immunoprecipitation, immunofluorescence, Western blotting","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunoprecipitation with multiple antibodies establishing complex composition, single lab","pmids":["15953647"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"The N-terminal 24 amino acids of RhopH2 (including its signal peptide) are sufficient to target GFP to the rhoptries under the rhoph2 promoter in transgenic P. falciparum, indicating that a rhoptry-targeting signal resides within this short N-terminal sequence and is not mediated by timing of expression alone.","method":"Transgenic parasite expression of GFP chimeras, immunofluorescence localization in live and fixed parasites","journal":"Parasitology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct transgenic targeting assay with GFP chimeras, single lab","pmids":["17175193"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"After erythrocyte invasion, the RhopH complex (including RhopH2) is transferred not only to the parasitophorous vacuole membrane but also to defined domains of the red blood cell cytoplasm, with possible transient association with Maurer's clefts.","method":"Immunofluorescence, subcellular fractionation, immuno-electron microscopy","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence and electron microscopy, single lab, two orthogonal methods","pmids":["18508137"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"An RhopH2 signal peptide–GFP chimera (containing the RhopH2 signal peptide plus the downstream five amino acids) is trafficked to the rhoptries via a Brefeldin A-sensitive pathway, indicating that rhoptry targeting of RhopH2 passes through the conventional secretory pathway and that the five residues downstream of signal cleavage contribute to the sorting signal.","method":"Brefeldin A treatment of transgenic parasites expressing RhopH2-GFP chimera, live-cell fluorescence imaging","journal":"Folia parasitologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological dissection of trafficking route with chimeric reporter, single lab","pmids":["23539954"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"Conditional knockdown of RhopH2 in P. falciparum disrupts the entire RhopH complex, interfering with organellar targeting and subsequent trafficking of both RhopH2 and other complex members; RhopH2 knockdown reduces uptake of solutes that use new permeability pathways (NPPs), depletes essential vitamins and cofactors, decreases de novo pyrimidine synthesis, and impairs parasite growth and replication. RhopH2 was shown by co-immunoprecipitation to interact with RhopH1, RhopH3, the erythrocyte cytoskeleton, and exported host-cell remodeling proteins.","method":"Conditional knockdown (TetR-aptamer system), solute uptake assays, metabolomics, co-immunoprecipitation, growth assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KD with multiple orthogonal functional readouts (transport, metabolomics, growth), co-IP, replicated findings consistent with independent study (PMID:28221136)","pmids":["28252383"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"RhopH2 is essential in P. falciparum: the rhoph2 gene cannot be knocked out; conditional knockdown of RhopH2 disrupts the entire RhopH complex (including organellar targeting), and the complex is required for channel-mediated nutrient uptake at the erythrocyte membrane. RhopH2 does not contribute directly to erythrocyte invasion (invasion was not affected upon RhopH2 knockdown). After export via the PTEX translocon, RhopH2 becomes part of a nutrient channel at the host membrane.","method":"Conditional knockdown, attempted gene knockout, PSAC transport assays, trafficking assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KD and failed KO with multiple orthogonal methods; consistent with independent study (PMID:28252383)","pmids":["28221136"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"Knockdown of RhopH2 expression reduces influx of Na+ into infected erythrocytes through new permeability pathways (NPPs), thereby protecting infected erythrocytes from lysis induced by PfATP4 inhibitors; this places RhopH2-dependent NPPs upstream of PfATP4-driven Na+ accumulation in the mechanism of action of PfATP4 inhibitors.","method":"RhopH2 conditional knockdown, Na+ influx assays, infected erythrocyte lysis assays with PfATP4 inhibitor MB14","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KD with specific functional readout, single lab, pathway placement by epistasis","pmids":["31311978"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"Live-cell FRET between CLAG3-mCerulean and RhopH2-mVenus demonstrates that RhopH2 and CLAG3 remain tightly associated throughout their trafficking: from synthesis and packaging in rhoptries, through deposition into the parasitophorous vacuole after invasion, to export and insertion at the erythrocyte membrane. Stoichiometric increases in exported RhopH2 and CLAG3 protein correlate with proportional increases in PSAC functional copy number.","method":"FRET live-cell imaging, gene editing (fluorescent tagging), whole-cell patch-clamp electrophysiology, fluorescence intensity quantification","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — live-cell FRET with orthogonal electrophysiology, direct subcellular localization with functional consequence, single rigorous study with multiple methods","pmids":["32900800"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"RhopH2 cannot be disrupted (complete knockout is not viable) under conditions where nutrient uptake is abolished, in contrast to CLAG3 which can be fully knocked out with only partial loss of transport. This places RhopH2 as essential for nutrient transport activity in a manner that cannot be compensated by CLAG paralog oligomerization.","method":"Attempted gene knockout by CRISPR/selection, PSAC transport assays, conditional knockdown","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — failed KO combined with conditional KD and transport assays, single