EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between parasite and host compartments. The PVM is the site of several essential transport activities but the basis for organization of this membrane system is unknown. We utilized the second-generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of the PVM, identifying known and novel candidate PVM proteins. Among the best represented hits were members of a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated the function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by profound changes in vacuole ultrastructure, including increased separation of the PVM and PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM organization.