RON3

RON3 (PF3D7_1252100) is a rhoptry bulb membrane protein of Plasmodium falciparum merozoites that governs establishment of the intraerythrocytic ring stage after invasion (PMID:21237287, PMID:37457963). Unlike the moving-junction RON proteins, it partitions into a distinct RON complex, co-immunoprecipitating with PfRON2 and PfRON4 but not with PfAMA1 (PMID:21237287), and additionally associates with the rhoptry-associated membrane antigen RAMA in immature schizonts (PMID:33537242). Recombinant RON3 binds erythrocytes directly and anti-RON3 antibodies inhibit merozoite invasion, indicating a role at the host-cell interface (PMID:25495792). RON3 is proteolytically processed into N- and C-terminal fragments, and its C-terminal fragment is the functionally critical species: conditional disruption or stage-specific knockdown before invasion blocks erythrocyte invasion and arrests development beyond the ring stage, abolishes PTEX-dependent export of parasite proteins (e.g., RESA) across the parasitophorous vacuolar membrane, and abrogates glucose uptake into the parasitophorous vacuolar space while only partially affecting plasmodial surface anion channel activity (PMID:31289187, PMID:37457963). Knockdown after invasion does not perturb growth, placing RON3's essential action at or immediately before ring-stage establishment (PMID:37457963). Rhoptry localization is conserved in P. berghei sporozoites (PMID:31552198).

1. 2011 Medium

   Establishing where RON3 resides and which proteins it partners with was needed to distinguish it from the moving-junction machinery; localization to the rhoptry bulb and interaction with RON2/RON4 but not AMA1 defined RON3 as part of a separate RON complex that persists into the parasitophorous vacuole at the ring stage.

   Evidence IFA, immunoelectron microscopy, and co-immunoprecipitation in P. falciparum merozoites

   PMID:21237287

   Open questions at the time

   • Whether the RON2/3/4 interactions are direct or bridged by other proteins is unresolved
   • Functional consequence of the complex not tested
   • Single lab
2. 2014 Medium

   To test whether RON3 engages the host cell, recombinant protein and antibody assays showed direct erythrocyte binding and antibody-mediated inhibition of invasion, giving the first functional link between RON3 and merozoite invasion.

   Evidence Erythrocyte-binding assay with recombinant RON3, invasion inhibition with anti-RON3 antibodies, IEM/IFA in P. falciparum

   PMID:25495792

   Open questions at the time

   • Erythrocyte receptor for RON3 not identified
   • Binding domain within RON3 not mapped
   • Antibody inhibition does not prove native binding is required in vivo
3. 2019 High

   Genetic loss-of-function was needed to define RON3's essential role; conditional disruption showed RON3 is required for PTEX-dependent protein export, glucose uptake into the PV space, and progression beyond the ring stage.

   Evidence Conditional gene disruption with RESA translocation, glucose uptake, and growth assays in P. falciparum

   PMID:31289187

   Open questions at the time

   • Molecular mechanism linking a rhoptry protein to PTEX and glucose transport is unexplained
   • Whether RON3 acts directly on transporters or indirectly is unknown
4. 2019 Medium

   Conservation of RON3 rhoptry localization across infective stages was addressed by showing rhoptry targeting in P. berghei sporozoites, extending RON3 biology beyond the merozoite.

   Evidence Immunoelectron microscopy in P. berghei sporozoites

   PMID:31552198

   Open questions at the time

   • Functional role of RON3 in sporozoites not tested
   • Partner interactions in sporozoites not examined
5. 2021 Medium

   Beyond the RON2/4 complex, an additional partner was sought; RON3 was found to associate with RAMA, expanding its interactome, though the interaction's directness is unresolved.

   Evidence Immunoprecipitation with LC-MS/MS and IFA in P. falciparum merozoites

   PMID:33537242

   Open questions at the time

   • Direct vs. indirect RON3-RAMA interaction not resolved
   • Functional significance of the RON3-RAMA complex untested
   • Single lab
6. 2023 High

   To pinpoint which part of RON3 and which timing underlie its essential functions, stage-specific knockdown of the processed C-terminal fragment showed this fragment is required before or at invasion for erythrocyte invasion, PTEX-dependent export, and glucose uptake, while PSAC activity is only partially affected.

   Evidence Conditional C-terminal knockdown with invasion, export, glucose uptake, and PSAC assays in P. falciparum

   PMID:37457963

   Open questions at the time

   • Protease and cleavage sites generating the fragments not identified
   • Mechanism by which the C-terminal fragment enables PTEX export and glucose transport unknown
   • Role of the N-terminal fragment not defined

• The molecular basis by which a rhoptry-derived protein deposited into the PVM controls both PTEX-mediated protein export and nutrient (glucose) transport remains unresolved.
• No structural model of RON3 or its fragments
• No identified direct molecular link between RON3 and PTEX components or glucose transporters
• Erythrocyte receptor and RON3 binding domain unidentified