Affinage · Toxoplasma gondii

RON2

rhoptry neck protein RON2 · UniProt B6KV60

Annotated
2026-09-11
29 papers in source corpus 18 papers cited in narrative 18 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RON2 is a rhoptry neck protein of Toxoplasma gondii that forms the parasite-derived core of the moving junction (MJ), the ring-shaped adhesion structure that drives host cell invasion (PMID:16244709). Upon secretion, RON2 is inserted as an integral membrane protein into the host cell membrane, where its C-terminus is displayed to the parasite surface while its partner proteins RON4/5/8 associate with the cytoplasmic face of the host membrane (PMID:19247437, PMID:21347343). The RON2 C-terminus binds directly to the microneme protein AMA1 on the parasite surface, bridging AMA1 to the rest of the MJ complex; two short segments (D3 and D4) flanking a hydrophobic stretch dock into the hydrophobic groove of the AMA1 ectodomain through a stepwise, high-buried-surface assembly that resists the mechanical forces of penetration, and this interaction is required for invasion in both T. gondii and P. falciparum (PMID:21347343, PMID:21347354, PMID:21778402). Within the MJ complex, RON5 is required for RON2 stability—its loss triggers complete RON2 degradation and abolishes invasion (PMID:24651769)—and the RON2/4/5 module actively recruits host CIN85, CD2AP, and the ESCRT-I components ALIX and TSG101 to the junction to support efficient invasion and virulence (PMID:28848228). The RON2-AMA1 interaction also protects AMA1 from rhomboid-mediated shedding and drives dephosphorylation of the AMA1 cytoplasmic tail, constituting an outside-in signal that promotes productive entry (PMID:27624124). The AMA-RON pairing is stage-specific and structurally plastic: sporozoites and bradyzoites deploy cognate paralogue pairs (sporoRON2/RON2L1 with sporoAMA1/AMA4) that bind exclusively to their partners and use distinct groove architectures, revealing a conserved but adaptable invasion strategy across the life cycle (PMID:26712012, PMID:23940612, PMID:36693095). RON2 expression is a direct transcriptional target of AP2XII-9 during the S/M phase (PMID:39207100). RON2-depleted parasites can generate a transient host cell membrane conductance increase prior to invasion but fail to invade productively, demonstrating that RON2 is not essential for generation of the conductance pathway but is required for productive invasion (PMID:39416144, PMID:40973829).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2005 High

    Establishing that rhoptry neck proteins physically join the microneme protein AMA1 at the moving junction answered how two distinct secretory organelles cooperate to build the invasion machinery.

    Evidence Co-IP, mass spectrometry, and immunofluorescence localization of RON2/RON4 during invasion in T. gondii

    PMID:16244709

    Open questions at the time
    • Did not define which RON provides the direct AMA1 contact
    • Membrane topology of the complex unresolved
  2. 2009 High

    Showing that RON2 inserts into the host membrane while RON4/5/8 coat its cytoplasmic face, and that RON2 binds AMA1 without other complex members, resolved the physical architecture and the direct AMA1 partner.

    Evidence Metabolic labelling, IP, differential permeabilization, and antibody loading into host cells; RON8 identified as an additional complex member in T. gondii

    PMID:19247437

    Open questions at the time
    • Region of RON2 mediating AMA1 binding not mapped
    • Mechanism of RON2 membrane insertion unknown
  3. 2011 High

    Mapping the AMA1-binding region to the RON2 C-terminus (D3/D4) and solving the AMA1-RON2 co-structure explained how the junction resists mechanical force and that this bridge is essential for invasion across apicomplexa.

    Evidence Deletion mapping, GST-fusion pulldowns, invasion/rhoptry-injection inhibition assays, and a 1.95 Å AMA1-RON2 crystal structure in T. gondii and P. falciparum

    PMID:21347343 PMID:21347354 PMID:21778402

    Open questions at the time
    • How D3/D4 binding is coupled to downstream RON4/5/8 function not defined
    • Contribution of the hydrophobic stretch to membrane insertion vs binding unclear
  4. 2013 High

    Discovering that sporozoites use a dedicated sporoRON2/sporoAMA1 pair with reciprocal binding specificity revealed that AMA-RON pairings are stage-specific rather than universal.

