2.1 A systematic review of PEXEL motif mutants reveals that a single point mutation of P2’ into a positively charged residue produces the most consistent export-blocking effect.
To better understand the contribution of the PEXEL P2’ position on protein export, we conducted a review of P2’ mutagenesis experiments that have been performed on various PEXEL proteins (Table S1). We sorted the mutants based on the amino acids incorporated into the P2’ position and linked it to the export phenotype, PEXEL processing pattern and the N-terminal acetylation (Table S1 ’PMV cleavage’ and ’N-acetylation’) reported for the corresponding mutant. This in general revealed that alanine (A) mutations in the P2’ position have a limited or uncertain effect on export (Table S1). For example when the P2’ position of KAHRP (knob-associated histidine rich protein, PF3D7_0202000) was exchanged with an A, the protein was not exported and was retained in the parasite 39. This was contrary to the study by 14, where the reporter was mostly trapped in the PV, despite both groups using very similar constructs (i.e. the first 69aa of KAHRP leader sequence fused to GFP). The same discrepancy was also observed with P2’ A mutations of STEVOR and GBP130 (PF3D7_1016300) (Table S1), Rather than regard these findings as contradictory, the different localisation of P2’ A mutants could arise because of the continuum from the ER to the PV that might be dependent on the parasite age and the different module(s) appended downstream of the PEXEL motif36,38 . P2’ A mutagenesis study on other PEXEL proteins, such as RESA (PF3D7_0102200), PfEMP3 (PF3D7_0201900), PF3D7_0532600, and murine P. berghei CP1 (PBANKA_1246500) did not result in any export defect (Table S1).
Mutation of the P2’ PEXEL position to positively charged amino acids appears to produce the most robust export-blocking effect on exported proteins tested in two different studies (Table S1)36,40. Mutation to basic amino acids would usually reverse the charge of the P2’ position, given that the two most common P2’ residues are glutamic and aspartic acids. Curiously, the P2’ lysine (K) mutant of REX3 (PF3D7_0936300) PEXEL also moderately inhibited PMV cleavage of the motif 36, suggesting that P2’ may also be involved in PEXEL processing, albeit to a lesser extent than P1 and P3.
P2’ mutations can also cause changes to the N-acetylation profile of the mature protein. However, N-terminal acetylation is not sufficient to determine the fate of exported proteins, as suggested previously 12,36,38. Whilst it is unclear whether PEXEL proteins without N-terminal acetylation can be exported or not, having an N-terminal acetylation by itself does not guarantee successful export, as can be seen in the case of P2’ A mutant of GBP130 and P2’ K of STEVOR (PF3D7_0631900) which fail to be exported despite being shown to be N-terminally acetylated (Table S1)14. We therefore conclude that substituting the conserved P2’ PEXEL reside with a basic amino acid would likely produce a more pronounced export-blocking phenotype in the parasite.