PROJECT SUMMARY/ABSTRACT Toxoplasma gondii has the remarkable ability to infect virtually any cell type of almost all warm-blooded animals and is arguably the most successful parasite on earth, having infected an estimated one-third of humans globally. While initial infection typically resolves without complication, the parasite is able to persist for the life of its host, and can re-emerge in the immunocompromised and immunosuppressed to cause fatal disease. Toxoplasma, like other apicomplexan parasites, must invade a host cell to survive and replicate. Once inside a host cell, the parasite survives and replicates within a specialized organelle called the parasitophorous vacuole. Disruption of the vacuole results in parasite death, and the parasite secretes a battery of proteins into the vacuole to facilitate its biogenesis and regulate trafficking of nutrients and effector proteins. A principle structure within the parasitophorous vacuole is the intravacuolar network of membranous tubules (the IVN), which is thought to act as a major trafficking apparatus. IVN biogenesis is formed by the direct action of oligomeric complexes of parasite proteins and mutants that disrupt the IVN show reduced virulence in animal models of infection. We have identified a parasite-specific protein kinase that regulates the membrane association of a subset of the proteins that associate with the parasitophorous vacuolar and IVN membranes, and deletion of this kinase results in vacuoles with aberrant IVN tubulation. While we have identified the kinase substrates and the sites of phosphorylation, the interactions that are regulated by this phosphorylation are unknown. The goal of the proposed studies is to determine the precise molecular mechanisms by which phosphorylation regulates the inter- and intra-molecular interactions that drive IVN biogenesis. First, we will determine how the components of the protein complexes that drive IVN biogenesis change as the complexes progress through the parasite secretory system and insert into the IVN membrane. We will use molecular genetic, cellular, and biochemical methods to determine the molecular mechanisms by which phosphorylation regulates these protein-protein interactions to facilitate IVN development. Furthermore, we will use innovative biophysical methods to generate the first structural models of these critical parasite protein complexes to determine the biophysical mechanism by which they induce IVN formation.