lab","pmids":["32069335"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"The soluble trafficking RhopH complex is assembled with 1:1:1 stoichiometry of CLAG3, RhopH2, and RhopH3. A 2.9 Å cryo-EM structure shows that RhopH2 and RhopH3 interact with CLAG3 over large surface areas but make minimal direct contacts with each other; CLAG3 is positioned centrally between them. The complex is stabilized by extensive intramolecular disulfide bonding and predicted transmembrane helices are shielded in the soluble form. Export to the erythrocyte membrane requires the PTEX translocon.","method":"Single-particle cryo-electron microscopy (2.9 Å), mass spectrometry, stoichiometry analysis, PTEX translocon conditional knockdown","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution cryo-EM structure combined with mass spectrometry and genetic epistasis (PTEX dependence), single rigorous study with multiple orthogonal methods","pmids":["33393463"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"Cryo-EM structure of the native, endogenous RhopH complex purified directly from parasite lysates shows RhopH2 bound to CLAG3.1 with a substantial interface; RhopH2 and RhopH3 share large binding surfaces with CLAG3.1 but make minimal contact with each other. The complex contains 13 intramolecular disulfide bonds overall, and the putative CLAG3.1 transmembrane helix is buried in a helical bundle, suggesting a large conformational change is required for membrane insertion.","method":"Cryo-electron microscopy (endogenous complex), mass spectrometry, cryoID program for subunit identification","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — independent atomic-resolution cryo-EM structure of endogenous complex, consistent with PMID:33393463","pmids":["34446549"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"Five exported proteins (identified by reciprocal immunoprecipitation with mass spectrometry) interact with all members of the RhopH complex including RhopH2 in the infected erythrocyte; these interactions suggest a cytoskeletal complex and a Maurer's clefts/J-dot complex associated with the RhopH complex, providing candidate trafficking intermediates. Knockdown of these 13 candidate interactors individually had no effect on sorbitol NPP uptake, indicating they are not individually required for NPP function.","method":"Reciprocal immunoprecipitation, mass spectrometry-based proteomics, NPP activity assay (sorbitol uptake), conditional knockdown","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal IP/MS identifying RhopH2-associated proteins, combined with functional KD assays, single lab","pmids":["33774908"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"After merozoite invasion, RhopH2 is released into the nascent parasitophorous vacuole (not directly into the host cell cytoplasm, unlike CLAG3). Export of RhopH2 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of the Clag3/RhopH2/RhopH3 complex, which is then incorporated into the host cell membrane concomitant with activation of nutrient uptake. The full RhopH complex is not pre-assembled during merozoite invasion; compartmentalization prevents premature channel assembly.","method":"Fluorescence microscopy (live and fixed), conditional knockdown, super-resolution microscopy, nutrient uptake assays","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization by multiple microscopy modalities plus functional assays establishing assembly sequence, single lab with multiple orthogonal methods","pmids":["35393572"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"finding":"In P. berghei, PbSR1 (a serine/arginine-rich splicing factor) co-immunoprecipitates with RhopH2, and live-cell imaging shows cytoplasmic co-localization of PbSR1 and RhopH2 in trophozoites and gametocytes but not in mature schizonts or merozoites.","method":"Immunoprecipitation coupled to mass spectrometry, live-cell fluorescence imaging of dual-tagged transgenic parasites, Western blot","journal":"Malaria journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP/MS with co-localization, single lab, functional significance of interaction not established","pmids":["41634752"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":"ANKA"}]}],"current_model":"RhopH2 (PF3D7_0929400) is an essential P. falciparum rhoptry protein that is synthesized during schizogony, targeted to the rhoptry basal bulb via its N-terminal 24 amino acids through a Brefeldin A-sensitive secretory pathway, and assembled as a 1:1:1 stoichiometric soluble complex with CLAG3 and RhopH3 (with RhopH2 and RhopH3 each binding CLAG3 over large interfaces but making minimal direct contact with each other, stabilized by extensive disulfide bonds); upon merozoite invasion, RhopH2 is discharged into the parasitophorous vacuole, exported across the vacuolar membrane via the PTEX translocon, and—after assembling with CLAG3 (which is discharged directly into the host cytoplasm)—inserts into the erythrocyte membrane to form the new permeability pathway (NPP/PSAC) ion and nutrient channel essential for parasite nutrient acquisition, pyrimidine synthesis, and survival, with RhopH2 knockdown abolishing NPP activity, disrupting the entire complex, and being lethal to the parasite."