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

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

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 GFP1-10 proteins. Scale bar: 2 μm.</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

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 GFP1-10 or cytoplasmic GFP1-10, 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 GFP1-10 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.</p

Transmembrane domain analysis.

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

Western blotting analysis of Pf332 expressing parasites.

(A) Western blots of parasites expressing the indicated proteins are shown. Blots were probed with anti-mCherry. (B) Western blots of parasites for comparison of expression levels of the indicated Pf332 proteins. The blots were probed with anti-mCherry (shown in red) and anti-plasmepsin V as a loading control (shown in green). (C) Western blots of parasites expressing the indicated proteins are shown. Blots were probed with anti-mCherry or anti-GFP as indicated. (TIF)</p

Analysis of MAHRP2 parasites.

(A)Western blots of parasites expressing the indicated MAHRP2 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 MAHRP2 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 MAHRP2: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 MAHRP2: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 MAHRP2:C-S11:DSLE and treated with DMSO are shown. Scale bar: 2 μm. (TIF)</p

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 GFP1-10 or cytoplasmic GFP1-10, 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 GFP1-10 or cytoplasmic GFP1-10, as indicated. Parasites were treated with 1μg/ml Brefeldin A for four hours prior to imaging. Scale bar: 2 μm.</p

Immunofluorescence and live-cell microscopy of Pf332 expressing parasites.

(A-D) Immunofluorescence labelling of parasites expressing the indicated mCherry tagged proteins. Intrinsic mCherry fluorescence of the proteins is shown in red. Labelling with anti-MAHRP1 or anti-plasmepsin V is shown in green. (E) Immunofluorescence labelling of parasites expressing ER-lumenal GFP1-10 only. Parasites were labelled with anti-GFP (red) and anti-plasmepsin V (green). (F) Phase contrast and fluorescence images of parasites expressing the Pf332:Int-mCherry:C-S11:DSLE proteins either alone or with the indicated GFP1-10 proteins. Scale bar: 2 μm. (TIF)</p