Toxoplasma gondii replicates within a protective organelle called the parasitophorous vacuole (PV). The PV is ﬁlled with a network of tubulated membranes, which are thought to facilitate trafﬁcking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network's functions. Phosphoproteomics data indicated that many PV resident proteins are phosphorylated, including ones that have roles is the biogenesis and maintenance of these tubular networks. Since phosphorylation is a common method to regulate protein functions we hypothesized that PV resident kinases must regulate PV/IVN functions. Protein kinases are enzymes that modify proteins with phosphate molecules. Using bioinformatics, we identiﬁed an unusual family of kinases (WNG kinases) that lack a structural motif, called the Gly-loop, that is absolutely required for the activity of all previously described kinases. In this work, I show that the most conserved WNG kinase, WNG1 as well as WNG2 are catalytically active. The WNG kinases are only found in certain intracellular parasites, such as the human pathogen Toxoplasma gondii. I identify the parasite secreted kinase WNG1 as a critical regulator of tubular membrane biogenesis. By solving the crystal structure of a pseudokinase in the WNG1 family, I show members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, I find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, I show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, my results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.
Membranes, Protein Kinases, Toxoplasma, Vacuoles
University of Texas Southwestern Medical Center
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