RAB5b

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).

1. 2003 Low

   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

   PMID:12657463

   Open questions at the time

   • Computational prediction only, with no functional or biochemical assay on RAB5b itself
   • Membrane-targeting mechanism unresolved at this stage
   • No localization or interaction data
2. 2014 High

   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

   PMID:24498355

   Open questions at the time

   • Does not identify the enzymes or GTPase regulators acting on RAB5b
   • Cargo specificity not directly demonstrated by loss-of-function
3. 2014 Medium

   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

   PMID:24498355

   Open questions at the time

   • Essential process downstream of RAB5b not defined by this genetic test alone
   • Molecular basis of non-redundancy not established
4. 2016 High

   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

   PMID:27316546

   Open questions at the time

   • Overexpression-based cargo readout rather than endogenous loss-of-function
   • Machinery bridging RAB5b to PfAK2 not yet identified
5. 2021 High

   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

   PMID:33604307

   Open questions at the time

   • Direct biochemical interaction versus complex membership not fully separated
   • Role of PfRab1b, though co-immunoprecipitated, in the RAB5b pathway unclear
6. 2022 Medium

   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

   PMID:35649358

   Open questions at the time

   • Mechanism of APPL1 capture and its downstream consequence for the parasite undefined
   • Single lab and single organismal readout
7. 2024 High

   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

   PMID:38820535

   Open questions at the time

   • 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

• 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.
• No GEF/GAP identified for RAB5b
• No structural model of RAB5b
• Unclear whether blood-stage and liver-stage roles use overlapping effectors