    Evidence Co-IP, interaction-domain studies, and stage-specific reciprocal invasion-inhibition assays in T. gondii

    PMID:23940612

    Open questions at the time
    • Structural basis of paralogue specificity not yet defined
    • In vivo role during natural sporozoite infection untested
  5. 2014 High

    Defining RON5 as essential for RON2 stability and showing paralogue upregulation upon AMA1 loss established both the assembly hierarchy of the complex and the plasticity of MJ composition.

    Evidence Conditional RON5 knockdown with invasion/evacuole/egress readouts; AMA1 gene disruption with conditional complementation in T. gondii

    PMID:24651769 PMID:24934579

    Open questions at the time
    • Molecular basis of RON5-dependent RON2 stabilization unknown
    • Functional capacity of compensating paralogues only partially characterized
  6. 2014 Medium

    Live imaging linked productive junction anchoring to host cortical actin remodeling, showing that a RON2-containing junction must be tethered to the host cortex for invasion to succeed.

    Evidence Live imaging of fluorescent RON2 and kinematic analysis of >1000 invasion events in toxofilin-mutant T. gondii

    PMID:25551479

    Open questions at the time
    • Direct RON2 contribution to anchoring vs bystander role unresolved
    • Molecular link between RON complex and host cortex not identified
  7. 2015 High

    Structural and thermodynamic dissection of the AMA1 DII loop and of the divergent AMA4-RON2L1 complex explained how binding selectivity is enforced and how paralogue pairs achieve distinct assembly architectures.

    Evidence Site-directed mutagenesis, ITC, and X-ray crystallography of TgAMA1ΔDIIloop-EtRON2 and apo/bound TgAMA4-RON2L1 in T. gondii

    PMID:25955165 PMID:26712012

    Open questions at the time
    • How gatekeeping is regulated during natural invasion unclear
    • Whether groove-less architectures generalize to other paralogues untested
  8. 2016 High

    Showing that RON2 D3 binding shields AMA1 from rhomboid cleavage and triggers cytoplasmic-tail dephosphorylation established the junction as a site of outside-in signaling that stabilizes AMA1 for productive entry.

    Evidence Peptide/cleavage assays and phosphomimetic/phosphoablative AMA1 (S527) mutants with invasion readouts in T. gondii

    PMID:27624124

    Open questions at the time
    • Kinase/phosphatase acting on the AMA1 tail not identified
    • Downstream effectors of the dephosphorylation signal unknown
  9. 2017 Medium

    Demonstrating recruitment of host CIN85, CD2AP, ALIX, and TSG101 by RON2/4/5, and the existence of a RON2/4/5-independent RON4L1 complex, revealed that the junction subverts conserved host machinery and that multiple MJ complexes may coexist.

    Evidence Co-IP, interaction mapping, parasite mutants, invasion assays, and mouse virulence; RON4L1 knockout and RON2/4/5-independent localization in T. gondii

    PMID:28848228 PMID:29263399

    Open questions at the time
    • Functional role of recruited host ESCRT components in membrane biology at the MJ not fully defined
    • Relationship between parallel RON complexes and single vs multiple junctions unresolved
  10. 2023 High

    Genetic dissection of bradyzoite invasion showed that chronic-stage parasites substitute AMA2/AMA4-RON2L1 for the canonical AMA1-RON2 pair, and that these pairs have distinct vaccine potential.

    Evidence Gene knockouts, invasion assays, and immunization/challenge experiments in mice with T. gondii

    PMID:36693095

    Open questions at the time
    • Regulatory switch controlling stage-specific paralogue expression unknown
    • Structural detail of the bradyzoite complexes incomplete
  11. 2024 Medium

    Electrophysiology of RON2-depleted parasites separated pore formation from productive invasion, showing RON2 is dispensable for the invasion-associated conductance but required for entry and for setting pore properties.

