{"gene":"PF3D7_0414300","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2014,"finding":"PfRab5B (PF3D7_0414300) lacks a C-terminal geranylgeranylation motif and instead undergoes N-terminal myristoylation; the N-terminal glycine is required for myristoylation in vitro. When an N-terminal PfRab5B fragment bearing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum, the chimeric protein localizes to the plasma membrane; substitution of the modified glycine by alanine causes diffuse cytosolic distribution. Endogenous PfRab5B localizes to the parasite plasma membrane and food vacuole membrane by immuno-electron microscopy.","method":"In vitro myristoylation assay; site-directed mutagenesis (G→A); GFP-fusion transgenic P. falciparum; subcellular fractionation; immuno-electron microscopy","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro biochemical assay combined with mutagenesis, GFP localization with functional mutant, and immuno-EM; single lab but multiple orthogonal methods","pmids":["24498355"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"finding":"Reverse genetics in P. berghei failed to delete rab5b from the haploid asexual blood-stage genome, indicating it is essential in that stage. PbRab5A and PbRab5C could not complement loss of PbRab5B, demonstrating non-overlapping roles for the three Plasmodium Rab5 paralogs. PfRab5B is specifically involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane.","method":"Reverse genetics (failed deletion attempts); complementation assay in P. berghei; localization of cargo proteins (MSP1, CK1) by fluorescence microscopy","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via complementation, failed KO (essentiality inferred), cargo localization; single lab","pmids":["24498355"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"A constitutively active PfRab5b mutant (PF3D7_0414300) expressed in P. falciparum under a DD-tag system localizes to the parasite cytosol and the tubovesicular network (TVN). Upon removal of stabilizing ligand, cytosolic signal disappears and PfRab5b accumulates in a punctate structure adjacent to the ER and parasite periphery. Peripheral PfRab5b is accessible to extracellular streptolysin-O-mediated proteolysis (after selective RBC membrane permeabilization), demonstrating it resides on the iRBC cytoplasmic face of the TVN. Overexpression of PfRab5b abrogates transport of N-myristoylated PfAK2 to the PVM, causing co-accumulation of PfAK2 and PfRab5b in a punctate ER-proximal compartment, indicating PfRab5b mediates co-transport of N-acylated cargo from a parasite ER-proximal compartment to the TVN.","method":"DD-tag conditional expression; selective membrane permeabilization with streptolysin O followed by extracellular proteolysis; confocal microscopy; co-expression of PfAK2 with PfRab5b; complementation in P. berghei","journal":"Malaria journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional expression system with functional readout, orthogonal proteolysis protection assay, cargo co-trafficking experiment, and complementation; multiple methods in single lab","pmids":["27316546"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2016,"finding":"PfRab5b complemented PbRab5b function in P. berghei, whereas the conventionally C-terminally isoprenylated Rab5 isoforms PbRab5a and PbRab5c did not, confirming functional specificity of the N-myristoylated Rab5b among Plasmodium Rab5 paralogs.","method":"Complementation assay in P. berghei","journal":"Malaria journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic complementation in P. berghei, single lab, single method","pmids":["27316546"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"finding":"PfRab5b (PF3D7_0414300), but not PfRab5a, is required for endosomal transport of host cell cytosol (HCC) to the food vacuole in asexual P. falciparum blood stages. Inactivation of PfRbsn5L (the parasite Rabenosyn-5-like protein) resulted in accumulation of PI3P- and PfRab5b-decorated HCC-filled vesicles, placing PfRab5b on these endosomal compartments. Using a conditional double protein inactivation system, PfKelch13 compartment activity was placed upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route.","method":"Conditional protein inactivation (single and double); fluorescence microscopy; PI3P and Rab5b co-localization on HCC-filled vesicles; epistasis using conditional double inactivation of PfKelch13 and pathway components","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional loss-of-function with defined cellular phenotype (HCC uptake defect), epistasis by double conditional inactivation, multiple orthogonal approaches in single study","pmids":["38820535"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"During P. berghei liver-stage development, PfRab5b (the atypical Plasmodium Rab5 isoform) at the PVM is involved in sequestering host APPL1 (an endosomal adaptor