Asady, B. | bioRxiv Science

bioRxiv2019

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

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.

microbiology↗

bioRxiv2018

An ER-resident calcium binding protein modulates egress of malaria parasites from erythrocytes

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum Endoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic lifecycle revealed that PfERC is essential for parasite egress but not required for protein trafficking or Ca2+ storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1. Using double mutant parasites, we show that PfERC is required for the proteolytic maturation of the essential aspartic protease, Plasmepsin X, which cleaves SUB1. Further, we show that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish the ER-resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.

cell biology↗