{"gene":"PF3D7_1310600","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2003,"finding":"RAB5b (PfRab5B/Pfrab5b) lacks a classical C-terminal geranylgeranylation motif, suggesting a novel membrane-targeting and regulatory mechanism distinct from canonical Rab GTPases. The gene is transcribed in infected erythrocytes.","method":"Bioinformatic sequence analysis, phylogenetic analysis, RT-PCR (transcription confirmed experimentally)","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational prediction with transcriptional confirmation only; no functional or biochemical assay performed on RAB5b specifically","pmids":["12657463"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"RAB5b (PfRab5B) is N-terminally myristoylated; the N-terminal glycine is required for myristoylation in vitro and for membrane targeting. An N-terminal PfRab5B fragment fused to GFP localizes to the parasite plasma membrane in transgenic P. falciparum; substitution of the modified glycine by alanine causes diffuse cytosolic localization. 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 expression in transgenic P. falciparum, subcellular fractionation, immuno-electron microscopy","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay with mutagenesis, live imaging, fractionation, and electron microscopy, multiple orthogonal methods in one study","pmids":["24498355"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"finding":"RAB5b is essential in asexual blood-stage P. berghei: rab5b could not be deleted from the haploid genome. Complementation experiments showed that PbRab5A or PbRab5C failed to rescue loss of PbRab5B function, indicating non-overlapping roles among the three Plasmodium Rab5 isoforms.","method":"Reverse genetics (attempted gene deletion in P. berghei), complementation analysis with Rab5 paralogs","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — failed deletion with genetic complementation test, single study with two orthogonal approaches (knockout attempt + complementation)","pmids":["24498355"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"finding":"PfRab5B is involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane, based on localization studies in transgenic parasites.","method":"Transgenic P. falciparum localization studies (GFP fusions), immuno-electron microscopy","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, localization-based inference of trafficking role without direct functional loss-of-function assay for MSP1/CK1 trafficking","pmids":["24498355"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"PfRab5B localizes to the cytoplasmic face of the tubovesicular network (TVN) in infected red blood cells. A constitutively active PfRab5B mutant localizes to the parasite cytosol and TVN. After ligand removal under a DD-tag system, cytosolic signal disappears and PfRab5B accumulates in puncta adjacent to the ER and parasite periphery. Peripheral PfRab5B is sensitive to extracellular proteolysis after streptolysin O treatment, confirming localization on the iRBC cytoplasmic face of TVN. PfRab5B co-localizes with N-myristoylated adenylate kinase 2 (PfAK2), and overexpression of PfRab5B abrogates transport of PfAK2 to the parasitophorous vacuole membrane, causing accumulation of PfAK2 in puncta with PfRab5B.","method":"Conditional expression (DD-tag), constitutively active mutant expression, selective membrane permeabilization (streptolysin O), extracellular proteolysis assay, confocal co-localization with PfAK2, complementation in P. berghei","journal":"Malaria journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (conditional expression, mutagenesis, selective permeabilization, proteolysis, co-localization) in one study establishing localization and functional consequence","pmids":["27316546"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2016,"finding":"PfRab5B complemented the function of PbRab5B in P. berghei, but the conventional C-terminally isoprenylated Rab5 isoforms (PbRab5A and PbRab5C) did not, confirming that RAB5b has a unique functional role not shared with the other Rab5 paralogs.","method":"Complementation analysis in P. berghei with different Rab5 isoforms","journal":"Malaria journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with multiple isoforms tested, single lab","pmids":["27316546"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2021,"finding":"PfRab5B physically associates with PfArf1 and PfRab1b, identified by co-immunoprecipitation and mass spectrometry. PfArf1 and PfRab1b co-localize with PfRab5B adjacent to the ER in early erythrocytic stages but localize