An ER-Inner Membrane Complex Bridge via VAP-VPS13A-DAT1 Drives Daughter Budding in Toxoplasma gondii

All alveolates including apicomplexa parasites contain an inner membrane complex (IMC) underneath the plasma membrane. The IMC is synthesized de novo during the daughter cell budding (endodyogeny) within the mother cell and serves as a crucial scaffold for supporting cytoskeletal structures and the glideosome machinery for the parasite locomotion. However, the mechanism(s) underlying the membrane biogenesis in the IMC are not well understood. Using clinically-relevant and globally-prevalent pathogenic protist model, Toxoplasma gondii, we identified the TgVAP-TgVPS13A-TgDAT1 complex bridging the IMC to the endoplasmic reticulum (ER) - the major site of phospholipid synthesis. Individual components of this complex play a crucial role in the IMC biogenesis, where the multi-modular TgVPS13A protein interacts with the lipid scramblase TgDAT1 in the IMC via its C-terminal VAB domain, and with the ER-resident TgVAP through its N-terminal region. DAT1 is recruited for the progeny formation sites during the early stages of budding. Conditional depletion of TgVPS13A, TgDAT1 or TgVAP results in collapse of the inner membrane complex, leading to parasite death, as visualized by endodyogeny-specific organelle markers. LactC2-GFP, a biosensor of phosphatidylserine and phosphatidylthreonine lipids made in the ER and enriched in the IMC, also mislocalizes upon protein depletion. In conclusion, we propose that TgVAP-TgVPS13A-TgDAT1 bridge the ER and IMC and mediate the inter-organelle transport of lipids, thus contributing to the organelle biogenesis and daughter budding in T. gondii. Author SummaryThe inner membrane complex (IMC) in apicomplexan parasites is essential for maintaining the structural stability of the parasite, as well as for its budding and motility. The IMC is composed of flattened vesicles, alveolins, and microtubules. However, the mechanism behind the biogenesis of these flattened vesicles remains unclear. In this study, we provide evidence that a protein complex formed by TgVAP, TgVPS13A, and TgDAT1 exists between the endoplasmic reticulum (ER) and the IMC. Phenotypic analysis indicates that the absence of any individual component of this protein complex disrupts the biogenesis of daughter IMCs, which in turn affects the budding of daughter parasites. We also demonstrated that TgDAT1 functions as a scramblase. Based on our findings, we propose a model in which the TgVAP-TgVPS13A-TgDAT1 complex mediates lipid transport from the ER to the nascent IMC, driving the expansion of the IMC membrane and the budding of daughter parasites in T. gondii. Furthermore, TgDAT1 may facilitate the transfer of lipids from the outer leaflet to the inner leaflet of the IMC sacs, promoting a balanced composition of the IMC membrane components.