    Evidence Sub-200 µs electrophysiological recordings and quantal conductance analysis of RON2-depleted RH parasites

    PMID:39416144 PMID:40973829

    Open questions at the time
    • Molecular identity of the conductance pathway/pore unknown
    • How RON2 modulates quantal conductance mechanistically unresolved
  12. 2024 Medium

    Identifying AP2XII-9 as a direct transcriptional regulator of RON2 placed the gene within a cell-cycle-linked program controlling rhoptry neck protein expression.

    Evidence CUT&Tag chromatin profiling and conditional AP2XII-9 knockdown with invasion/egress assays in T. gondii

    PMID:39207100

    Open questions at the time
    • Full transcriptional network co-regulating RON2 not mapped
    • Direct vs indirect effects on RON2 protein levels not fully separated

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
AMA1 (MICRONEME PROTEIN, DIRECT BINDING PARTNER)AMA4AP2XII-9 (TRANSCRIPTIONAL REGULATOR)HOST ALIXHOST CD2APHOST CIN85HOST TSG101 (ESCRT-I)RON4RON5 (REQUIRED FOR RON2 STABILITY)RON8SPOROAMA1
Complex memberships
Moving junction complex RON2/RON4/RON5/RON8RON2-AMA1 complexRON2L1-AMA4 complexRON4L1-containing MJ complex (RON2/4/5-independent)sporoRON2(RON2L2)-sporoAMA1 complex

