Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium -secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.
Obligate intracellular malaria parasites depend on protein secretion to penetrate, subvert, and exit their host cells. Despite this key reliance on the secretory pathway, none of the conditional mutagenesis tools available in Plasmodium spp. exploit the unique trafficking features of secreted proteins. Here we report knockER, a novel DiCre-mediated knock sideways approach that sequesters secreted proteins in the ER via conditional fusion of a KDEL ER-retrieval sequence to the target protein C-terminus. We applied knockER to a diverse set of reporters and endogenous P. falciparum and P. berghei proteins targeted to the rhoptries, dense granules, parasite vacuole and apicoplast and show conditional ER sequestration is not generally toxic, enabling loss-of-function studies. Taking advantage of the unique ability to redistribute secreted protein pools from their terminal destination to the ER, we employed knockER to study components of the PTEX translocon in an attempt to separate HSP101 vacuolar function in protein export from a recently proposed role in cargo recognition at the ER. Strikingly, while KDEL-fusion produced similar levels of ER retrieval for both HSP101 and the translocon adaptor PTEX150, parasite growth and effector export were completely unaffected by HSP101 retention while ER retrieval of PTEX150 produced a lethal export defect, indicating vacuolar HSP101 levels are uniquely maintained in excess. Intriguingly, redistribution of HSP101 to the ER did not further sensitize parasites to TetR-DOZI-mediated knockdown compared to parasites containing normal vacuolar levels of HSP101, suggesting PV-localized function does not fully account for HSP101 contribution to parasite fitness and consistent with an important role for HSP101 in the ER. Collectively, our work provides a novel tool for dissecting secreted protein function with sub-compartmental resolution that should be widely amenable to genetically tractable eukaryotes.
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