to distinct ER subdomains. PfArf1 (not PfRab1b) specifically regulates the trafficking of PfAK2 (the PfRab5B-dependent cargo) from the ER to the parasitophorous vacuole.","method":"Co-immunoprecipitation, mass spectrometry, immunofluorescence, super-resolution microscopy, expression of active/inactive PfArf1 and PfRab1b mutants with functional trafficking readout","journal":"Frontiers in cellular and infection microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with MS identification of interactors, super-resolution imaging, and functional genetic dissection with active/inactive mutants in one study","pmids":["33604307"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"During liver-stage infection, an atypical Plasmodium Rab5 isoform (Rab5b) mediates enrichment of host APPL1 (an endosomal adaptor protein) at the parasitophorous vacuole membrane (PVM). Expression of a GTPase-deficient P. berghei Rab5b mutant (PbRab5b_Q91L) rescued PVM APPL1 signal and parasite size when host endosomal GTPase activity was compromised, linking RAB5b GTPase activity to APPL1 retention at the PVM.","method":"GTPase-deficient mutant expression (PbRab5b_Q91L) in P. berghei liver-stage parasites, immunofluorescence for APPL1 at PVM, parasite size measurement","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GTPase-deficient mutant with defined molecular phenotype (APPL1 localization) and organismal readout, single lab","pmids":["35649358"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"finding":"PfRab5b (but not PfRab5a) is required for endosomal transport of host cell cytosol (HCC) to the food vacuole. PfRab5b functions together with PfRbsn5L (a rabenosyn-5-like protein) and PfVPS45 in this endosomal pathway. Inactivation of PfRbsn5L caused accumulation of PI3P- and PfRab5b-decorated HCC-filled vesicles, characteristic of arrested endosomal compartments. The PfKelch13 compartment at the parasite plasma membrane acts upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route.","method":"Conditional protein inactivation (conditional double protein inactivation system), live imaging of HCC uptake, PI3P and PfRab5b co-localization, epistasis analysis with PfKelch13 compartment","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional inactivation with defined molecular phenotype, pathway epistasis established with multiple proteins, orthogonal imaging readouts, single study with rigorous controls","pmids":["38820535"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PfRab5B (PF3D7_1310600) is an N-terminally myristoylated Rab GTPase that lacks canonical C-terminal geranylgeranylation; myristoylation at the N-terminal glycine is required for its membrane targeting to the parasite plasma membrane, food vacuole membrane, and the cytoplasmic face of the tubovesicular network (TVN) in infected erythrocytes. RAB5b is essential in asexual blood stages and has non-redundant roles compared to PfRab5A and PfRab5C. It functions in endosomal transport of host cell cytosol to the food vacuole, acting together with PfRbsn5L and PfVPS45 downstream of the PfKelch13 compartment, and regulates trafficking of N-myristoylated cargo (PfAK2) from the ER to the TVN in partnership with PfArf1. In liver-stage parasites, RAB5b GTPase activity mediates sequestration of host APPL1 at the parasitophorous vacuole membrane."},"narrative":{"mechanistic_narrative":"RAB5b (PF3D7_1310600) is an atypical, essential Rab GTPase of Plasmodium that organizes endosomal transport routes and cargo trafficking in the blood- and liver-stage parasite [PMID:24498355, PMID:38820535]. Unlike canonical Rab GTPases, it lacks a C-terminal geranylgeranylation motif and is instead N-terminally myristoylated; the modified N-terminal glycine is required for its membrane targeting, and its mutation abolishes membrane association [PMID:24498355]. This modification directs RAB5b to the parasite plasma membrane and food vacuole membrane, and to the cytoplasmic face of the tubovesicular network in infected erythrocytes [PMID:24498355, PMID:27316546]. RAB5b is non-redundant with the conventional isoprenylated isoforms PfRab5A and PfRab5C, which cannot rescue its loss, whereas PfRab5B itself complements the P. berghei ortholog [PMID:24498355, PMID:27316546]. Functionally, RAB5b mediates endosomal delivery of host cell cytosol to the food vacuole, acting with PfRbsn5L and PfVPS45 downstream of the PfKelch13 compartment [PMID:38820535], and controls trafficking of the N-myristoylated cargo PfAK2 in concert with its physical partner PfArf1 [PMID:27316546, PMID:33604307]. In liver-stage parasites, its GTPase