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

Abstract

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.

Keywords: PEXEL; PTEX; Plasmodium falciparum; malaria; plasmepsin; protein export; signal peptide.

Fig 1

Minimal reporter constructs and their localization. (A) Schematic representation of minimal reporter constructs. (i), (ii), and (iii) are PEXEL reporters and (iv) is a PV-resident reporter. Different segments of PEXEL reporters are delineated by dashed lines and labeled at the bottom for (iv). Residue numbers starting from the nascent N-terminus are printed within each segment and the PEXEL motif residues are highlighted in bold red. (B) Compartment fractionation strategy. RBCM, red blood cell membrane; PVM, parasitophorous vacuole membrane; PPM, parasite plasma membrane. (C) Representative western blots of different fractions from RBCs infected with P. falciparum expressing the reporters from A. Primary antibodies that were used to probe the blots are labeled at the left. The bottom band (marked with asterisks) in the anti-GFP blots is the free GFP band devoid of mature reporter portions. Each construct was tested at least three times.

Fig 2

Investigation of the role of the PEXEL motif in protein export to the RBC. (A) Schematic representation of KAHRP and SERA5 fusion reporters (ii and iii). Original reporters (i and iv) are also shown for reference. (B) Representative western blots of different fractions from RBCs infected with P. falciparum expressing the reporters from A. Primary antibodies that were used to probe the blots are labeled at the left. The experiment was performed twice. (C) Representative western blots of different fractions from RBCs infected with P. falciparum expressing KAHRP and GBP130 reporters with alanine substituted semi-conserved fifth positions of the PEXEL motif. Each construct was tested twice.

Fig 3

Processing, export, and comparison of the protein level of different EMP3 reporters. (A) Schematic representation of the EMP3 reporter constructs. An alternative mature N-terminus for the last construct is marked at the bottom (B) Representative western blots of different fractions from RBCs infected with P. falciparum expressing the reporters from A. Primary antibodies that were used to probe the blots are labeled at the left. Note the presence of an alternative mature form (marked with a purple arrow) for the last reporter in the parasite fraction. (C) Normalized western blot quantification of the standard mature forms of the reporters. Signals were combined from each fraction and then normalized to the PM V signal from the parasite fraction. * denotes a P ≤ 0.05 and ** denotes P ≤ 0.01 in Fisher’s LSD test. The P-value for the one-way ANOVA was 0.0113. Mean and standard deviations from two or three biological replicates are shown along with individual data points.

Fig 4

Primary sequences and putative structures of PEXEL and PV-resident proteins. (A) Amino acid frequency plots of 59 experimentally validated exported PEXEL proteins starting from the first position of the PEXEL motif to the tenth position of the mature N-terminus. (B) Frequency plot of AlphaFold structural predictions of the same residues shown in panel (A), Where “A” denotes alpha-helical, “B” denotes beta-sheet, and “C” denotes random coil. (C) Amino acid frequency plots of 13 experimentally validated PV-resident proteins starting from the −3 position of the signal peptide cleavage site to the tenth position of the mature N-terminus. (D) Frequency plot of AlphaFold structural predictions of the same residues shown in panel (A), with the letters denoting the same structural conformation as in panel (B).

Fig 5

Investigation of the role of alpha-helical mature N-terminus in protein export. (A) Representative western blots of different fractions of the KAHRP and EMP3 reporters with proline insertions at the third or the sixth position of their mature N-terminal region. The experiment was performed twice. (B) CD spectra of two small peptides, one of which takes an alpha-helical conformation in vitro whereas the other one loses the conformation due to alanine to proline substitution. (C) Representative western blots of different fractions from RBCs infected with P. falciparum expressing two artificially designed PEXEL reporters with the mature N-terminal sequences shown in the construct name. The experiment was performed twice.

Fig 6

A model showing the trafficking of P. falciparum secretory proteins. Secretory proteins are targeted to the ER by their hydrophobic stretch and then cleaved by PM V at the PEXEL motif or by SP at the signal cleavage site, followed by acetylation. The cleavage liberates mature N-termini that can be export-competent (shown in red) or incompetent (shown in black). There might be other organelle-targeting signals also in the mature proteins that direct them to their respective target organelle. Mature proteins secreted into the PV are recognized and loaded into the PTEX translocon based on the export competency of their mature N-terminus.