Evidence

Reading pass · 18 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2005 RON2 (together with RON4 and RON5) was identified as a rhoptry neck protein that associates with the microneme protein AMA1 at the moving junction during host cell invasion, forming a complex derived from two distinct secretory organelles (micronemes and rhoptry necks). Co-immunoprecipitation, mass spectrometry, immunofluorescence localization during invasion PLoS pathogens High 16244709
2009 RON2 and AMA1 can interact directly in the absence of other MJ complex members (RON4, RON5, RON8), as shown by metabolic labelling and immunoprecipitation. Additionally, RON2 is targeted to the host cell membrane upon secretion, where RON4/5/8 associate with the cytoplasmic face of the host cell membrane. Metabolic labelling, immunoprecipitation, differential permeabilization, antibody loading into host cell PLoS pathogens High 19247437
2009 RON8 was identified as an additional member of the RON2/4/5 complex at the moving junction. All RON proteins (RON2, RON4, RON5, RON8) are present at the MJ during invasion and undergo proteolytic maturation during trafficking to the rhoptries. Biochemical fractionation, Co-immunoprecipitation, immunofluorescence during invasion PLoS pathogens High 19247437
2011 RON2 is inserted as an integral membrane protein in the host cell membrane and directly binds AMA1 through a specific region near its C-terminus. This RON2-AMA1 interaction is critical for invasion by both T. gondii and P. falciparum, demonstrated by invasion inhibition assays using recombinant proteins. Interaction assays with native and recombinant proteins, invasion inhibition assays, membrane topology analysis PLoS pathogens High 21347343
2011 The C-terminus of TgRON2 contains two short segments (D3 and D4) flanking a hydrophobic stretch that independently associate with the ectodomain of TgAMA1. Pre-incubation with D3 peptide (fused to GST) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins, demonstrating that the RON2 C-terminus forms the crucial bridge between AMA1 and the rest of the MJ complex. Deletion mapping, GST-fusion pulldown, invasion inhibition assay, rhoptry injection assay PLoS pathogens High 21347354
2011 Crystal structure of TgAMA1 complexed with a RON2 peptide was determined at 1.95 Å resolution, revealing that RON2 binds the hydrophobic groove of AMA1 through a stepwise assembly mechanism creating extensive buried surface area, enabling resistance to mechanical forces during invasion. X-ray crystallography at 1.95 Å resolution Science (New York, N.Y.) High 21778402
2012 RON5 plays a key role in organization of the MJ RON complex: conditional knockdown of RON5 causes complete degradation of RON2 and mistargeting of RON4 within the secretory pathway, demonstrating that RON5 is required for RON2 stability. Loss of RON5 also abolishes host cell invasion and prevents evacuole formation, but does not impact egress. Conditional knockdown (tetracycline-regulatable), immunofluorescence, invasion assay, evacuole assay, egress assay PLoS pathogens High 24651769
2014 AMA1 interacts with RON2 to maintain MJ structural integrity in T. gondii and is required for parasite internalization. Disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues (paralogues) that cooperate to support residual invasion, demonstrating plasticity in MJ composition. Reverse genetics (gene disruption), conditional complementation, invasion assays, immunofluorescence Nature communications High 24934579
2014 When Toxoplasma lacks toxofilin and cannot locally remodel cortical actin, the RON2-containing moving junction fails to anchor to the host cell cortex and travels retrogradely, demonstrating that junction anchoring requires host cortex remodeling for efficient invasion. RON2 molecules are eventually posteriorly capped and cleaved/released in failed invasion events. Live imaging of fluorescently-tagged RON2, kinematic analysis of >1000 invasion events, toxofilin mutant parasites BMC biology Medium 25551479
2015 The AMA1 DII loop governs ligand binding selectivity for RON2; removing the mobile portion of the DII loop (TgAMA1ΔDIIloop) dramatically increases binding affinity for non-cognate RON2 peptides (e.g., EtRON2). Crystal structure of TgAMA1ΔDIIloop with EtRON2 identified spatially distinct anchor points in the AMA1 groove, supporting a model where the DII loop acts as a structural gatekeeper. Site-directed mutagenesis, isothermal titration calorimetry, X-ray crystallography PloS one High 25955165
2015 TgAMA4 (a sporozoite-expressed AMA paralogue) forms a high-affinity functional complex with its coevolved partner TgRON2L1. Crystal structures of TgAMA4 in apo and TgRON2L1-bound forms revealed an unexpected architecture lacking both a deep surface groove and a key surface loop present in AMA1-RON2 complexes, defining a distinct assembly mechanism. Co-crystallization, X-ray crystallography (apo and bound forms), alanine scanning mutagenesis, binding affinity measurements Proceedings of the National Academy of Sciences of the United States of America High 26712012
2016 Binding of TgRON2 domain 3 (D3) peptide to TgAMA1 protects AMA1 from rhomboid protease-mediated cleavage of its transmembrane domain, stabilizing AMA1 at the moving junction. Parasites expressing a hypercleavable AMA1 invade less efficiently. Additionally, TgAMA1-TgRON2 interaction leads to dephosphorylation of S527 on the AMA1 cytoplasmic tail, and a phosphomimetic S527D allele causes invasion defects, revealing outside-in signaling through the MJ. Peptide treatment assays, cleavage assays, phosphomimetic/phosphoablative AMA1 mutants, invasion efficiency assays mBio High 27624124
2017 RON2, RON4, and RON5 cooperate to actively recruit host cell proteins CIN85, CD2AP, and ESCRT-I components ALIX and TSG101 to the moving junction during invasion. Parasite mutants unable to recruit these host proteins show inefficient invasion in culture and attenuated virulence in mice, demonstrating that the RON complex subverts conserved host machinery. Co-immunoprecipitation, interaction mapping, parasite mutagenesis, invasion assays, mouse virulence model Nature microbiology High 28848228
2013 Sporozoites express paralogue sporoRON2 (RON2L2) that localizes to the apical region and interacts specifically and exclusively with sporoAMA1 (not with generic AMA1), while generic RON2 interacts only with generic AMA1. Pretreatment with domain 3 of sporoRON2 inhibits sporozoite but not tachyzoite invasion, and vice versa for generic RON2 D3, demonstrating stage-specific AMA-RON pairings. Co-immunoprecipitation, structural studies of interacting domains, invasion inhibition assays with stage-specific parasites PloS one High 23940612
2023 Bradyzoites invade host cells in an MJ-dependent fashion but use paralogue proteins AMA2 and AMA4 (with AMA4 partnering RON2L1) rather than canonical AMA1-RON2. AMA1-RON2 complex immunization induces strong protection against both acute and chronic infection, while AMA4-RON2L1 more selectively targets chronic infection. Reverse genetics (gene knockout), invasion assays, immunization/challenge experiments in mice Proceedings of the National Academy of Sciences of the United States of America High 36693095
2017 RON4L1 is an additional member of the MJ complex that is targeted to the cytosolic face of the host membrane upon secretion. Critically, RON4L1 trafficking to the MJ is not affected by the absence of RON2, RON4, and RON5, suggesting the co-existence of independent MJ complexes in tachyzoites. Immunofluorescence, gene knockout (RON4L1 KO), invasion and virulence assays, triple RON2/4/5-independent localization experiments Scientific reports Medium 29263399
2024 RON2-depleted parasites generate a transient host cell membrane conductance increase (invasion pore) prior to invasion but fail to invade, demonstrating that RON2 is not essential for generation of the conductance pathway/pore but is required for productive invasion. Without RON2, the quantal conductance mean differs significantly (0.19 nS vs 0.26 nS for wild-type), implicating RON2 in modulating pore characteristics. Electrophysiological recordings at sub-200 µs resolution, RON2-depleted parasites, time-series quantal conductance analysis bioRxiv (preprint) / EMBO reports Medium 39416144 40973829
2024 The transcription factor TgAP2XII-9 directly targets RON2 (along with RON4 and RON8) gene loci as shown by CUT&Tag, and its deficiency leads to significant downregulation of rhoptry neck proteins including RON2, resulting in invasion and egress defects, establishing RON2 as a transcriptional target of AP2XII-9 during S/M phase. CUT&Tag chromatin profiling, conditional knockdown, invasion and egress assays mBio Medium 39207100