activity drives sequestration of host APPL1 at the parasitophorous vacuole membrane [PMID:35649358].","teleology":[{"year":2003,"claim":"Established that RAB5b diverges from canonical Rab GTPases, lacking the C-terminal geranylgeranylation motif and raising the question of how it associates with membranes.","evidence":"Bioinformatic and phylogenetic sequence analysis with RT-PCR confirmation of transcription in infected erythrocytes","pmids":["12657463"],"confidence":"Low","gaps":["Computational prediction only, with no functional or biochemical assay on RAB5b itself","Membrane-targeting mechanism unresolved at this stage","No localization or interaction data"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Resolved how an isoprenylation-deficient Rab reaches membranes by showing RAB5b is N-terminally myristoylated, and mapped its endogenous location to the plasma membrane and food vacuole.","evidence":"In vitro myristoylation assay, G→A site-directed mutagenesis, GFP-fusion expression in transgenic P. falciparum, subcellular fractionation, and immuno-electron microscopy","pmids":["24498355"],"confidence":"High","gaps":["Does not identify the enzymes or GTPase regulators acting on RAB5b","Cargo specificity not directly demonstrated by loss-of-function"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2014,"claim":"Demonstrated RAB5b is essential and functionally non-redundant, since it could not be deleted and the paralogs PbRab5A/PbRab5C failed to compensate.","evidence":"Attempted gene deletion in haploid P. berghei blood stages plus complementation with Rab5 paralogs","pmids":["24498355"],"confidence":"Medium","gaps":["Essential process downstream of RAB5b not defined by this genetic test alone","Molecular basis of non-redundancy not established"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2016,"claim":"Localized RAB5b to the cytoplasmic face of the tubovesicular network and provided the first functional cargo link by showing it controls PfAK2 trafficking to the parasitophorous vacuole membrane.","evidence":"Conditional DD-tag expression, constitutively active mutant, streptolysin O permeabilization with extracellular proteolysis, and confocal co-localization with PfAK2","pmids":["27316546"],"confidence":"High","gaps":["Overexpression-based cargo readout rather than endogenous loss-of-function","Machinery bridging RAB5b to PfAK2 not yet identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2021,"claim":"Identified physical partners of RAB5b and assigned PfArf1 as the specific regulator of the RAB5b-dependent PfAK2 trafficking route.","evidence":"Reciprocal Co-IP with mass spectrometry, super-resolution microscopy, and active/inactive PfArf1 and PfRab1b mutants with a trafficking readout","pmids":["33604307"],"confidence":"High","gaps":["Direct biochemical interaction versus complex membership not fully separated","Role of PfRab1b, though co-immunoprecipitated, in the RAB5b pathway unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"Extended RAB5b function to the liver stage, linking its GTPase activity to retention of host APPL1 at the parasitophorous vacuole membrane and parasite growth.","evidence":"GTPase-deficient PbRab5b_Q91L mutant in P. berghei liver-stage parasites with APPL1 immunofluorescence and parasite size measurement","pmids":["35649358"],"confidence":"Medium","gaps":["Mechanism of APPL1 capture and its downstream consequence for the parasite undefined","Single lab and single organismal readout"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2024,"claim":"Placed RAB5b within a defined endosomal pathway, showing it is specifically required for host cell cytosol transport to the food vacuole together with PfRbsn5L and PfVPS45, downstream of the PfKelch13 compartment.","evidence":"Conditional double protein inactivation, live imaging of host cytosol uptake, PI3P/PfRab5b co-localization, and epistasis with the PfKelch13 compartment","pmids":["38820535"],"confidence":"High","gaps":["Nucleotide-state regulation of RAB5b in this pathway not resolved","GEF/GAP proteins acting on RAB5b unidentified","Relationship between this HCC route and the PfAK2/PfArf1 route not integrated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How RAB5b's nucleotide cycle is controlled, which GEFs/GAPs act on it, and whether its distinct trafficking roles (host cytosol import, PfAK2/PfArf1 route, liver-stage APPL1 capture) share common machinery remain unresolved.","evidence":"No timeline discovery addresses upstream nucleotide regulators or integration of the separate RAB5b-dependent routes","pmids":[],"confidence":"Low","gaps":["No