},"narrative":{"mechanistic_narrative":"RhopH2 (PF3D7_0929400) is an essential ~140 kDa Plasmodium falciparum rhoptry protein, synthesized during schizogony and localized to the apical rhoptry organelles of merozoites, that functions as an obligatory subunit of the high-molecular-mass RhopH complex driving nutrient acquisition at the infected erythrocyte membrane [PMID:3887156, PMID:11738712, PMID:28221136]. It is encoded by a single-copy gene and, together with RhopH3, is shared across all RhopH complex variants, whereas the CLAG (RhopH1) subunit varies between complexes [PMID:12615335, PMID:15953647]. RhopH2 is targeted to the rhoptry basal bulb via a signal within its N-terminal 24 amino acids that routes it through a Brefeldin A-sensitive secretory pathway [PMID:15049814, PMID:17175193, PMID:23539954]. Structurally, RhopH2 assembles with CLAG3 and RhopH3 in a 1:1:1 soluble complex in which RhopH2 binds CLAG3 over a large interface but makes minimal direct contact with RhopH3, with the assembly stabilized by extensive intramolecular disulfide bonding and transmembrane helices shielded in the soluble state [PMID:33393463, PMID:34446549]. Following merozoite invasion RhopH2 is discharged into the nascent parasitophorous vacuole, exported into the host cytoplasm via the PTEX translocon, and only then assembles with CLAG3 and inserts into the erythrocyte membrane to form the new permeability pathway (NPP/PSAC), a compartmentalized sequence that prevents premature channel assembly [PMID:32900800, PMID:33393463, PMID:35393572]. RhopH2 is essential and cannot be knocked out; its conditional knockdown collapses the entire complex, abolishes channel-mediated solute uptake and de novo pyrimidine synthesis, and is lethal, while leaving erythrocyte invasion unaffected [PMID:28252383, PMID:28221136, PMID:32069335]. Because RhopH2-dependent NPPs mediate Na+ influx, their loss protects infected cells from PfATP4-inhibitor-induced lysis, placing RhopH2 upstream of PfATP4-driven Na+ accumulation [PMID:31311978].","teleology":[{"year":1985,"claim":"Establishing RhopH2 as a discrete rhoptry-resident protein defined it as an apical organellar factor rather than a surface antigen, framing its role in the invasion/post-invasion program.","evidence":"Monoclonal antibody affinity purification with immunofluorescence and immunoelectron microscopy in P. falciparum","pmids":["3887156"],"confidence":"High","gaps":["Molecular function undefined at this stage","No partners identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1993,"claim":"Detection of RhopH2 in a low-density pre-rhoptry vesicle before its appearance in mature rhoptries revealed a staggered biogenesis pathway for the protein.","evidence":"Sucrose-gradient subcellular fractionation and immunoelectron microscopy in P. falciparum","pmids":["8433718"],"confidence":"Medium","gaps":["Trafficking machinery for vesicle maturation not defined","Timing relative to complex assembly unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"claim":"Identifying RhopH2 as a single-copy, schizont-transcribed gene conserved across species and defining the intact complex transfer to ring stages linked its biology to the post-invasion period.","evidence":"Gene cloning, cDNA screening and sequence analysis in P. falciparum and P. yoelii, with complex purification (also #2)","pmids":["12615335","11738712"],"confidence":"High","gaps":["Function of the transferred complex not yet established","No mechanism for erythrocyte transfer"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium yoelii","strain":""}]},{"year":2005,"claim":"Defining complex composition showed RhopH2 (with RhopH3) is a constant subunit shared across variant complexes that each carry a single CLAG paralog, clarifying the combinatorial architecture.","evidence":"Immunoprecipitation, immunofluorescence and Western blotting with anti-CLAG antibodies in P. falciparum (with co-localization #5)","pmids":["15953647","15049814"],"confidence":"Medium","gaps":["Functional consequence of CLAG variation not resolved","Stoichiometry not yet quantified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Mapping a rhoptry-targeting signal to the N-terminal 24 residues and demonstrating Brefeldin A sensitivity established that RhopH2 is sorted through the conventional secretory pathway.","evidence":"Transgenic GFP-chimera targeting assays and Brefeldin A treatment in P. falciparum (with earlier chimera mapping #7)","pmids":["23539954","17175193"],"confidence":"Medium","gaps":["Specific sorting receptor/adaptor unidentified","How signal directs basal-bulb specificity unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"claim":"Conditional knockdown and failed knockout established RhopH2 as essential and required for the whole complex, directly linking it to channel-mediated nutrient uptake, pyrimidine synthesis and parasite survival while excluding a direct invasion role.","evidence":"TetR-aptamer conditional knockdown, attempted knockout, solute-uptake assays, metabolomics and co-IP in P. falciparum (two concordant studies)","pmids":["28252383","28221136"],"confidence":"High","gaps":["Molecular basis of channel formation not defined","Whether RhopH2 itself forms the pore unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"Placing RhopH2-dependent NPPs upstream of PfATP4-driven Na+ accumulation defined the transport pathway's position in the mechanism of a drug class.","evidence":"RhopH2 conditional knockdown with Na+ influx and PfATP4-inhibitor lysis assays in P. falciparum","pmids":["31311978"],"confidence":"Medium","gaps":["Direct ion selectivity of RhopH2-containing channel not established","Epistasis based on functional readouts, not structure"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Atomic-resolution cryo-EM of recombinant and endogenous complexes defined the 1:1:1 architecture with RhopH2 and RhopH3 bridged centrally by CLAG3, disulfide-stabilized and with membrane helices shielded, explaining why soluble trafficking precedes a conformational change for membrane insertion.","evidence":"Single-particle cryo-EM (2.9 Å) with mass spectrometry and PTEX conditional knockdown, plus independent endogenous-complex cryo-EM in P. falciparum","pmids":["33393463","34446549"],"confidence":"High","gaps":["Membrane-inserted conformation not visualized","Trigger for the conformational change unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"Demonstrating that RhopH2 is discharged into the parasitophorous vacuole and only assembles with CLAG3 after export established a compartmentalized, sequential assembly that prevents premature channel formation.","evidence":"Live, fixed and super-resolution microscopy with conditional knockdown and nutrient-uptake assays in P. falciparum (with FRET trafficking #13)","pmids":["35393572","32900800"],"confidence":"High","gaps":["Signals gating assembly timing unknown","How RhopH2 crosses PTEX in an unfolded state unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"claim":"A candidate stage-specific interaction between RhopH2 and the splicing factor PbSR1 in P. berghei raises the possibility of non-canonical RhopH2 associations, though its functional significance is undefined.","evidence":"Co-IP/MS and live-cell co-localization of dual-tagged transgenic P. berghei ANKA parasites","pmids":["41634752"],"confidence":"Low","gaps":["Functional significance of the interaction not established","Single co-IP/MS in a different species, not validated in P. falciparum","Cytoplasmic co-localization inconsistent with known rhoptry role"],"attribution":[{"species":"Plasmodium berghei","strain":"ANKA"}]},{"year":null,"claim":"How the RhopH2-containing complex physically forms an ion/nutrient-conducting channel in the erythrocyte membrane, and what triggers the conformational change unshielding its transmembrane helices, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No membrane-inserted structure","Pore-forming residues of RhopH2 unidentified","Trigger for soluble-to-membrane transition unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["RhopH complex (CLAG3/RhopH1–RhopH2–RhopH3, 1:1:1 stoichiometry)","New permeability pathway / PSAC channel at erythrocyte membrane"],"partners":["CLAG3 (RHOPH1)","RHOPH3","CLAG9","PTEX TRANSLOCON (EXPORT DEPENDENCE)","ERYTHROCYTE CYTOSKELETON AND EXPORTED HOST-REMODELING PROTEINS","PBSR1 (P. BERGHEI, LOW CONFIDENCE)"],"other_free_text":["Essential ~140 kDa protein encoded by a single-copy gene on chromosome 9, transcribed during schizogony; cannot be knocked out.","N-terminal 24 residues carry a rhoptry basal-bulb targeting signal routed through a Brefeldin A-sensitive secretory pathway.","Assembles as a soluble disulfide-stabilized complex with membrane helices shielded, requiring a conformational change for membrane insertion.","Complex assembly is spatially/temporally compartmentalized: RhopH2 enters the parasitophorous vacuole and assembles with CLAG3 only after PTEX-mediated export.","RhopH2 knockdown does not affect erythrocyte invasion."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"3887156","id":"PMC_3887156","title":"Isolation of a Plasmodium falciparum rhoptry protein.","date":"1985","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/3887156","citation_count":103,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"28252383","id":"PMC_28252383","title":"Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate.","date":"2017","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/28252383","citation_count":92,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RhopH2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"28221136","id":"PMC_28221136","title":"An essential dual-function complex mediates erythrocyte invasion and channel-mediated nutrient uptake in malaria parasites.","date":"2017","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/28221136","citation_count":90,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RhopH2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"12968029","id":"PMC_12968029","title":"Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites. A role for microbial raft proteins in apicomplexan vacuole biogenesis.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12968029","citation_count":80,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RhopH2"],"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":["RhopH2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15049814","id":"PMC_15049814","title":"The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion.","date":"2004","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/15049814","citation_count":70,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RhopH2"],"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":["RhopH2"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18508137","id":"PMC_18508137","title":"The RhopH complex is transferred to the host cell cytoplasm following red blood cell invasion by Plasmodium 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surface, as determined by monoclonal antibody affinity purification and immunolocalization.\",\n      \"method\": \"Monoclonal antibody affinity chromatography, immunofluorescence, immunoelectron microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct protein purification and immunolocalization, replicated in multiple subsequent studies\",\n      \"pmids\": [\"3887156\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"During rhoptry biogenesis, RhopH2 (Rhop-H2) is detected in a low-density vesicle (pre-rhoptry compartment, peak density ~1.12 g/ml) at the four-nucleus schizont stage, before assembly into mature rhoptry vesicles of higher density (~1.16 g/ml) at the 16-nucleus stage, indicating a staggered assembly pathway.\",\n      \"method\": \"Subcellular fractionation by sucrose gradient, immunoelectron microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation with direct localization, single lab, two orthogonal methods\",\n      \"pmids\": [\"8433718\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RhopH2 is a stoichiometric component of the high molecular mass RhopH complex (together with RhopH1/CLAG and RhopH3) in both P. falciparum and P. yoelii, as shown by monoclonal antibody purification of the intact complex.\",\n      \"method\": \"Monoclonal antibody purification, mass spectrometry, Edman degradation peptide sequencing\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical purification of complex replicated across two species and multiple labs\",\n      \"pmids\": [\"11738712\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RhopH2 is a single-copy gene product located on chromosome 9 of P. falciparum, transcribed during schizogony, with nine introns, and the encoded protein shares high amino acid sequence similarity with its P. yoelii ortholog; the intact RhopH complex (including RhopH2) is synthesized during schizogony and transferred to ring-stage parasites after erythrocyte invasion.