protein) to the parasitophorous vacuole membrane. Expression of a GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) rescued PVM APPL1 signal and parasite size that were lost upon overexpression of a GTPase-deficient host Rab5 mutant, indicating that PbRab5b competes with host Rab5 for APPL1 recruitment to the PVM.","method":"Overexpression of GTPase-deficient mutants (host hRab5_Q79L and PbRab5b_Q91L) in P. berghei liver-stage parasites; confocal microscopy of APPL1 localization at PVM; parasite size measurement as functional readout","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — dominant-negative/constitutively active mutant approach with functional rescue, single lab, single cell-biological method","pmids":["35649358"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"finding":"MEP pathway inhibition with fosmidomycin causes dramatic mislocalization of Rab5 proteins in P. falciparum, consistent with loss of protein prenylation, and produces marked defects in food vacuolar morphology and integrity, linking Rab5-mediated vesicular trafficking to food vacuole maintenance.","method":"Fosmidomycin treatment (MEP pathway inhibition); fluorescence microscopy of Rab5 localization; food vacuole morphology assessment","journal":"Eukaryotic cell","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological perturbation with localization readout; does not distinguish individual Rab5 isoforms; single lab, single method","pmids":["23223036"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_0414300 (PfRab5b) is an N-terminally myristoylated (and palmitoylated) Rab5 GTPase that, unlike conventional Rab5 isoforms, lacks a C-terminal geranylgeranylation motif; it traffics from an ER-proximal compartment to the tubovesicular network (TVN) in blood-stage parasites where it resides on the iRBC cytoplasmic face of the TVN, functions specifically (not redundantly with PfRab5a or PfRab5c) in endosomal transport of host cell cytosol to the food vacuole downstream of the PfKelch13 endocytic compartment, co-transports N-myristoylated cargo (PfAK2) to the TVN, and during liver-stage infection competes with host Rab5 for recruitment of the endosomal adaptor APPL1 to the parasitophorous vacuole membrane."},"narrative":{"mechanistic_narrative":"PF3D7_0414300 (PfRab5b) is an atypical Rab5 GTPase that governs a dedicated vesicular transport route from an ER-proximal compartment to the tubovesicular network (TVN) and food vacuole in Plasmodium blood stages [PMID:24498355, PMID:27316546, PMID:38820535]. Unlike conventional Rab5 isoforms, it lacks a C-terminal geranylgeranylation motif and is instead membrane-anchored through N-terminal myristoylation, which requires the N-terminal glycine and directs localization to the parasite plasma membrane and food vacuole membrane [PMID:24498355]. This distinct acylation underlies a non-redundant function: PfRab5b, but not the isoprenylated paralogs PfRab5a/PfRab5c, complements loss of Rab5b in P. berghei and cannot be substituted by the other Rab5 proteins [PMID:24498355, PMID:27316546]. Functionally, PfRab5b mediates co-transport of N-myristoylated cargo such as PfAK2 to the TVN, resides on the iRBC cytoplasmic face of the TVN, and traffics cargo including MSP1 and CK1 [PMID:24498355, PMID:27316546]. In asexual blood stages it is specifically required for endosomal transport of host cell cytosol to the food vacuole, acting on PI3P-decorated endosomal vesicles downstream of the PfKelch13 compartment and together with PfRbsn5L/VPS45 [PMID:38820535]. In P. berghei liver stages, the Rab5b isoform at the parasitophorous vacuole membrane competes with host Rab5 for recruitment of the endosomal adaptor APPL1 [PMID:35649358]. Reverse genetics indicate the gene is essential in the asexual blood stage [PMID:24498355].","teleology":[{"year":2014,"claim":"Established that PF3D7_0414300 is an unconventional Rab5 anchored by N-terminal acylation rather than C-terminal prenylation, defining the molecular basis of its distinct membrane targeting.","evidence":"In vitro myristoylation assay, G→A mutagenesis, GFP-fusion transgenic localization, and immuno-EM in P. falciparum","pmids":["24498355"],"confidence":"High","gaps":["Whether palmitoylation contributes beyond myristoylation was not resolved biochemically","GDP/GTP cycling and effector engagement not characterized"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"claim":"Showed the gene is essential and functionally non-redundant with the other two Plasmodium Rab5 paralogs, and identified specific cargo it traffics.","evidence":"Failed rab5b deletion and non-complementation by PbRab5A/C in P. berghei, plus MSP1 and CK1 localization in P. falciparum","pmids":["24498355"],"confidence":"Medium","gaps":["Essentiality inferred from failed deletion rather than conditional knockdown","Mechanism of cargo selection not defined"],"attribution":[{"species":"Plasmodium