Files
Abstract
Toxoplasma gondii is an obligate intracellular parasite that infects up to one-third of the world’s population and causes Toxoplasmosis. Infection is life long and can even be life threatening to immunocompromised individuals and to the fetus of an infected mother. During acute infection, the parasite proliferates asexually by engaging in the lytic cycle, which involves the parasite invading, replicating, and egressing from host cells. T. gondii can infect virtually any nucleated cell, destroying the cells upon egress. During the lytic cycle, the parasite is exposed to dramatic ionic changes in its surrounding environment. The parasite survives these changes by employing several mechanisms to help it resist ionic stress, one of these being the evolutionary acquisition of the plant-like vacuole (PLV). The membrane of this organelle features the Vacuolar Proton-ATPase (V-ATPase), a multi-subunit enzyme responsible for transporting protons into the lumen of the PLV. Previous work from our lab has characterized this enzyme as being essential to the parasite lytic cycle. I performed co-immunoprecipitations to identify constitutive interactors of the V-ATPase in T. gondii. These studies identified a potentially novel subunit of the T. gondii V-ATPase. To study transient interactors of the complex, I utilized a novel, mutated form of the bacterial biotin ligase (BirA), termed TurboID. A hypothetical protein with homology to a yeast V-ATPase assembly factor, Vma21, was discovered. Depletion of this protein resulted in degradation of subunit a1 of the V-ATPase and subsequent functional defects in V-ATPase activity. This work identifies a potentially novel subunit of the T. gondii V-ATPase and also sheds light on conserved trafficking, regulatory and assembly mechanisms of the V-ATPase found in T. gondii.