De novo mapping of the apicomplexan Ca ²⁺ -responsive proteome

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 transition between the replicative and lytic phases of the infectious cycle. 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 T. 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 (PP ₁ ) as a Ca ²⁺ -responsive enzyme that relocalized to the parasite apex upon Ca ²⁺ store release. Conditional depletion of PP ₁ revealed that the phosphatase regulates Ca ²⁺ uptake to promote parasite motility. PP ₁ may thus be partly responsible for Ca ²⁺ -regulated serine/threonine phosphatase activity in apicomplexan parasites.