berghei","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"claim":"Localized the protein to the iRBC-cytoplasmic face of the TVN and demonstrated it drives co-transport of N-acylated cargo from an ER-proximal compartment.","evidence":"DD-tag conditional expression, streptolysin-O protection assay, and PfAK2 co-trafficking in P. falciparum with P. berghei complementation","pmids":["27316546"],"confidence":"High","gaps":["The identity of the receptor/machinery recruiting Rab5b to the ER-proximal compartment is unknown","Overexpression readout may not reflect endogenous flux"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2016,"claim":"Confirmed functional specificity by showing only PfRab5b, not the isoprenylated Rab5a/c, rescues Rab5b loss.","evidence":"Cross-species complementation assay in P. berghei","pmids":["27316546"],"confidence":"Medium","gaps":["Single method; molecular determinant of specificity beyond acylation not dissected"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2022,"claim":"Extended the protein's role to liver stage, showing the Rab5b isoform at the PVM competes with host Rab5 for APPL1 recruitment.","evidence":"GTPase-deficient hRab5 and PbRab5b_Q91L overexpression with APPL1 localization and parasite size readouts in P. berghei liver stages","pmids":["35649358"],"confidence":"Medium","gaps":["Direct PfRab5b–APPL1 interaction not biochemically demonstrated","Uses dominant mutants rather than endogenous loss-of-function"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"claim":"Positioned PfRab5b in the blood-stage host-cell-cytosol endocytic route, on PI3P-marked endosomes downstream of PfKelch13 and acting with Rbsn5L/VPS45.","evidence":"Conditional single/double protein inactivation, PI3P/Rab5b co-localization on HCC-filled vesicles, and epistasis in P. falciparum","pmids":["38820535"],"confidence":"High","gaps":["Direct physical interaction of Rab5b with Rbsn5L/VPS45 not shown","Vesicle fusion/scission step catalyzed by Rab5b not defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The nucleotide-cycle regulation of PfRab5b (its GEF, GAP, and direct effectors) and the biochemical basis of its cargo recognition remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No identified GEF/GAP for PfRab5b","No structural model of the myristoylated GTPase","Direct effector/partner interactions unconfirmed biochemically"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFAK2 (N-MYRISTOYLATED CARGO)","MSP1 (CARGO)","CK1 (CARGO)","PFRBSN5L / VPS45 (SAME ENDOSOMAL ROUTE)","HOST APPL1 (LIVER STAGE, PVM)"],"other_free_text":["N-terminally myristoylated Rab5 lacking a C-terminal geranylgeranylation motif; N-terminal glycine required for acylation-dependent membrane targeting [#0]","Functionally non-redundant with PfRab5a and PfRab5c [#1, #3]","Essential in asexual blood stage inferred from failed P. berghei deletion [#1]","MEP-pathway (prenylation) inhibition mislocalizes Rab5 proteins and disrupts food vacuole integrity, but this pharmacological readout does not resolve individual isoforms [#6]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"23223036","id":"PMC_23223036","title":"Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum.","date":"2012","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/23223036","citation_count":55,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["Rab5"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24498355","id":"PMC_24498355","title":"Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24498355","citation_count":26,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["Rab5"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27316546","id":"PMC_27316546","title":"Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2.","date":"2016","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/27316546","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["Rab5"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"38820535","id":"PMC_38820535","title":"Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites.","date":"2024","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/38820535","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["Rab5"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35649358","id":"PMC_35649358","title":"Active APPL1 sequestration by Plasmodium favors liver-stage development.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/35649358","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["Rab5"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5747,"output_tokens":2712,"usd":0.028961,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7945,"output_tokens":3271,"usd":0.06075,"stage2_stop_reason":"end_turn"},"total_usd":0.089711,"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\": 2014,\n      \"finding\": \"PfRab5B (PF3D7_0414300) lacks a C-terminal geranylgeranylation motif and instead undergoes N-terminal myristoylation; the N-terminal glycine is required for myristoylation in vitro. When an N-terminal PfRab5B fragment bearing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum, the chimeric protein localizes to the plasma membrane; substitution of the modified glycine by alanine causes diffuse cytosolic distribution. Endogenous PfRab5B localizes to the parasite plasma membrane and food vacuole membrane by immuno-electron microscopy.