A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

Abstract

The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identify here a novel protein that localizes to discrete punctae that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis shows that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then use deletion analyses to identify a domain in the N-terminus of the protein that is critical for both localization and function. Finally, we use the protein as bait for in vivo biotinylation which identifies several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, this data reveals an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggests that the sutures punctae are likely a portal for trafficking cargo across the IMC.