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.