GEF/GAP identified for RAB5b","No structural model of RAB5b","Unclear whether blood-stage and liver-stage roles use overlapping effectors"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PFARF1 (PHYSICAL INTERACTOR, CO-IP/MS; REGULATES RAB5B-DEPENDENT PFAK2 TRAFFICKING) [#6]","PFRAB1B (CO-IMMUNOPRECIPITATED WITH RAB5B) [#6]","PFRBSN5L (FUNCTIONS WITH RAB5B IN HOST CYTOSOL ENDOSOMAL ROUTE) [#8]","PFVPS45 (FUNCTIONS WITH RAB5B IN HOST CYTOSOL ENDOSOMAL ROUTE) [#8]","PFAK2 (N-MYRISTOYLATED CARGO WHOSE TRAFFICKING IS RAB5B-DEPENDENT) [#4]"],"other_free_text":["N-terminally myristoylated and lacks canonical C-terminal geranylgeranylation; the N-terminal glycine is required for membrane targeting [#0, #1]","Essential in asexual blood stages and non-redundant with PfRab5A/PfRab5C [#2, #5]","Acts downstream of the PfKelch13 compartment in host cell cytosol uptake to the food vacuole [#8]","GTPase activity mediates host APPL1 enrichment at the parasitophorous vacuole membrane in liver-stage parasites [#7]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"12657463","id":"PMC_12657463","title":"The Plasmodium falciparum family of Rab GTPases.","date":"2003","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/12657463","citation_count":74,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfRAB5b"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15275124","id":"PMC_15275124","title":"Rab GTPases and the unusual secretory pathway of plasmodium.","date":"1997","source":"Parasitology today (Personal ed.)","url":"https://pubmed.ncbi.nlm.nih.gov/15275124","citation_count":47,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"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":["RAB5b","PfRAB5b","PbRAB5b"],"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":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RAB5b","PfRAB5b"],"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":["RAB5b","PfRAB5b","PbRAB5b"],"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":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RAB5b","PbRAB5b"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33604307","id":"PMC_33604307","title":"Rab5b-Associated Arf1 GTPase Regulates Export of N-Myristoylated Adenylate Kinase 2 From the Endoplasmic Reticulum in Plasmodium falciparum.","date":"2021","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33604307","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["RAB5b","PfRAB5b"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6994,"output_tokens":3061,"usd":0.033448,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8551,"output_tokens":3445,"usd":0.06444,"stage2_stop_reason":"end_turn"},"total_usd":0.097888,"stage1_batch_id":"msgbatch_011BPjN2BL33F8cJXD1r7V7T","stage2_batch_id":"msgbatch_01Qwg6vP7qxkpL6Gsc7tajsb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"RAB5b (PfRab5B/Pfrab5b) lacks a classical C-terminal geranylgeranylation motif, suggesting a novel membrane-targeting and regulatory mechanism distinct from canonical Rab GTPases. The gene is transcribed in infected erythrocytes.\",\n      \"method\": \"Bioinformatic sequence analysis, phylogenetic analysis, RT-PCR (transcription confirmed experimentally)\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational prediction with transcriptional confirmation only; no functional or biochemical assay performed on RAB5b specifically\",\n      \"pmids\": [\"12657463\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAB5b (PfRab5B) is N-terminally myristoylated; the N-terminal glycine is required for myristoylation in vitro and for membrane targeting. An N-terminal PfRab5B fragment fused to GFP localizes to the parasite plasma membrane in transgenic P. falciparum; substitution of the modified glycine by alanine causes diffuse cytosolic localization. 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 expression in transgenic P. falciparum, subcellular fractionation, immuno-electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay with mutagenesis, live imaging, fractionation, and electron microscopy, multiple orthogonal methods in one study\",\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\": \"RAB5b is essential in asexual blood-stage P. berghei: rab5b could not be deleted from the haploid genome. Complementation experiments showed that PbRab5A or PbRab5C failed to rescue loss of PbRab5B function, indicating non-overlapping roles among the three Plasmodium Rab5 isoforms.