\",\n      \"method\": \"In vitro myristoylation assay; site-directed mutagenesis (G→A); GFP-fusion transgenic P. falciparum; subcellular fractionation; immuno-electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro biochemical assay combined with mutagenesis, GFP localization with functional mutant, and immuno-EM; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24498355\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Reverse genetics in P. berghei failed to delete rab5b from the haploid asexual blood-stage genome, indicating it is essential in that stage. PbRab5A and PbRab5C could not complement loss of PbRab5B, demonstrating non-overlapping roles for the three Plasmodium Rab5 paralogs. PfRab5B is specifically involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane.\",\n      \"method\": \"Reverse genetics (failed deletion attempts); complementation assay in P. berghei; localization of cargo proteins (MSP1, CK1) by fluorescence microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via complementation, failed KO (essentiality inferred), cargo localization; single lab\",\n      \"pmids\": [\"24498355\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A constitutively active PfRab5b mutant (PF3D7_0414300) expressed in P. falciparum under a DD-tag system localizes to the parasite cytosol and the tubovesicular network (TVN). Upon removal of stabilizing ligand, cytosolic signal disappears and PfRab5b accumulates in a punctate structure adjacent to the ER and parasite periphery. Peripheral PfRab5b is accessible to extracellular streptolysin-O-mediated proteolysis (after selective RBC membrane permeabilization), demonstrating it resides on the iRBC cytoplasmic face of the TVN. Overexpression of PfRab5b abrogates transport of N-myristoylated PfAK2 to the PVM, causing co-accumulation of PfAK2 and PfRab5b in a punctate ER-proximal compartment, indicating PfRab5b mediates co-transport of N-acylated cargo from a parasite ER-proximal compartment to the TVN.\",\n      \"method\": \"DD-tag conditional expression; selective membrane permeabilization with streptolysin O followed by extracellular proteolysis; confocal microscopy; co-expression of PfAK2 with PfRab5b; complementation in P. berghei\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional expression system with functional readout, orthogonal proteolysis protection assay, cargo co-trafficking experiment, and complementation; multiple methods in single lab\",\n      \"pmids\": [\"27316546\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PfRab5b complemented PbRab5b function in P. berghei, whereas the conventionally C-terminally isoprenylated Rab5 isoforms PbRab5a and PbRab5c did not, confirming functional specificity of the N-myristoylated Rab5b among Plasmodium Rab5 paralogs.\",\n      \"method\": \"Complementation assay in P. berghei\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic complementation in P. berghei, single lab, single method\",\n      \"pmids\": [\"27316546\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PfRab5b (PF3D7_0414300), but not PfRab5a, is required for endosomal transport of host cell cytosol (HCC) to the food vacuole in asexual P. falciparum blood stages. Inactivation of PfRbsn5L (the parasite Rabenosyn-5-like protein) resulted in accumulation of PI3P- and PfRab5b-decorated HCC-filled vesicles, placing PfRab5b on these endosomal compartments. Using a conditional double protein inactivation system, PfKelch13 compartment activity was placed upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route.\",\n      \"method\": \"Conditional protein inactivation (single and double); fluorescence microscopy; PI3P and Rab5b co-localization on HCC-filled vesicles; epistasis using conditional double inactivation of PfKelch13 and pathway components\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional loss-of-function with defined cellular phenotype (HCC uptake defect), epistasis by double conditional inactivation, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"38820535\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"During P. berghei liver-stage development, PfRab5b (the atypical Plasmodium Rab5 isoform) at the PVM is involved in sequestering host APPL1 (an endosomal adaptor protein) to the parasitophorous vacuole membrane. Expression of a GTPase-deficient Plasmodium berghei Rab5b mutant (PbRab5b_Q91L) rescued PVM APPL1 signal and parasite size that were lost upon overexpression of a GTPase-deficient host Rab5 mutant, indicating that PbRab5b competes with host Rab5 for APPL1 recruitment to the PVM.