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

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 pentamericPlasmodiumexport element (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 theP. falciparumblood-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', RIL↓DE). 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.

Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites

“…Based on this, we propose a model in which the N-terminal region of a protein with a small, polar residue at the extreme N-terminus is recognized by the PTEX (potentially HSP101 or an accessory factor) and thereby targets these for export, regardless of the process by which the N-terminus has been generated. Proteins that feature a large, charged residue at the N-terminus, such as many surface proteins, PV proteins, and PVM proteins that are processed by signal peptidase and, as shown here and by Fierro et al ( 72 ), a subset of PM V substrates are not recognized and hence are not exported, thereby preventing improper removal of proteins from either the surface of the parasite or the PV or clogging the PTEX with parasite surface proteins. The fate of proteins that feature at their N-terminus an amino acid that is present in exported and surface proteins may be influenced by the residues that follow the N-terminal residue, as these also play an important role in determining the localization of parasite proteins ( 31 , 35 , 73 ).…”

“…However, PV6 is the first native PM V substrate identified that has a demonstrated essential function in the PV. A recent study by Fierro et al revealed that the P. falciparum protein UIS2 is also cleaved by PM V but is not exported ( 72 ); notably, the N-terminal residue of this protein after processing is a D, whereas the N-terminal residue of the Plasmodium berghei orthology is a Q (export of this ortholog was not determined in the blood stages, however) ( 45 , 72 ). These findings indicate that processing of a protein by PM V does not inevitably result in protein export and that processing by PM V and recognition of proteins for export seem to be two separate events that are unlikely to be mechanistically coupled.…”

Comparative analysis of codon usage patterns of Plasmodium helical interspersed subtelomeric (PHIST) proteins

“…PHIST is a unique protein family of Plasmodium falciparum and the PHIST proteins are characterized by a conserved domain of approximately 150 amino acids that are predicted to form four consecutive alpha helices. Some members of this family consist of an export signal sequence, the PEXEL motif, and the PHIST domain, while others also contain a DnaJ domain and tryptophan residue ( Fierro et al, 2023 ; Hasan et al, 2023 ). Besides, the PHIST family includes different subgroups, such as PHISTa, PHISTa-like/PHIST, PHISTb, PHISTb-DnaJ, and PHISTc, which plays crucial roles in the parasite’s biology, including host cell invasion, immune evasion, and modulation of the host immune response ( Warncke et al, 2016 ; Kumar et al, 2019 ; Mutisya et al, 2020 ; Yang et al, 2020 ).…”