The mature N-termini of Plasmodium effector proteins confer specificity of export

The intraerythrocytic malaria parasite Plasmodium falciparum exports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the endoplasmic reticulum (ER) by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Plasmodium Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV-resident proteins, and are selectively translocated out into the RBC. The mature N-terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export or whether any means of generating the mature N-terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N-terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N-terminus is necessary for export. IMPORTANCE: Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.