Source papers

Stage 0 corpus · 29 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS pathogens 327 16244709
2011 The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites. PLoS pathogens 268 21347343
2009 Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS pathogens 238 19247437
2011 The moving junction of apicomplexan parasites: a key structure for invasion. Cellular microbiology 227 21535344
2005 The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion. Cellular microbiology 172 16309467
2011 Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science (New York, N.Y.) 155 21778402
2012 Transcriptomic analysis of toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts. PloS one 140 22347997
2014 Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites. Nature communications 128 24934579
2011 The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex. PLoS pathogens 105 21347354
2018 A systematic review of Toxoplasma gondii antigens to find the best vaccine candidates for immunization. Microbial pathogenesis 58 30399440
2017 Efficient invasion by Toxoplasma depends on the subversion of host protein networks. Nature microbiology 50 28848228
2014 RON5 is critical for organization and function of the Toxoplasma moving junction complex. PLoS pathogens 47 24651769
2013 Toxoplasma gondii sporozoites invade host cells using two novel paralogues of RON2 and AMA1. PloS one 46 23940612
2014 The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force. BMC biology 32 25551479
2015 Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA-RON2 pair. Proceedings of the National Academy of Sciences of the United States of America 29 26712012
2015 Protective immunity induced by peptides of AMA1, RON2 and RON4 containing T-and B-cell epitopes via an intranasal route against toxoplasmosis in mice. Parasites & vectors 28 25582167
2023 Invasion of Toxoplasma gondii bradyzoites: Molecular dissection of the moving junction proteins and effective vaccination targets. Proceedings of the National Academy of Sciences of the United States of America 24 36693095
2016 Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail. mBio 20 27624124
2012 Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii. PloS one 18 22427839
2017 RON4L1 is a new member of the moving junction complex in Toxoplasma gondii. Scientific reports 16 29263399
2015 An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1) Governs Ligand Binding Selectivity. PloS one 16 25955165
2016 Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1). Genomics & informatics 11 27445648
2024 Cell cycle-regulated transcription factor AP2XII-9 is a key activator for asexual division and apicoplast inheritance in Toxoplasma gondii tachyzoite. mBio 8 39207100
2022 Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation. International journal for parasitology. Drugs and drug resistance 7 36512904
2017 The moving junction protein RON4, although not critical, facilitates host cell invasion and stabilizes MJ members. Parasitology 6 28662729
2018 Deciphering ophthalmic adaptive inhibitors targeting RON4 of Toxoplasma gondii: An integrative in silico approach. Life sciences 5 30326220
2025 The invasion pore induced by Toxoplasma gondii. EMBO reports 2 40973829
2025 In silico evaluation of Toxoplasma gondii rhoptry neck proteins (TgRONs) for potential immunogenic epitopes. EXCLI journal 1 40881926
2024 The invasion pore induced by Toxoplasma gondii. bioRxiv : the preprint server for biology 1 39416144