The Toxoplasma Proteins MIC2 and M2AP Form a Hexameric Complex Necessary for Intracellular Survival

Toxoplasma gondii parasites gain entry into host cells through a process that depends on apically stored adhesins that are strategically released during invasion. One of these adhesins, microneme protein 2 (MIC2), is a type one transmembrane protein that binds to an accessory protein known as MIC2-associated protein (M2AP). Together the MIC2·M2AP complex participates in host cell attachment and invasion. The short cytoplasmic C-domain of MIC2 is implicated in protein trafficking and mediating an association with the parasite cytoskeleton. To define the role of the cytoplasmic domain of MIC2, proteins lacking the C-domain were expressed in transgenic T. gondii. Surprisingly, protein trafficking and secretion were not affected. We hypothesized that mutant mic2 lacking the C-domain might be escorted to the micronemes by association with endogenous wild-type MIC2 possessing functional transmembrane and cytoplasmic domains. To investigate this interaction, native blue gels and gel filtration were employed to identify a stable macromolecular MIC2·M2AP complex of ∼450 kDa. Our findings reveal that MIC2 and M2AP proteins form stable hexamers consisting of three αβ dimers. Resolution of this complex has implications for how MIC2·M2AP associates with host cell receptors and the cytoskeleton to facilitate parasite motility and invasion.