\",\n      \"method\": \"Gene identification by peptide sequencing, oligonucleotide primer-based cloning, cDNA library screening with mAb 4E10, sequence analysis\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct gene characterization with protein-level validation, replicated across species\",\n      \"pmids\": [\"12615335\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RhopH2 resides in detergent-resistant membrane (DRM) rafts within the rhoptry organelles, and upon erythrocyte invasion it is inserted into the newly formed parasitophorous vacuole membrane alongside rhoptry stomatin.\",\n      \"method\": \"Detergent-resistant membrane fractionation, immunofluorescence co-localization, immunoelectron microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DRM fractionation combined with immunolocalization, single lab, two orthogonal methods\",\n      \"pmids\": [\"12968029\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RhopH2 co-localizes with Clag9 exclusively to the basal bulb region of rhoptries (not the apical ducts) in merozoites, and Clag9 is detected in the RhopH complex purified with anti-RhopH2 antibodies, confirming RhopH2 co-complex membership; after invasion, RhopH2 and Clag9 share identical ring-stage localization.\",\n      \"method\": \"Immunofluorescence co-localization, immunoelectron microscopy, immunoprecipitation with anti-RhopH2 antibody\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal immunoprecipitation and co-localization, single lab, two orthogonal methods\",\n      \"pmids\": [\"15049814\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The RhopH complex immunoprecipitated with anti-Clag9 antibody does not contain Clag2 or Clag3.1, indicating that individual RhopH complexes contain only single RhopH1/Clag gene products, while RhopH2 and RhopH3 (encoded by single-copy genes) are shared across all complex variants.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence, Western blotting\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunoprecipitation with multiple antibodies establishing complex composition, single lab\",\n      \"pmids\": [\"15953647\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal 24 amino acids of RhopH2 (including its signal peptide) are sufficient to target GFP to the rhoptries under the rhoph2 promoter in transgenic P. falciparum, indicating that a rhoptry-targeting signal resides within this short N-terminal sequence and is not mediated by timing of expression alone.\",\n      \"method\": \"Transgenic parasite expression of GFP chimeras, immunofluorescence localization in live and fixed parasites\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct transgenic targeting assay with GFP chimeras, single lab\",\n      \"pmids\": [\"17175193\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"After erythrocyte invasion, the RhopH complex (including RhopH2) is transferred not only to the parasitophorous vacuole membrane but also to defined domains of the red blood cell cytoplasm, with possible transient association with Maurer's clefts.\",\n      \"method\": \"Immunofluorescence, subcellular fractionation, immuno-electron microscopy\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunofluorescence and electron microscopy, single lab, two orthogonal methods\",\n      \"pmids\": [\"18508137\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"An RhopH2 signal peptide–GFP chimera (containing the RhopH2 signal peptide plus the downstream five amino acids) is trafficked to the rhoptries via a Brefeldin A-sensitive pathway, indicating that rhoptry targeting of RhopH2 passes through the conventional secretory pathway and that the five residues downstream of signal cleavage contribute to the sorting signal.\",\n      \"method\": \"Brefeldin A treatment of transgenic parasites expressing RhopH2-GFP chimera, live-cell fluorescence imaging\",\n      \"journal\": \"Folia parasitologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological dissection of trafficking route with chimeric reporter, single lab\",\n      \"pmids\": [\"23539954\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Conditional knockdown of RhopH2 in P. falciparum disrupts the entire RhopH complex, interfering with organellar targeting and subsequent trafficking of both RhopH2 and other complex members; RhopH2 knockdown reduces uptake of solutes that use new permeability pathways (NPPs), depletes essential vitamins and cofactors, decreases de novo pyrimidine synthesis, and impairs parasite growth and replication. RhopH2 was shown by co-immunoprecipitation to interact with RhopH1, RhopH3, the erythrocyte cytoskeleton, and exported host-cell remodeling proteins.\",\n      \"method\": \"Conditional knockdown (TetR-aptamer system), solute uptake assays, metabolomics, co-immunoprecipitation, growth assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KD with multiple orthogonal functional readouts (transport, metabolomics, growth), co-IP, replicated findings consistent with independent study (PMID:28221136)\",\n      \"pmids\": [\"28252383\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RhopH2 is essential in P. falciparum: the rhoph2 gene cannot be knocked out; conditional knockdown of RhopH2 disrupts the entire RhopH complex (including organellar targeting), and the complex is required for channel-mediated nutrient uptake at the erythrocyte membrane. RhopH2 does not contribute directly to erythrocyte invasion (invasion was not affected upon RhopH2 knockdown). After export via the PTEX translocon, RhopH2 becomes part of a nutrient channel at the host membrane.