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

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

Fluorescence microscopy analysis of split-GFP expressing parasites.

(A-B) Cartoon representation of plasmepsin V with a C-terminal S11 tag (plasmepsinV:3xHA:C-S11), and phase contrast and green fluorescence images of parasites expressing GFP1-10 fragments together with plasmepsinV:3xHA:C-S11 are shown. (C-D) Images of parasites co-expressing cytoplasmic mCherry that has a C-terminal S11 tag with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10 are shown. For increased clarity and comparison to figures in the main text, two brightness ranges are shown for each image, as indicated. For GFP and mCherry images in the main text brightness settings of 0–1000 and 0–800 were used, respectively. In the images shown here, 0–1000 and 0–800 are shown for GFP and mCherry, respectively, but a brightness setting of 0–4095 is also shown for both channels. (E-F) Images of parasites co-expressing ER-lumenal mCherry (ER-lumenal mCherry comprises the N-terminal signal peptide derived from PF3D7_0827900, mCherry, a C-terminal S11 tag, and a STREP tag, followed by an SDEL sequence) with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10, are shown. (TIF)</p

High contrast GFP images of MAHRP2-expressing parasites.

(A-C) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Images are identical to those in the main text Fig 6 except that high contrast images of the GFP channel are shown. Contrast settings for GFP images are set at 0–200 to show weak GFP signal. Scale bar: 2 μm. (TIF)</p

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.</p

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.</p

High contrast GFP images of SBP1-expressing parasites.

(A-F) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Images are identical to those in the main text Figs 4 and 5 except that high contrast images of the GFP channel are shown. Contrast settings for GFP images are set at 0–200 to show weak GFP signal. Scale bar: 2 μm. (TIF)</p

Analysis of SBP1 parasites.

(A) Western blots of parasites expressing the indicated SBP1 proteins are shown. Blots were probed with anti-mCherry or anti-GFP antibodies as indicated. (B) Western blot of parasites for comparison of expression levels of the indicated SBP1 proteins. The blots were probed with anti-mCherry (shown in red) and anti-plasmepsin V as a loading control (shown in green). (C) Immunofluorescence labelling of parasites expressing mCherry tagged SBP1:C-S11:DSLE. Intrinsic mCherry fluorescence of the proteins is shown in red. Labelling with anti-MAHRP1 is shown in green. (D) Immunofluorescence labelling of parasites expressing mCherry tagged SBP1:C-S11:DSLE and treated with Brefeldin A. Intrinsic mCherry fluorescence of the proteins is shown in red. Labelling with anti-plasmepsin V is shown in green. (E) Phase contrast and fluorescence images of parasites expressing mCherry tagged SBP1:C-S11:DSLE and treated with DMSO are shown. Scale bar: 2 μm. (TIF)</p

Mass spectrometry methods.

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.</div

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 GFP1-10 or cytoplasmic GFP1-10, as indicated. (B) Images of parasites expressing MAHRP2:C-S11:DSLE expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, 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 GFP1-10 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 GFP1-10 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 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.</p

Characterisation of GFP_1-10 protein expression in parasites.

Characterisation of GFP1-10 protein expression in parasites.</p