Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the "Vacuolar Compartment/Plant-Like Vacuole" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T . gondii . The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T . gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite’s endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in Δ TgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T . gondii .

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca concentration is tightly regulated at the physiological cytosolic concentration of 50-100 nm. The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high-performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy-terminus tagging plasmid containing the previously characterized "spaghetti monster-HA" (smHA) or "spaghetti monster-MYC" (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.

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

Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites. IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.