\",\n      \"method\": \"Conditional knockdown, attempted gene knockout, PSAC transport assays, trafficking assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KD and failed KO with multiple orthogonal methods; consistent with independent study (PMID:28252383)\",\n      \"pmids\": [\"28221136\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Knockdown of RhopH2 expression reduces influx of Na+ into infected erythrocytes through new permeability pathways (NPPs), thereby protecting infected erythrocytes from lysis induced by PfATP4 inhibitors; this places RhopH2-dependent NPPs upstream of PfATP4-driven Na+ accumulation in the mechanism of action of PfATP4 inhibitors.\",\n      \"method\": \"RhopH2 conditional knockdown, Na+ influx assays, infected erythrocyte lysis assays with PfATP4 inhibitor MB14\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KD with specific functional readout, single lab, pathway placement by epistasis\",\n      \"pmids\": [\"31311978\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Live-cell FRET between CLAG3-mCerulean and RhopH2-mVenus demonstrates that RhopH2 and CLAG3 remain tightly associated throughout their trafficking: from synthesis and packaging in rhoptries, through deposition into the parasitophorous vacuole after invasion, to export and insertion at the erythrocyte membrane. Stoichiometric increases in exported RhopH2 and CLAG3 protein correlate with proportional increases in PSAC functional copy number.\",\n      \"method\": \"FRET live-cell imaging, gene editing (fluorescent tagging), whole-cell patch-clamp electrophysiology, fluorescence intensity quantification\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — live-cell FRET with orthogonal electrophysiology, direct subcellular localization with functional consequence, single rigorous study with multiple methods\",\n      \"pmids\": [\"32900800\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RhopH2 cannot be disrupted (complete knockout is not viable) under conditions where nutrient uptake is abolished, in contrast to CLAG3 which can be fully knocked out with only partial loss of transport. This places RhopH2 as essential for nutrient transport activity in a manner that cannot be compensated by CLAG paralog oligomerization.\",\n      \"method\": \"Attempted gene knockout by CRISPR/selection, PSAC transport assays, conditional knockdown\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — failed KO combined with conditional KD and transport assays, single lab\",\n      \"pmids\": [\"32069335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The soluble trafficking RhopH complex is assembled with 1:1:1 stoichiometry of CLAG3, RhopH2, and RhopH3. A 2.9 Å cryo-EM structure shows that RhopH2 and RhopH3 interact with CLAG3 over large surface areas but make minimal direct contacts with each other; CLAG3 is positioned centrally between them. The complex is stabilized by extensive intramolecular disulfide bonding and predicted transmembrane helices are shielded in the soluble form. Export to the erythrocyte membrane requires the PTEX translocon.\",\n      \"method\": \"Single-particle cryo-electron microscopy (2.9 Å), mass spectrometry, stoichiometry analysis, PTEX translocon conditional knockdown\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution cryo-EM structure combined with mass spectrometry and genetic epistasis (PTEX dependence), single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"33393463\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the native, endogenous RhopH complex purified directly from parasite lysates shows RhopH2 bound to CLAG3.1 with a substantial interface; RhopH2 and RhopH3 share large binding surfaces with CLAG3.1 but make minimal contact with each other. The complex contains 13 intramolecular disulfide bonds overall, and the putative CLAG3.1 transmembrane helix is buried in a helical bundle, suggesting a large conformational change is required for membrane insertion.\",\n      \"method\": \"Cryo-electron microscopy (endogenous complex), mass spectrometry, cryoID program for subunit identification\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — independent atomic-resolution cryo-EM structure of endogenous complex, consistent with PMID:33393463\",\n      \"pmids\": [\"34446549\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Five exported proteins (identified by reciprocal immunoprecipitation with mass spectrometry) interact with all members of the RhopH complex including RhopH2 in the infected erythrocyte; these interactions suggest a cytoskeletal complex and a Maurer's clefts/J-dot complex associated with the RhopH complex, providing candidate trafficking intermediates. Knockdown of these 13 candidate interactors individually had no effect on sorbitol NPP uptake, indicating they are not individually required for NPP function.\",\n      \"method\": \"Reciprocal immunoprecipitation, mass spectrometry-based proteomics, NPP activity assay (sorbitol uptake), conditional knockdown\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal IP/MS identifying RhopH2-associated proteins, combined with functional KD assays, single lab\",\n      \"pmids\": [\"33774908\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"After merozoite invasion, RhopH2 is released into the nascent parasitophorous vacuole (not directly into the host cell cytoplasm, unlike CLAG3). Export of RhopH2 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of the Clag3/RhopH2/RhopH3 complex, which is then incorporated into the host cell membrane concomitant with activation of nutrient uptake. The full RhopH complex is not pre-assembled during merozoite invasion; compartmentalization prevents premature channel assembly.\",\n      \"method\": \"Fluorescence microscopy (live and fixed), conditional knockdown, super-resolution microscopy, nutrient uptake assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization by multiple microscopy modalities plus functional assays establishing assembly sequence, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35393572\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In P. berghei, PbSR1 (a serine/arginine-rich splicing factor) co-immunoprecipitates with RhopH2, and live-cell imaging shows cytoplasmic co-localization of PbSR1 and RhopH2 in trophozoites and gametocytes but not in mature schizonts or merozoites.