Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

Abstract

Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export.

The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans Is Cleaved before Secretion

“…However, the RxLR motif is not recognized and cleaved by the ER-located plasmepsin V protease of P. falciparum (Boddey et al, 2016). Furthermore, despite the phylogenetic relationship between plasmepsin V from P. falciparum and PiAP12 (Kay et al, 2011), we could not detect any proteolytic activity against recombinant AVR3a 22-147 for PiAP12 nor for any of the other 10 aspartic protease domains ( Figure 2C).…”

“…A fusion protein containing the effector core domain of AVR3a from P. infestans coupled to the PEXEL sequence of HRPII (histidine rich protein II) from Plasmodium falciparum is delivered into the plant by P. infestans (Grouffaud et al, 2008). The equivalent experiment in Plasmodium, the delivery of HRPII with the RxLR leader of AVR3a, was also reported to be successful (Bhattacharjee et al, 2006), although this observation was not reproducible in a recent study (Boddey et al, 2016). Nevertheless, RxLR motifs reside within flexible protein regions (Yaeno et al, 2011;Wawra et al, 2012b;Sun et al, 2013) and could be easily accessed by endopeptidases and modified by acetyltransferases.…”

“…This sequence element is cleaved by endoplasmic reticulum-located proteases and the N terminus becomes acetylated (Ac-Xaa-Glu/Asp/Gln) (Chang et al, 2008;Boddey et al, 2009Boddey et al, , 2010Russo et al, 2010). The conserved spatial position of the PEXEL motif and its very rapid proteolytic modification are necessary for the export of effector proteins and seem to work as an internal signal for a specialized effector export pathway (Marti and Spielmann, 2013;Boddey et al, 2016). Recently, a similar motif (TEXEL) was found in effector proteins of Toxoplasma gondii and reported to play a crucial role in effector maturation and sorting during the infection process (Hammoudi et al, 2015;Coffey et al, 2015;Curt-Varesano et al, 2016).…”

Role of the ER and Golgi in protein export by Apicomplexa

“…Export uses a two-step mechanism where an N-terminal signal peptide (SP) directs entry into the ER, followed by recognition and cleavage of a pentameric amino acid sequence called the Plasmodium export element (PEXEL) [10,11], usually located 15-30 amino acids from the SP [25,26,27 ]. PEXEL processing is performed by the ER membrane-resident aspartyl protease Plasmepsin V (PMV) [14,15] (Figure 2a).…”

“…An alternate mechanism has been proposed where the PEXEL binds phosphatidylinositol-3-phosphate (PI(3)P) in the ER, which is an unprecedented location for this lipid, independent of PMV activity [31]. However, subsequent studies have failed to confirm PI(3)P in the Plasmodium ER or detect PEXEL-lipid binding [27 ]. Exported cargo traffic through the Golgi [32] and out of the parasite, where they are unfolded [33] and translocated across the PVM through the Plasmodium translocon of exported proteins (PTEX) (Figure 2a) [16,17 ,18 ].…”