Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca 2+ -responsive pathways.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca 2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca 2+ -responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca 2+ -responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca 2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca 2+ -responsive proteins, such as parasite Ca 2+ -dependent protein kinases. Our approach also identified numerous Ca 2+ -responsive proteins that are not predicted to bind Ca 2+ , yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca 2+ -responsive enzyme that relocalized to the parasite apex upon Ca 2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca 2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca 2+ -regulated serine/threonine phosphatase activity in apicomplexan parasites.