\",\n      \"method\": \"Immunoprecipitation coupled to mass spectrometry, live-cell fluorescence imaging of dual-tagged transgenic parasites, Western blot\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP/MS with co-localization, single lab, functional significance of interaction not established\",\n      \"pmids\": [\"41634752\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"ANKA\"}]\n    }\n  ],\n  \"current_model\": \"RhopH2 (PF3D7_0929400) is an essential P. falciparum rhoptry protein that is synthesized during schizogony, targeted to the rhoptry basal bulb via its N-terminal 24 amino acids through a Brefeldin A-sensitive secretory pathway, and assembled as a 1:1:1 stoichiometric soluble complex with CLAG3 and RhopH3 (with RhopH2 and RhopH3 each binding CLAG3 over large interfaces but making minimal direct contact with each other, stabilized by extensive disulfide bonds); upon merozoite invasion, RhopH2 is discharged into the parasitophorous vacuole, exported across the vacuolar membrane via the PTEX translocon, and—after assembling with CLAG3 (which is discharged directly into the host cytoplasm)—inserts into the erythrocyte membrane to form the new permeability pathway (NPP/PSAC) ion and nutrient channel essential for parasite nutrient acquisition, pyrimidine synthesis, and survival, with RhopH2 knockdown abolishing NPP activity, disrupting the entire complex, and being lethal to the parasite.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RhopH2 (PF3D7_0929400) is an essential ~140 kDa Plasmodium falciparum rhoptry protein, synthesized during schizogony and localized to the apical rhoptry organelles of merozoites, that functions as an obligatory subunit of the high-molecular-mass RhopH complex driving nutrient acquisition at the infected erythrocyte membrane [#0, #2, #11]. It is encoded by a single-copy gene and, together with RhopH3, is shared across all RhopH complex variants, whereas the CLAG (RhopH1) subunit varies between complexes [#3, #6]. RhopH2 is targeted to the rhoptry basal bulb via a signal within its N-terminal 24 amino acids that routes it through a Brefeldin A-sensitive secretory pathway [#5, #7, #9]. Structurally, RhopH2 assembles with CLAG3 and RhopH3 in a 1:1:1 soluble complex in which RhopH2 binds CLAG3 over a large interface but makes minimal direct contact with RhopH3, with the assembly stabilized by extensive intramolecular disulfide bonding and transmembrane helices shielded in the soluble state [#15, #16]. Following merozoite invasion RhopH2 is discharged into the nascent parasitophorous vacuole, exported into the host cytoplasm via the PTEX translocon, and only then assembles with CLAG3 and inserts into the erythrocyte membrane to form the new permeability pathway (NPP/PSAC), a compartmentalized sequence that prevents premature channel assembly [#13, #15, #18]. RhopH2 is essential and cannot be knocked out; its conditional knockdown collapses the entire complex, abolishes channel-mediated solute uptake and de novo pyrimidine synthesis, and is lethal, while leaving erythrocyte invasion unaffected [#10, #11, #14]. Because RhopH2-dependent NPPs mediate Na+ influx, their loss protects infected cells from PfATP4-inhibitor-induced lysis, placing RhopH2 upstream of PfATP4-driven Na+ accumulation [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1985,\n      \"claim\": \"Establishing RhopH2 as a discrete rhoptry-resident protein defined it as an apical organellar factor rather than a surface antigen, framing its role in the invasion/post-invasion program.\",\n      \"evidence\": \"Monoclonal antibody affinity purification with immunofluorescence and immunoelectron microscopy in P. falciparum\",\n      \"pmids\": [\"3887156\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular function undefined at this stage\", \"No partners identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Detection of RhopH2 in a low-density pre-rhoptry vesicle before its appearance in mature rhoptries revealed a staggered biogenesis pathway for the protein.\",\n      \"evidence\": \"Sucrose-gradient subcellular fractionation and immunoelectron microscopy in P. falciparum\",\n      \"pmids\": [\"8433718\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trafficking machinery for vesicle maturation not defined\", \"Timing relative to complex assembly unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identifying RhopH2 as a single-copy, schizont-transcribed gene conserved across species and defining the intact complex transfer to ring stages linked its biology to the post-invasion period.\",\n      \"evidence\": \"Gene cloning, cDNA screening and sequence analysis in P. falciparum and P. yoelii, with complex purification (also #2)\",\n      \"pmids\": [\"12615335\", \"11738712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the transferred complex not yet established\", \"No mechanism for erythrocyte transfer\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium yoelii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defining complex composition showed RhopH2 (with RhopH3) is a constant subunit shared across variant complexes that each carry a single CLAG paralog, clarifying the combinatorial architecture.\",\n      \"evidence\": \"Immunoprecipitation, immunofluorescence and Western blotting with anti-CLAG antibodies in P. falciparum (with co-localization #5)\",\n      \"pmids\": [\"15953647\", \"15049814\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of CLAG variation not resolved\", \"Stoichiometry not yet quantified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping a rhoptry-targeting signal to the N-terminal 24 residues and demonstrating Brefeldin A sensitivity established that RhopH2 is sorted through the conventional secretory pathway.