MIC6

TgMIC6 is a type I transmembrane micronemal protein of Toxoplasma gondii that organizes and traffics a heterotrimeric adhesive complex used for host cell invasion (PMID:11157983). Through its lumenal EGF-like domains it recruits the soluble adhesin MIC1, which in turn binds MIC4 as passive cargo, and MIC6's cytoplasmic tail supplies the sorting signal that targets the assembled MIC1-MIC4-MIC6 complex to the micronemes (PMID:11157983). Structural work resolved this recruitment at atomic resolution: two analogous sites within the EGF2 and EGF3 domains of MIC6 each engage the C-terminal galectin-like domain of MIC1, and this MIC1 interaction stabilizes MIC6 and provides quality control for exit from early secretory compartments (PMID:16166092, PMID:18818666). During transport MIC6 is proteolytically matured at its N-terminus, and upon microneme discharge at the parasite surface it is shed by regulated intramembrane cleavage within its transmembrane domain at a conserved apicomplexan motif by the rhomboid protease ROM4 (PMID:11861763, PMID:11927542, PMID:25846828). The MIC6 cytoplasmic tail links the adhesive complex to the actomyosin invasion motor by binding aldolase and the parasite cytoskeleton via a critical tryptophan residue W348, although this aldolase interaction is dispensable for invasion (PMID:19308454, PMID:20545864, PMID:25223060). Beyond its role in invasion, MIC6 is a secreted virulence effector: its EGF domains activate host EGFR-Akt-mTOR signaling to suppress autophagy-mediated killing of the parasite, and knockout reduces virulence in vivo (PMID:24367261, PMID:39116491).

1. 2001 High

   Established that MIC6 is not merely a surface adhesin but the transmembrane escorter that traffics the soluble adhesins MIC1 and MIC4 to the micronemes, defining the architecture of an adhesive complex.

   Evidence Reciprocal gene disruption of MIC1/MIC4/MIC6, domain mapping and secretory pathway analysis in T. gondii

   PMID:11157983

   Open questions at the time

   • Molecular identity of the EGF domain(s) mediating MIC1 binding not yet resolved
   • Cytoplasmic tail sorting signal not mapped to specific residues
2. 2002 High

   Defined MIC6 as a type I membrane protein subject to two distinct proteolytic events — N-terminal maturation and surface intramembrane cleavage — separating trafficking maturation from adhesin shedding.

   Evidence Site-specific mutagenesis of cleavage sites, pulse-chase, fractionation and fusion protein assays in T. gondii

   PMID:11861763

   Open questions at the time

   • Protease responsible for intramembrane cleavage not identified
   • Functional consequence of shedding for invasion not tested
3. 2002 High

   Localized the intramembrane cleavage to a conserved IA↓GG motif within the transmembrane domain and showed the cleavage machinery (MPP1) is constitutively active at the surface and recognizes the TM domain independent of the ectodomain.

   Evidence Site-directed TM-domain mutagenesis and chimeric SAG1-TM fusion cleavage assays in T. gondii

   PMID:11927542

   Open questions at the time

   • MPP1 enzyme not yet identified as a specific protease
   • Regulation of cleavage timing during discharge unresolved
4. 2005 Medium

   Identified rhomboid proteases as candidate MPP1 enzymes for microneme TM-domain cleavage, narrowing the search for the physiological MIC6 sheddase.

   Evidence Heterologous rhomboid cleavage assays with chimeric MIC TM-domain substrates in T. gondii

   PMID:15913633

   Open questions at the time

   • MIC6 transmembrane domain specifically not tested (only MIC2/MIC12)
   • In vivo protease responsible for MIC6 shedding not established
5. 2005 High

   Provided the structural and functional basis for complex quality control by showing the MIC1 galectin-like domain binds and stabilizes MIC6, coupling assembly to secretory exit.