\",\n      \"method\": \"Reverse genetics (attempted gene deletion in P. berghei), complementation analysis with Rab5 paralogs\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — failed deletion with genetic complementation test, single study with two orthogonal approaches (knockout attempt + complementation)\",\n      \"pmids\": [\"24498355\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PfRab5B is involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane, based on localization studies in transgenic parasites.\",\n      \"method\": \"Transgenic P. falciparum localization studies (GFP fusions), immuno-electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, localization-based inference of trafficking role without direct functional loss-of-function assay for MSP1/CK1 trafficking\",\n      \"pmids\": [\"24498355\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PfRab5B localizes to the cytoplasmic face of the tubovesicular network (TVN) in infected red blood cells. A constitutively active PfRab5B mutant localizes to the parasite cytosol and TVN. After ligand removal under a DD-tag system, cytosolic signal disappears and PfRab5B accumulates in puncta adjacent to the ER and parasite periphery. Peripheral PfRab5B is sensitive to extracellular proteolysis after streptolysin O treatment, confirming localization on the iRBC cytoplasmic face of TVN. PfRab5B co-localizes with N-myristoylated adenylate kinase 2 (PfAK2), and overexpression of PfRab5B abrogates transport of PfAK2 to the parasitophorous vacuole membrane, causing accumulation of PfAK2 in puncta with PfRab5B.\",\n      \"method\": \"Conditional expression (DD-tag), constitutively active mutant expression, selective membrane permeabilization (streptolysin O), extracellular proteolysis assay, confocal co-localization with PfAK2, complementation in P. berghei\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (conditional expression, mutagenesis, selective permeabilization, proteolysis, co-localization) in one study establishing localization and functional consequence\",\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 the function of PbRab5B in P. berghei, but the conventional C-terminally isoprenylated Rab5 isoforms (PbRab5A and PbRab5C) did not, confirming that RAB5b has a unique functional role not shared with the other Rab5 paralogs.\",\n      \"method\": \"Complementation analysis in P. berghei with different Rab5 isoforms\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with multiple isoforms tested, single lab\",\n      \"pmids\": [\"27316546\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PfRab5B physically associates with PfArf1 and PfRab1b, identified by co-immunoprecipitation and mass spectrometry. PfArf1 and PfRab1b co-localize with PfRab5B adjacent to the ER in early erythrocytic stages but localize to distinct ER subdomains. PfArf1 (not PfRab1b) specifically regulates the trafficking of PfAK2 (the PfRab5B-dependent cargo) from the ER to the parasitophorous vacuole.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, immunofluorescence, super-resolution microscopy, expression of active/inactive PfArf1 and PfRab1b mutants with functional trafficking readout\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with MS identification of interactors, super-resolution imaging, and functional genetic dissection with active/inactive mutants in one study\",\n      \"pmids\": [\"33604307\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"During liver-stage infection, an atypical Plasmodium Rab5 isoform (Rab5b) mediates enrichment of host APPL1 (an endosomal adaptor protein) at the parasitophorous vacuole membrane (PVM). Expression of a GTPase-deficient P. berghei Rab5b mutant (PbRab5b_Q91L) rescued PVM APPL1 signal and parasite size when host endosomal GTPase activity was compromised, linking RAB5b GTPase activity to APPL1 retention at the PVM.\",\n      \"method\": \"GTPase-deficient mutant expression (PbRab5b_Q91L) in P. berghei liver-stage parasites, immunofluorescence for APPL1 at PVM, parasite size measurement\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GTPase-deficient mutant with defined molecular phenotype (APPL1 localization) and organismal readout, single lab\",\n      \"pmids\": [\"35649358\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PfRab5b (but not PfRab5a) is required for endosomal transport of host cell cytosol (HCC) to the food vacuole. PfRab5b functions together with PfRbsn5L (a rabenosyn-5-like protein) and PfVPS45 in this endosomal pathway. Inactivation of PfRbsn5L caused accumulation of PI3P- and PfRab5b-decorated HCC-filled vesicles, characteristic of arrested endosomal compartments. The PfKelch13 compartment at the parasite plasma membrane acts upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route.