Affinage · Toxoplasma gondii

PPM3C

protein phosphatase 2C domain-containing protein · UniProt A0A125YZH2

Annotated
2026-09-08
2 papers in source corpus 2 papers cited in narrative 5 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TgPPM3C is a secreted PP2C-class serine/threonine phosphatase of Toxoplasma gondii that acts within the parasitophorous vacuole (PV) to control selective export of parasite effectors into the host cell (PMID:33370417). It was identified as a MYR1-interacting protein localized to the PV (PMID:32075880), and phosphoproteomics of knockout parasites shows altered phosphorylation of vacuolar substrates including GRA16, GRA28, and MYR1, consistent with TgPPM3C dephosphorylating these PV proteins (PMID:33370417). Functionally, loss of TgPPM3C specifically blocks export of the GRA16 and GRA28 effectors across the PV membrane while sparing other MYR1-dependent effectors (PMID:33370417), and phosphomimetic substitution of two GRA16 serines reproduces the export defect, establishing that TgPPM3C-mediated dephosphorylation of GRA16 is a required step for its translocation into the host cell (PMID:33370417). This effector export is required for pathogenesis: TgPPM3C-deficient parasites are avirulent in acute mouse infection and form fewer brain cysts during chronic infection (PMID:33370417). Direct in vitro reconstitution of TgPPM3C phosphatase activity on its substrates has not been characterized in the available corpus.

Mechanistic history

Synthesis pass · year-by-year structured walk · 5 steps
  1. 2020 Medium

    Establishing what protein partners MYR1 in the PV identified TgPPM3C, connecting a phosphatase to the known MYR1-dependent effector export machinery.

    Evidence Affinity purification of MYR1 with mass spectrometry in T. gondii TGGT1

    PMID:32075880

    Open questions at the time
    • Single method (affinity purification/MS) without reciprocal orthogonal validation
    • Interaction stoichiometry and whether binding is direct not established
    • No functional consequence of the MYR1 interaction defined in this study
  2. 2020 High

    Knockout defined TgPPM3C as a secreted PP2C-domain phosphatase whose loss selectively blocks export of GRA16 and GRA28 but not other MYR1-dependent effectors, showing it acts as a substrate-selective export determinant.

    Evidence CRISPR knockout with complementation and effector export/localization assays in T. gondii

    PMID:33370417

    Open questions at the time
    • Basis of substrate selectivity for GRA16/GRA28 over other effectors unresolved
    • Whether TgPPM3C phosphatase activity per se is required not directly tested by catalytic-dead mutant here
  3. 2020 Medium

    Phosphoproteomics of the knockout revealed hyperphosphorylation of vacuolar substrates (GRA16, GRA28, MYR1), linking the export defect to a loss of dephosphorylation activity in the PV.

    Evidence Phosphoproteomic mass spectrometry of KO parasite cultures

    PMID:33370417

    Open questions at the time
    • No in vitro phosphatase reconstitution demonstrating direct dephosphorylation of these substrates
    • Cannot distinguish direct substrates from indirect phosphorylation changes
  4. 2020 High

    Phosphomimetic mutagenesis of two GRA16 serines recapitulated the export block, causally establishing that dephosphorylation of GRA16 is required for its translocation across the PV membrane.

    Evidence Site-directed phosphomimetic serine substitutions in GRA16 with export assay in T. gondii

    PMID:33370417

    Open questions at the time
    • Whether the same phospho-switch mechanism governs GRA28 export not shown
    • Kinase counteracting TgPPM3C on these sites not identified
  5. 2020 Medium

    In vivo infection linked the molecular export defect to pathogenesis, showing TgPPM3C is required for acute virulence and efficient chronic cyst formation.

    Evidence Mouse acute virulence and chronic brain cyst burden assays with KO parasites

    PMID:33370417

    Open questions at the time
    • Which exported effector(s) drive the virulence phenotype not dissected
    • Host pathway downstream of GRA16/GRA28 export in vivo not mapped

Open questions

Synthesis pass · forward-looking unresolved questions
  • Direct biochemical demonstration of TgPPM3C catalytic activity on GRA16/GRA28 and the structural basis of its substrate selectivity remain open.
  • No purified-enzyme phosphatase assay against defined substrates
  • No structure or catalytic-dead complementation defining the active site's role in export
  • Mechanism coupling dephosphorylation to membrane translocation unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
GRA16 (SUBSTRATE; DEPHOSPHORYLATION REQUIRED FOR EXPORT) [#2, #3]GRA28 (SUBSTRATE; EXPORT-DEPENDENT ON TGPPM3C) [#1, #2]MYR1 (INTERACTOR; PV-LOCALIZED) [#0]

Evidence

Reading pass · 5 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2020 PPM3C (TGGT1_270320) was identified as a MYR1-interacting protein localized to the parasitophorous vacuole (PV) of Toxoplasma gondii, discovered via affinity purification of MYR1 followed by mass spectrometry. Affinity purification / co-immunoprecipitation with MYR1, mass spectrometry mSphere Medium 32075880
2020 TgPPM3C is a secreted protein containing a PP2C-class serine/threonine phosphatase domain that localizes to the parasitophorous vacuole; parasites lacking TgPPM3C are defective specifically in the export of GRA16 and GRA28 effectors into the host cell, but not other MYR1-dependent effectors. Gene knockout (CRISPR/KO), effector export assays (GRA16, GRA28 localization), complementation PLoS pathogens High 33370417
2020 Phosphoproteomic analysis of TgPPM3C-deleted parasites revealed altered phosphorylation of secreted vacuolar proteins including GRA16, GRA28, and MYR1, indicating TgPPM3C functions as a phosphatase that dephosphorylates these PV substrates. Phosphoproteomics (mass spectrometry) of KO parasite cultures PLoS pathogens Medium 33370417
2020 Phosphomimetic mutation of two serine residues in GRA16 caused GRA16 export defects, establishing that dephosphorylation of GRA16 by TgPPM3C is a critical step required for its translocation across the PV membrane into the host cell. Site-directed mutagenesis (phosphomimetic serine substitutions in GRA16), effector export assay PLoS pathogens High 33370417
2020 TgPPM3C-deficient parasites are avirulent during acute infection in mice and form fewer cysts in mouse brain during chronic infection, indicating TgPPM3C-mediated effector export is required for full parasite virulence in vivo. Mouse infection assay (acute virulence, chronic cyst burden) with KO parasites PLoS pathogens Medium 33370417

Source papers

Stage 0 corpus · 2 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2020 Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells. mSphere 53 32075880
2020 Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export. PLoS pathogens 20 33370417