   Evidence Crystal structure of MIC1 C-terminal domain with binding, immunofluorescence and invasion assays in T. gondii

   PMID:16166092

   Open questions at the time

   • MIC6 residues contacting MIC1 not yet defined
   • Stoichiometry of the assembled complex not addressed
6. 2008 High

   Resolved the MIC6-MIC1 interface at atomic resolution, revealing two analogous MIC1-binding sites in the EGF2 and EGF3 domains of MIC6 and defining a new mode of EGF domain recognition.

   Evidence NMR solution structure of the MIC6-MIC1 complex with resonance assignments, plus immunofluorescence and invasion assays in T. gondii

   PMID:18818666 PMID:19636898 PMID:19636901

   Open questions at the time

   • Whether both EGF sites act cooperatively in vivo not resolved
   • Contribution of each site to receptor recognition versus complex stability not disentangled
7. 2009 Medium

   Connected the adhesive complex to the invasion motor by showing the MIC6 cytoplasmic tail binds aldolase and the parasite cytoskeleton.

   Evidence GST pulldown of the MIC6 cytoplasmic tail with tachyzoite lysate and recombinant aldolase in T. gondii

   PMID:19308454

   Open questions at the time

   • Single-lab pulldown without genetic confirmation of functional relevance
   • Aldolase-binding residues not yet mapped
8. 2010 Medium

   Tested the functional necessity of the aldolase link and found it dispensable for invasion, distinguishing MIC6 from AMA1 and pointing to ectodomain-mediated interactions for some trafficking steps.

   Evidence Mutagenesis of aldolase-binding residues with invasion and rhomboid cleavage assays in T. gondii

   PMID:20545864

   Open questions at the time

   • Identity of the ectodomain-interacting trafficking partners unknown
   • Redundant motor-linkage mechanisms not identified
9. 2014 Medium

   Pinpointed the aldolase-binding determinant to tryptophan W348 in the MIC6 cytoplasmic tail.

   Evidence Site-directed W348V mutagenesis with GST pulldown against parasite lysate and recombinant aldolase in T. gondii

   PMID:25223060

   Open questions at the time

   • In vitro binding assay only; in vivo consequence of W348V not established
   • Single method set from a single lab
10. 2015 High

    Identified ROM4 as the physiological rhomboid protease that sheds MIC6 (and MIC2), linking surface intramembrane cleavage to the switch from attachment to productive invasion.

    Evidence Cre-mediated ROM4 knockout and CRISPR ROM5 knockout with substrate cleavage, motility and invasion assays in T. gondii

    PMID:25846828

    Open questions at the time

    • Whether ROM4 is the sole MIC6 sheddase not fully excluded
    • Structural basis of ROM4 substrate selection for MIC6 not addressed
11. 2013 High

    Revealed a second, effector role for MIC6 beyond adhesion: its EGF domains directly activate host EGFR-Akt signaling to block autophagic targeting of the vacuole and prevent parasite killing.

    Evidence MIC1/MIC3 knockouts deficient in EGF-MIC secretion, recombinant MIC6 stimulation, EGFR/Akt readouts, LC3 and parasite killing assays in T. gondii

    PMID:24367261

    Open questions at the time

    • Direct MIC6-EGFR binding interface not mapped
    • Whether the same EGF sites used for MIC1 binding engage EGFR unresolved
12. 2024 Medium

    Established MIC6 as an in vivo virulence factor in the Chinese 1 WH3 strain, acting through EGFR-Akt-mTOR-mediated autophagy suppression and contributing to evasion of CD40L-dependent immunity.

    Evidence CRISPR/Cas9 MIC6 knockout in WH3, mouse infection, cytokine and proliferation assays, and CD40L depletion in T. gondii WH3

    PMID:39116491

    Open questions at the time

    • No direct biochemical confirmation of the signaling mechanism in this strain
    • Relative contribution of adhesion versus immune-evasion roles to virulence not separated

• How MIC6's EGF domains partition between their intramolecular role in MIC1 binding/complex assembly and their extracellular role in host EGFR activation remains unresolved.
• No structural map of a MIC6-EGFR interaction
• Ectodomain trafficking partners inferred but unidentified
• Whether shed versus membrane-bound MIC6 mediates EGFR signaling not determined