\",\n      \"method\": \"Conditional protein inactivation (conditional double protein inactivation system), live imaging of HCC uptake, PI3P and PfRab5b co-localization, epistasis analysis with PfKelch13 compartment\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional inactivation with defined molecular phenotype, pathway epistasis established with multiple proteins, orthogonal imaging readouts, single study with rigorous controls\",\n      \"pmids\": [\"38820535\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PfRab5B (PF3D7_1310600) is an N-terminally myristoylated Rab GTPase that lacks canonical C-terminal geranylgeranylation; myristoylation at the N-terminal glycine is required for its membrane targeting to the parasite plasma membrane, food vacuole membrane, and the cytoplasmic face of the tubovesicular network (TVN) in infected erythrocytes. RAB5b is essential in asexual blood stages and has non-redundant roles compared to PfRab5A and PfRab5C. It functions in endosomal transport of host cell cytosol to the food vacuole, acting together with PfRbsn5L and PfVPS45 downstream of the PfKelch13 compartment, and regulates trafficking of N-myristoylated cargo (PfAK2) from the ER to the TVN in partnership with PfArf1. In liver-stage parasites, RAB5b GTPase activity mediates sequestration of host APPL1 at the parasitophorous vacuole membrane.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAB5b (PF3D7_1310600) is an atypical, essential Rab GTPase of Plasmodium that organizes endosomal transport routes and cargo trafficking in the blood- and liver-stage parasite [#2, #8]. Unlike canonical Rab GTPases, it lacks a C-terminal geranylgeranylation motif and is instead N-terminally myristoylated; the modified N-terminal glycine is required for its membrane targeting, and its mutation abolishes membrane association [#1]. This modification directs RAB5b to the parasite plasma membrane and food vacuole membrane, and to the cytoplasmic face of the tubovesicular network in infected erythrocytes [#1, #4]. RAB5b is non-redundant with the conventional isoprenylated isoforms PfRab5A and PfRab5C, which cannot rescue its loss, whereas PfRab5B itself complements the P. berghei ortholog [#2, #5]. Functionally, RAB5b mediates endosomal delivery of host cell cytosol to the food vacuole, acting with PfRbsn5L and PfVPS45 downstream of the PfKelch13 compartment [#8], and controls trafficking of the N-myristoylated cargo PfAK2 in concert with its physical partner PfArf1 [#4, #6]. In liver-stage parasites, its GTPase activity drives sequestration of host APPL1 at the parasitophorous vacuole membrane [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that RAB5b diverges from canonical Rab GTPases, lacking the C-terminal geranylgeranylation motif and raising the question of how it associates with membranes.\",\n      \"evidence\": \"Bioinformatic and phylogenetic sequence analysis with RT-PCR confirmation of transcription in infected erythrocytes\",\n      \"pmids\": [\"12657463\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational prediction only, with no functional or biochemical assay on RAB5b itself\", \"Membrane-targeting mechanism unresolved at this stage\", \"No localization or interaction data\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved how an isoprenylation-deficient Rab reaches membranes by showing RAB5b is N-terminally myristoylated, and mapped its endogenous location to the plasma membrane and food vacuole.\",\n      \"evidence\": \"In vitro myristoylation assay, G\\u2192A site-directed mutagenesis, GFP-fusion expression in transgenic P. falciparum, subcellular fractionation, and immuno-electron microscopy\",\n      \"pmids\": [\"24498355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the enzymes or GTPase regulators acting on RAB5b\", \"Cargo specificity not directly demonstrated by loss-of-function\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated RAB5b is essential and functionally non-redundant, since it could not be deleted and the paralogs PbRab5A/PbRab5C failed to compensate.\",\n      \"evidence\": \"Attempted gene deletion in haploid P. berghei blood stages plus complementation with Rab5 paralogs\",\n      \"pmids\": [\"24498355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Essential process downstream of RAB5b not defined by this genetic test alone\", \"Molecular basis of non-redundancy not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Localized RAB5b to the cytoplasmic face of the tubovesicular network and provided the first functional cargo link by showing it controls PfAK2 trafficking to the parasitophorous vacuole membrane.