Running title: Toxoplasma V-H + -ATPase 2 HIGHLIGHTS • The V-H + -ATPase localizes to the plasma membrane and pumps protons outside the cell • The V-H + -ATPase localizes to pro-rhoptries in dividing parasites and to the plant-like vacuole in extracellular tachyzoites. • The Vacuolar-H + -ATPase supports the maturation of rhoptry and microneme proteins. • Loss of V-H + -ATPase activity leads to hyperaccumulation of immature rhoptry and microneme proteins and defective microneme distribution and secretion. SUMMARY Vacuolar-proton ATPases (V-H + -ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-H + -ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-H + -ATPase complex and discovered a novel dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-H + -ATPase subunits localize to the plasma membrane and to acidic vesicles and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-H + -ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a novel role for V-H + -ATPases in regulating virulence pathways. 3

SUMMARYVacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways.

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V‐H+‐ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrates in acidocalcisomes. This was supported by the co‐localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V‐ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content, impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

The universal role of calcium (Ca ) as a second messenger in cells depends on a large number of Ca -binding proteins (CBP), which are able to bind Ca through specific domains. Many CBPs share a type of Ca -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca measurements showed that Δtg216620 cells did not respond to extracellular Ca as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca influx while TgGT1_280480 may be playing a different role in the rhoptries.