Abstract Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting Motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N-terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream ∼10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position which constitutes the extreme N-terminal residue following PEXEL cleavage (P1’, RILτDE). Using a series of chimeric reporter fusions, we show that Asp at P1’ is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N-terminus mediates export, not PEXEL processing per se . Prompted by this observation, we further show that PEXEL sequences in the N-termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export. Importance Host erythrocyte remodeling by malaria parasite exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum , most exported proteins undergo proteolytic maturation via recognition of the pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) which exposes a mature N-terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

PEXEL is a proteolytic maturation site for both exported and non-exportedPlasmodiumproteins