Keywords.
Plasmodium falciparum , malaria, protein export, PTEX, virulence, PEXEL, chaperone.
Introduction
The pathogenesis of malaria lies upon the ability of Plasmodiumparasites, the causative agent of the disease, to propagate asexually within red blood cells (RBCs) while avoiding the immune mechanisms of its host. To accomplish this, the parasite exports hundreds of its proteins into its host cell to transform it into a hospitable niche and to avoid host immunity 1-5. The intraerythrocytic stage of the parasite resides within a membranous compartment called the parasitophorous vacuole (PV) 6,7. Consequently, parasite proteins destined for export must traverse two membranes, the parasite plasma membrane and the encasing parasitophorous vacuole membrane (PVM), to reach the host RBC. The Plasmodium t ranslocon of ex ported proteins (PTEX) mediates the translocation of proteins across the PVM 8 and is the only protein channel known to reside at the PVM. PTEX is comprised of three core components, HSP101, PTEX150, and EXP2, that are all essential for parasite survival, making it an attractive drug target candidate8-10.
Most parasite proteins destined for export into the RBC begin their journey with entry into the endoplasmic reticulum (ER) via the Sec61 translocon, followed by cleavage of the protein within the ER by the aspartyl protease, plasmepsin V (PMV) 11-13. The cleavage step occurs within a pentameric amino acid motif near the N-terminus of the exported protein destined for export, termed thePlasmodium export element (PEXEL) 1,2,14. The resulting mature proteins then travel via the vesicular transport pathway to the parasite plasma membrane where they are secreted into the parasitophorous vacuole 15. Here the exported proteins are unfolded and translocated across the PVM and into the host-cell compartment in an ATP-dependent manner by PTEX 16. It is thought the AAA+ ATPase HSP101 first engages and unfolds cargo proteins in an ATP-dependent manner and threads them through a tetradecamer membrane-spanning channel consisting of the scaffold protein PTEX150 and the pore-forming protein EXP217-19. However, the information that is contained within proteins destined for export that allows HSP101 to specifically recognise these proteins prior to the unfolding step is still unknown20,21.
Historically, the N-terminal region of exported proteins (and the protein’s transmembrane domain in some cases) have been found to be sufficient to mediate the trafficking of proteins into the RBC compartment 22-26. This N-terminal region usually contains a recessed signal peptide to promote entry of the proteins into the parasite’s ER 27 or a hydrophobic stretch of amino acids that serve the same function 28. The discovery of the five-amino acid PEXEL motif in the N-terminal region of many exported proteins was the first export-specific signal1,2 that was proven to be predictive for exported proteins 29 and allowed the identification of >450 putative exported proteins in P. falciparum4. Interestingly, however, the presence of the PEXEL motif alone does not guarantee export, as its location within a protein’s primary structure 30 and the presence of ~12 amino acids downstream of the motif have also been shown to be required to achieve efficient export 24. Furthermore, PEXEL-negative exported proteins (PNEPs) are also present in the Plasmodium exportome 5,31,32, suggesting that PEXEL is not strictly required for passage through PTEX.
The function of the PEXEL motif has been relatively well studied and its consensus sequence is RxLxE/D/Q where x can be almost any amino acid. The position P3 R and P1 L residues are the most conserved of the PEXEL motif and in P. falciparum have been shown to be necessary for efficient cleavage by PMV12-14. It is assumed therefore that P1and P3 facilitate PEXEL cleavage and thus help to release the exported proteins from the ER membrane where they are originally anchored following ER import 11. In comparison, the purpose of the last conserved P2’ (E/Q/D) residue of PEXEL is less certain. Some evidence suggests that following cleavage, the P1’ and P2’ resides which now cap the mature PEXEL protein, play a role in promoting export across the PVM 1,14,33. After cleavage, the exposed xE/Q/D motif becomes N-terminally acetylated34 resulting in an Ac- xE/Q/D cap that was thought to serve as a ’barcode’ or a recognition motif for HSP101 engagement in the PV 35. Contrary to this, however, other studies have found that P2’ amino acid also influences the efficiency of PEXEL processing or does not influence export at all36-38.
Here, we present evidence that the P2’ residue has a dual function. For some PEXEL proteins, P2’ mutations greatly reduce PMV cleavage increasing protein retention in the ER and reducing export. For other proteins, P2’ mutations do not inhibit PMV cleavage as much and PEXEL proteins can reach the PV but are translocated less efficiently. We also provide evidence that the length of the region downstream of the PEXEL motif universally regulates the degree of cargo interaction with HSP101 and ultimately affects protein export across the PVM.
Results