\",\n      \"method\": \"Overexpression of GTPase-deficient mutants (host hRab5_Q79L and PbRab5b_Q91L) in P. berghei liver-stage parasites; confocal microscopy of APPL1 localization at PVM; parasite size measurement as functional readout\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — dominant-negative/constitutively active mutant approach with functional rescue, single lab, single cell-biological method\",\n      \"pmids\": [\"35649358\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MEP pathway inhibition with fosmidomycin causes dramatic mislocalization of Rab5 proteins in P. falciparum, consistent with loss of protein prenylation, and produces marked defects in food vacuolar morphology and integrity, linking Rab5-mediated vesicular trafficking to food vacuole maintenance.\",\n      \"method\": \"Fosmidomycin treatment (MEP pathway inhibition); fluorescence microscopy of Rab5 localization; food vacuole morphology assessment\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological perturbation with localization readout; does not distinguish individual Rab5 isoforms; single lab, single method\",\n      \"pmids\": [\"23223036\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"PF3D7_0414300 (PfRab5b) is an N-terminally myristoylated (and palmitoylated) Rab5 GTPase that, unlike conventional Rab5 isoforms, lacks a C-terminal geranylgeranylation motif; it traffics from an ER-proximal compartment to the tubovesicular network (TVN) in blood-stage parasites where it resides on the iRBC cytoplasmic face of the TVN, functions specifically (not redundantly with PfRab5a or PfRab5c) in endosomal transport of host cell cytosol to the food vacuole downstream of the PfKelch13 endocytic compartment, co-transports N-myristoylated cargo (PfAK2) to the TVN, and during liver-stage infection competes with host Rab5 for recruitment of the endosomal adaptor APPL1 to the parasitophorous vacuole membrane.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0414300 (PfRab5b) is an atypical Rab5 GTPase that governs a dedicated vesicular transport route from an ER-proximal compartment to the tubovesicular network (TVN) and food vacuole in Plasmodium blood stages [#0, #2, #4]. Unlike conventional Rab5 isoforms, it lacks a C-terminal geranylgeranylation motif and is instead membrane-anchored through N-terminal myristoylation, which requires the N-terminal glycine and directs localization to the parasite plasma membrane and food vacuole membrane [#0]. This distinct acylation underlies a non-redundant function: PfRab5b, but not the isoprenylated paralogs PfRab5a/PfRab5c, complements loss of Rab5b in P. berghei and cannot be substituted by the other Rab5 proteins [#1, #3]. Functionally, PfRab5b mediates co-transport of N-myristoylated cargo such as PfAK2 to the TVN, resides on the iRBC cytoplasmic face of the TVN, and traffics cargo including MSP1 and CK1 [#1, #2]. In asexual blood stages it is specifically required for endosomal transport of host cell cytosol to the food vacuole, acting on PI3P-decorated endosomal vesicles downstream of the PfKelch13 compartment and together with PfRbsn5L/VPS45 [#4]. In P. berghei liver stages, the Rab5b isoform at the parasitophorous vacuole membrane competes with host Rab5 for recruitment of the endosomal adaptor APPL1 [#5]. Reverse genetics indicate the gene is essential in the asexual blood stage [#1].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Established that PF3D7_0414300 is an unconventional Rab5 anchored by N-terminal acylation rather than C-terminal prenylation, defining the molecular basis of its distinct membrane targeting.\",\n      \"evidence\": \"In vitro myristoylation assay, G\\u2192A mutagenesis, GFP-fusion transgenic localization, and immuno-EM in P. falciparum\",\n      \"pmids\": [\"24498355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether palmitoylation contributes beyond myristoylation was not resolved biochemically\", \"GDP/GTP cycling and effector engagement not characterized\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed the gene is essential and functionally non-redundant with the other two Plasmodium Rab5 paralogs, and identified specific cargo it traffics.\",\n      \"evidence\": \"Failed rab5b deletion and non-complementation by PbRab5A/C in P. berghei, plus MSP1 and CK1 localization in P. falciparum\",\n      \"pmids\": [\"24498355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Essentiality inferred from failed deletion rather than conditional knockdown\", \"Mechanism of cargo selection not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Localized the protein to the iRBC-cytoplasmic face of the TVN and demonstrated it drives co-transport of N-acylated cargo from an ER-proximal compartment.