Abstract 1984 Towards a Biochemical Understanding of Toxoplasma GRA chaperoning

Toxoplasma gondii is an obligate intracellular parasite that can infect all warm-blooded animals. It owes much of its success to the battery of secreted effectors that allow it to develop its replicative niche, the parasitophorous vacuole (PV), and evade host immune clearance. The proteins secreted from Toxoplasma's dense granules, termed GRAs, are a class of effectors that carry out these functions in the PV, at the PV membrane (PVM), and in the host cytosol. A subset of these GRAs is responsible for reshaping the PVM into a tubulovesicular network that is necessary for parasite virulence. We refer to this membranous superstructure as the intravacuolar network (IVN). While the exact role of the IVN is undefined, genetic phenotypes have implicated that it mediates diverse processes including nutrient uptake and effector export across the PVM. Interestingly, despite having typical traits of integral membrane proteins, the GRAs that build and decorate the IVN are trafficked through the parasite secretory system as soluble, heteromultimeric complexes. This regulation of membrane insertion and switch in solubility suggest that there are chaperoning interactions that keep these GRAs out of surrounding membranes until they reach the IVN. Our group previously demonstrated that the kinase WNG1 is essential for correct membrane integration for a subset of IVN GRAs. We hypothesize that phosphorylation by WNG1 regulates the release of these IVN GRAs from their chaperoning interactions at the PVM, allowing them to insert into the membrane and develop the IVN. The goal of my project is to define the inter- and intra-molecular interactions that regulate membrane association for these GRAs. To determine the role of WNG1 phosphorylation on these IVN GRAs, we generated parasite strains in which endogenous WNG1 phosphosites were mutated to alanine. Interestingly, while we observed a similar shift in membrane insertion as in the Δwng1 strain, these experiments identified a subcomplex between two IVN GRAs. To identify additional WNG1 dependent chaperoning interactions, I am using immunoprecipitation of soluble versus membrane bound GRAs coupled with mass spectrometry. Taken together, the data from these experiments will provide mechanistic details on both Toxoplasma dense granule trafficking in the PV and IVN biogenesis. Using this information, I aim to develop an in vitro system that reconstitutes the release of chaperoning and insertion into the membrane for the IVN GRAs. I will further use this system to determine structures of both the chaperoned complexes and the GRA oligomers that form the IVN tubules.