Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences

Abstract

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.

Fig 1. Export of a Pf332 model protein into the infected red blood cell.

(A-F) Phase contrast and fluorescence images of parasites expressing the indicated proteins. Scale bar: 2 μm.

Fig 2. ER-lumenal localisation of ER-retained Pf332.

(A-C) Phase contrast and fluorescence images of parasites expressing the indicated Pf332 proteins either alone or with the indicated GFP_1-10 proteins. Scale bar: 2 μm.

Fig 3. The putative TM segment of Pf332 translocates into the ER lumen.

(A-B) Phase contrast and fluorescence images of parasites expressing the indicated proteins. (C) Phase and fluorescence images of parasites expressing REX3_AQLSE:Pf332:_C-S11:DSLE alone, or co-expressing either ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. Scale bar: 2 μm. (D) Western blot of parasites expressing the indicated proteins (probed with an anti-mCherry antibody). (E) Cartoon representation of plasmepsin V cleavage of the PEXEL sequence in REX3_RQLSE:Pf332:_C-S11:SDEL within the ER and recognition of the C-terminal SDEL sequence in the Golgi lumen.

Fig 4. ER-lumenal location of SBP1 in Brefeldin A-treated parasites.

(A-B) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. (C-F) Phase contrast and fluorescence images of Brefeldin A-treated parasites expressing the indicated proteins. Proteins were expressed alone, co-expressed with ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. Parasites were treated with 1μg/ml Brefeldin A for four hours prior to imaging. Scale bar: 2 μm.

Fig 5. Export of SBP1 is perturbed by a C-terminal ER-retention sequence.

(A-B) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. Scale bar: 2 μm. (C) The fraction of total mCherry fluorescence located within the parasite is shown for parasite lines expressing the indicated SBP1 and GFP_1-10 proteins. Forty individual trophozoite stage parasites, from two independent experiments, were analysed for each parasite line. Data points for individual parasites, mean and standard deviation are shown. P-values were determined using a one-way ANOVA test, P < 0.0001 = ****. (D-E) For parasites expressing the indicated SBP1 proteins, the total mCherry fluorescence and total GFP fluorescence levels are plotted (for both channels this corresponds to the fluorescence in the infected red blood cell and the parasite). Forty individual trophozoite stage parasites, from two independent experiments, were analysed for each parasite line.

Fig 6. ER-lumenal location of the C-terminus of MAHRP2.

(A) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. (B) Images of parasites expressing MAHRP2:_C-S11:DSLE expressed alone, co-expressed with ER-lumenal GFP_1-10 or cytoplasmic GFP_1-10, as indicated. Parasites were treated with 1μg/ml Brefeldin A for four hours prior to imaging. (C) Images of parasites expressing MAHRP2:_C-S11:SDEL and the indicated GFP_1-10 proteins, are shown. Scale bar: 2 μm. (D) The fraction of total mCherry fluorescence located within the parasite is shown for each parasite line expressing the indicated MAHRP2 and GFP_1-10 proteins. Forty individual trophozoite stage parasites, from four independent experiments, were analysed for each parasite line. Data points for individual parasites, mean, and standard deviation are shown. P-values were determined using a one-way ANOVA test, P < 0.0001 = ****. (E) For parasites expressing the indicated MAHRP2 and GFP_1-10 proteins, the total mCherry fluorescence and total GFP fluorescence levels are plotted. Forty individual trophozoite stage parasites, from four independent experiments, were analysed for each parasite line.

Fig 7. A model of the ways by which exported proteins can traverse the parasite secretory pathway.

RBC PM, red blood cell plasma membrane; PVM, parasitophorous vacuole membrane; PPM, parasite plasma membrane; ER, endoplasmic reticulum. Hydrophobic targeting sequences (signal sequences or transmembrane segments) are shown in red.