The Toxoplasma Vacuolar H+-ATPase regulates intracellular pH, and impacts the maturation of essential secretory proteins

Abstract

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

Biogenesis and discharge of the rhoptries: Key organelles for entry and hijack of host cells by the Apicomplexa

“…Genetic disruption or chemical inhibition of these maturases lead to severe blocks in invasion and egress (Nasamu et al., 2017; Pino et al., 2017). Of relevance, parasite depleted in vacuolar‐proton ATPases (V‐H+‐ATPases) exhibited a delay in ASP3‐dependent maturation and defective rhoptry function (Stasic et al., 2019), probably due to the absence of proper pre‐rhoptry acidification (Yakubu et al., 2018) needed for protease activity and condensation of the organelle content. Another class of proteins has been recently described as being important for secretory organelles maturation and morphology.…”

An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion

“…Despite its importance, the VAC has not been well characterized. Only a few proteins have been localized to the VAC/PLV [4,10,14–17]. The VAC exists as a prominent intact organelle during initial infection and subsequently fragments during intracellular replication, based on staining of TgCPL, a major luminal protease in the VAC [4].…”

Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V‐H+‐ATPase in Toxoplasma gondii

“…These results indicate that proton transport at the plasma membrane regulates Ca 2+ influx, possibly by controlling the parasite plasma membrane potential (Stasic et al., 2019) or by coupling H + ejection with Ca 2+ influx (Schwiening et al., 1993b) by the plasma membrane Ca 2+ /H + counter‐transporting ATPase TgA1 (Luo et al., 2001).…”

“…Previous work localized the V‐ATPase to the plasma membrane, PLV, and immature rhoptries of T. gondii (Stasic et al., 2019). Additional vesicles were also observed that were not identified.…”