\",\n      \"evidence\": \"Transgenic GFP-chimera targeting assays and Brefeldin A treatment in P. falciparum (with earlier chimera mapping #7)\",\n      \"pmids\": [\"23539954\", \"17175193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific sorting receptor/adaptor unidentified\", \"How signal directs basal-bulb specificity unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Conditional knockdown and failed knockout established RhopH2 as essential and required for the whole complex, directly linking it to channel-mediated nutrient uptake, pyrimidine synthesis and parasite survival while excluding a direct invasion role.\",\n      \"evidence\": \"TetR-aptamer conditional knockdown, attempted knockout, solute-uptake assays, metabolomics and co-IP in P. falciparum (two concordant studies)\",\n      \"pmids\": [\"28252383\", \"28221136\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of channel formation not defined\", \"Whether RhopH2 itself forms the pore unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Placing RhopH2-dependent NPPs upstream of PfATP4-driven Na+ accumulation defined the transport pathway's position in the mechanism of a drug class.\",\n      \"evidence\": \"RhopH2 conditional knockdown with Na+ influx and PfATP4-inhibitor lysis assays in P. falciparum\",\n      \"pmids\": [\"31311978\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ion selectivity of RhopH2-containing channel not established\", \"Epistasis based on functional readouts, not structure\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Atomic-resolution cryo-EM of recombinant and endogenous complexes defined the 1:1:1 architecture with RhopH2 and RhopH3 bridged centrally by CLAG3, disulfide-stabilized and with membrane helices shielded, explaining why soluble trafficking precedes a conformational change for membrane insertion.\",\n      \"evidence\": \"Single-particle cryo-EM (2.9 Å) with mass spectrometry and PTEX conditional knockdown, plus independent endogenous-complex cryo-EM in P. falciparum\",\n      \"pmids\": [\"33393463\", \"34446549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane-inserted conformation not visualized\", \"Trigger for the conformational change unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrating that RhopH2 is discharged into the parasitophorous vacuole and only assembles with CLAG3 after export established a compartmentalized, sequential assembly that prevents premature channel formation.\",\n      \"evidence\": \"Live, fixed and super-resolution microscopy with conditional knockdown and nutrient-uptake assays in P. falciparum (with FRET trafficking #13)\",\n      \"pmids\": [\"35393572\", \"32900800\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals gating assembly timing unknown\", \"How RhopH2 crosses PTEX in an unfolded state unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"A candidate stage-specific interaction between RhopH2 and the splicing factor PbSR1 in P. berghei raises the possibility of non-canonical RhopH2 associations, though its functional significance is undefined.\",\n      \"evidence\": \"Co-IP/MS and live-cell co-localization of dual-tagged transgenic P. berghei ANKA parasites\",\n      \"pmids\": [\"41634752\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Functional significance of the interaction not established\", \"Single co-IP/MS in a different species, not validated in P. falciparum\", \"Cytoplasmic co-localization inconsistent with known rhoptry role\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"ANKA\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the RhopH2-containing complex physically forms an ion/nutrient-conducting channel in the erythrocyte membrane, and what triggers the conformational change unshielding its transmembrane helices, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No membrane-inserted structure\", \"Pore-forming residues of RhopH2 unidentified\", \"Trigger for soluble-to-membrane transition unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0022857\", \"supporting_discovery_ids\": [10, 11, 12, 14]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [15, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020008\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11, 13, 18]},\n      {\"term_id\": \"GO:0020002\", \"supporting_discovery_ids\": [4, 8, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0015850\", \"supporting_discovery_ids\": [10, 11, 12]},\n      {\"term_id\": \"GO:0006221\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0006886\", \"supporting_discovery_ids\": [7, 9, 15, 18]}\n    ],\n    \"complexes\": [\n      \"RhopH complex (CLAG3/RhopH1–RhopH2–RhopH3, 1:1:1 stoichiometry)\",\n      \"New permeability pathway / PSAC channel at erythrocyte membrane\"\n    ],\n    \"partners\": [\n      \"CLAG3 (RhopH1)\",\n      \"RhopH3\",\n      \"Clag9\",\n      \"PTEX translocon (export dependence)\",\n      \"erythrocyte cytoskeleton and exported host-remodeling proteins\",\n      \"PbSR1 (P. berghei, Low confidence)\"\n    ],\n    \"other_free_text\": [\n      \"Essential ~140 kDa protein encoded by a single-copy gene on chromosome 9, transcribed during schizogony; cannot be knocked out.\",\n      \"N-terminal 24 residues carry a rhoptry basal-bulb targeting signal routed through a Brefeldin A-sensitive secretory pathway.\",\n      \"Assembles as a soluble disulfide-stabilized complex with membrane helices shielded, requiring a conformational change for membrane insertion.\",\n      \"Complex assembly is spatially/temporally compartmentalized: RhopH2 enters the parasitophorous vacuole and assembles with CLAG3 only after PTEX-mediated export.\",\n      \"RhopH2 knockdown does not affect erythrocyte invasion.\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0929400","name":null,"product":"high molecular weight rhoptry protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0830200","name":null,"product":"high molecular weight rhoptry protein 2","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0727900","name":null,"product":"high molecular weight rhoptry protein 2, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"RhopH2","evaluation":null}