\",\n      \"evidence\": \"Conditional DD-tag expression, constitutively active mutant, streptolysin O permeabilization with extracellular proteolysis, and confocal co-localization with PfAK2\",\n      \"pmids\": [\"27316546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Overexpression-based cargo readout rather than endogenous loss-of-function\", \"Machinery bridging RAB5b to PfAK2 not yet identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified physical partners of RAB5b and assigned PfArf1 as the specific regulator of the RAB5b-dependent PfAK2 trafficking route.\",\n      \"evidence\": \"Reciprocal Co-IP with mass spectrometry, super-resolution microscopy, and active/inactive PfArf1 and PfRab1b mutants with a trafficking readout\",\n      \"pmids\": [\"33604307\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical interaction versus complex membership not fully separated\", \"Role of PfRab1b, though co-immunoprecipitated, in the RAB5b pathway unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended RAB5b function to the liver stage, linking its GTPase activity to retention of host APPL1 at the parasitophorous vacuole membrane and parasite growth.\",\n      \"evidence\": \"GTPase-deficient PbRab5b_Q91L mutant in P. berghei liver-stage parasites with APPL1 immunofluorescence and parasite size measurement\",\n      \"pmids\": [\"35649358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of APPL1 capture and its downstream consequence for the parasite undefined\", \"Single lab and single organismal readout\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed RAB5b within a defined endosomal pathway, showing it is specifically required for host cell cytosol transport to the food vacuole together with PfRbsn5L and PfVPS45, downstream of the PfKelch13 compartment.\",\n      \"evidence\": \"Conditional double protein inactivation, live imaging of host cytosol uptake, PI3P/PfRab5b co-localization, and epistasis with the PfKelch13 compartment\",\n      \"pmids\": [\"38820535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nucleotide-state regulation of RAB5b in this pathway not resolved\", \"GEF/GAP proteins acting on RAB5b unidentified\", \"Relationship between this HCC route and the PfAK2/PfArf1 route not integrated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAB5b's nucleotide cycle is controlled, which GEFs/GAPs act on it, and whether its distinct trafficking roles (host cytosol import, PfAK2/PfArf1 route, liver-stage APPL1 capture) share common machinery remain unresolved.\",\n      \"evidence\": \"No timeline discovery addresses upstream nucleotide regulators or integration of the separate RAB5b-dependent routes\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No GEF/GAP identified for RAB5b\", \"No structural model of RAB5b\", \"Unclear whether blood-stage and liver-stage roles use overlapping effectors\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005525\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0020020\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006897\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0016192\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PfArf1 (physical interactor, Co-IP/MS; regulates RAB5b-dependent PfAK2 trafficking) [#6]\",\n      \"PfRab1b (co-immunoprecipitated with RAB5b) [#6]\",\n      \"PfRbsn5L (functions with RAB5b in host cytosol endosomal route) [#8]\",\n      \"PfVPS45 (functions with RAB5b in host cytosol endosomal route) [#8]\",\n      \"PfAK2 (N-myristoylated cargo whose trafficking is RAB5b-dependent) [#4]\"\n    ],\n    \"other_free_text\": [\n      \"N-terminally myristoylated and lacks canonical C-terminal geranylgeranylation; the N-terminal glycine is required for membrane targeting [#0, #1]\",\n      \"Essential in asexual blood stages and non-redundant with PfRab5A/PfRab5C [#2, #5]\",\n      \"Acts downstream of the PfKelch13 compartment in host cell cytosol uptake to the food vacuole [#8]\",\n      \"GTPase activity mediates host APPL1 enrichment at the parasitophorous vacuole membrane in liver-stage parasites [#7]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1310600","name":null,"product":"ras-related protein Rab-5B","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1409100","name":null,"product":"ras-related protein Rab-5B, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1411000","name":null,"product":"ras-related protein Rab-5B, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"RAB5b","evaluation":null}