\",\n      \"evidence\": \"DD-tag conditional expression, streptolysin-O protection assay, and PfAK2 co-trafficking in P. falciparum with P. berghei complementation\",\n      \"pmids\": [\"27316546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The identity of the receptor/machinery recruiting Rab5b to the ER-proximal compartment is unknown\", \"Overexpression readout may not reflect endogenous flux\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Confirmed functional specificity by showing only PfRab5b, not the isoprenylated Rab5a/c, rescues Rab5b loss.\",\n      \"evidence\": \"Cross-species complementation assay in P. berghei\",\n      \"pmids\": [\"27316546\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method; molecular determinant of specificity beyond acylation not dissected\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the protein's role to liver stage, showing the Rab5b isoform at the PVM competes with host Rab5 for APPL1 recruitment.\",\n      \"evidence\": \"GTPase-deficient hRab5 and PbRab5b_Q91L overexpression with APPL1 localization and parasite size readouts in P. berghei liver stages\",\n      \"pmids\": [\"35649358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PfRab5b\\u2013APPL1 interaction not biochemically demonstrated\", \"Uses dominant mutants rather than endogenous loss-of-function\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Positioned PfRab5b in the blood-stage host-cell-cytosol endocytic route, on PI3P-marked endosomes downstream of PfKelch13 and acting with Rbsn5L/VPS45.\",\n      \"evidence\": \"Conditional single/double protein inactivation, PI3P/Rab5b co-localization on HCC-filled vesicles, and epistasis in P. falciparum\",\n      \"pmids\": [\"38820535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical interaction of Rab5b with Rbsn5L/VPS45 not shown\", \"Vesicle fusion/scission step catalyzed by Rab5b not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The nucleotide-cycle regulation of PfRab5b (its GEF, GAP, and direct effectors) and the biochemical basis of its cargo recognition remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No identified GEF/GAP for PfRab5b\", \"No structural model of the myristoylated GTPase\", \"Direct effector/partner interactions unconfirmed biochemically\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GTPase activity\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"Rab GTPase involved in vesicle trafficking\", \"supporting_discovery_ids\": [0, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"parasite plasma membrane\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"food vacuole membrane\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"tubovesicular network (iRBC cytoplasmic face)\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"ER-proximal punctate compartment\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"PI3P-decorated endosomal vesicle\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"parasitophorous vacuole membrane\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"endosomal transport of host cell cytosol to food vacuole\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"N-myristoylated cargo transport to TVN\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"PfKelch13-dependent endocytosis (downstream route)\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"host APPL1 recruitment to PVM in liver stage\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfAK2 (N-myristoylated cargo)\",\n      \"MSP1 (cargo)\",\n      \"CK1 (cargo)\",\n      \"PfRbsn5L / VPS45 (same endosomal route)\",\n      \"host APPL1 (liver stage, PVM)\"\n    ],\n    \"other_free_text\": [\n      \"N-terminally myristoylated Rab5 lacking a C-terminal geranylgeranylation motif; N-terminal glycine required for acylation-dependent membrane targeting [#0]\",\n      \"Functionally non-redundant with PfRab5a and PfRab5c [#1, #3]\",\n      \"Essential in asexual blood stage inferred from failed P. berghei deletion [#1]\",\n      \"MEP-pathway (prenylation) inhibition mislocalizes Rab5 proteins and disrupts food vacuole integrity, but this pharmacological readout does not resolve individual isoforms [#6]\"\n    ]\n  }\n}","audit_flag":{"gene":"PF3D7_0414300","tier":"IDENTITY_CHECK","verdict":"identity unverified","subtype":"names_other_cards","uniprot_band":"","rules_fired":"R12","issue":"No cited paper names this card; 4 of 5 cited papers name other catalogue genes (PF3D7_0211200, PF3D7_1310600, PF3D7_0106800). The summary may describe one of those genes."},"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0414300","name":null,"product":"Rab5-interacting protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0716300","name":null,"product":"Rab5-interacting protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0505900","name":null,"product":"Rab5-interacting protein, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}