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Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte.

Boddey JA, Moritz RL, Simpson RJ, Cowman AF - Traffic (2008)

Bottom Line: The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner.Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell.Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Melbourne, Australia.

ABSTRACT
The intracellular survival of Plasmodium falciparum within human erythrocytes is dependent on export of parasite proteins that remodel the host cell. Most exported proteins require a conserved motif (RxLxE/Q/D), termed the Plasmodium export element (PEXEL) or vacuolar targeting sequence (VTS), for targeting beyond the parasitophorous vacuole membrane and into the host cell; however, the precise role of this motif in export is poorly defined. We used transgenic P. falciparum expressing chimeric proteins to investigate the function of the PEXEL motif for export. The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner. Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell. Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell. This work defines the role of each residue in the PEXEL for export into the P. falciparum-infected erythrocyte.

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GBP130 chimaeras localise to the endoplasmic reticulum in addition to the parasitophorous vacuole or erythrocyte cytosolImages captured by immunofluorescence microscopy show substantial colocalisation of GBP130 chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild-type PEXEL chimaera is efficiently exported to the erythrocyte cytosol (uppermost panels). All mutants show some accumulation within the parasitophorous vacuole and low-level fluorescence in the erythrocyte cytosol (lower panels), and this is evident, upon careful examination, in previously published images of these chimaeras (19). The presence of GBP130RILE>A in the saponin supernatant in Figure 4 suggests localisation predominantly in the parasitophorous vacuole (signal sequence processed). This is confirmed by immunofluorescence microscopy (compare GBP130RILE>A in this figure to KAHRPRLQ>A in Figure 6, the latter of which retains the signal sequence and localises more in the ER as a result). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.
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fig07: GBP130 chimaeras localise to the endoplasmic reticulum in addition to the parasitophorous vacuole or erythrocyte cytosolImages captured by immunofluorescence microscopy show substantial colocalisation of GBP130 chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild-type PEXEL chimaera is efficiently exported to the erythrocyte cytosol (uppermost panels). All mutants show some accumulation within the parasitophorous vacuole and low-level fluorescence in the erythrocyte cytosol (lower panels), and this is evident, upon careful examination, in previously published images of these chimaeras (19). The presence of GBP130RILE>A in the saponin supernatant in Figure 4 suggests localisation predominantly in the parasitophorous vacuole (signal sequence processed). This is confirmed by immunofluorescence microscopy (compare GBP130RILE>A in this figure to KAHRPRLQ>A in Figure 6, the latter of which retains the signal sequence and localises more in the ER as a result). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.

Mentions: To confirm the effect of PEXEL mutations on subcellular localisation of the KAHRP and GBP130 chimaerae, we used immunofluorescence analysis (IFA) to colocalise them with known markers of the ER and parasitophorous vacuole (Figures 6 and 7). Consistent with previous results (19), we observed accumulation of each PEXEL mutant chimaera in the parasitophorous vacuole, as shown by colocalisation with the parasitophorous vacuole membrane protein EXP-2 (Figures 6 and 7) (26). We also observed colocalisation with the endoplasmic reticulum calcium-binding protein ERC (Figures 6 and 7) (27) consistent with the ER as a transit organelle during export. We observed strong fluorescence of the KAHRPR>A, KAHRPL>A and KAHRPRLQ>A GFP chimaeras in the ER but decreased levels for the KAHRPQ>A GFP chimaera (Figure 6). This may be attributed to accumulation at the ER membrane of the subspecies of each mutant chimaera that retained the signal peptide. If so, this would suggest that the proteins were retained in a folded state.


Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte.

Boddey JA, Moritz RL, Simpson RJ, Cowman AF - Traffic (2008)

GBP130 chimaeras localise to the endoplasmic reticulum in addition to the parasitophorous vacuole or erythrocyte cytosolImages captured by immunofluorescence microscopy show substantial colocalisation of GBP130 chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild-type PEXEL chimaera is efficiently exported to the erythrocyte cytosol (uppermost panels). All mutants show some accumulation within the parasitophorous vacuole and low-level fluorescence in the erythrocyte cytosol (lower panels), and this is evident, upon careful examination, in previously published images of these chimaeras (19). The presence of GBP130RILE>A in the saponin supernatant in Figure 4 suggests localisation predominantly in the parasitophorous vacuole (signal sequence processed). This is confirmed by immunofluorescence microscopy (compare GBP130RILE>A in this figure to KAHRPRLQ>A in Figure 6, the latter of which retains the signal sequence and localises more in the ER as a result). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682620&req=5

fig07: GBP130 chimaeras localise to the endoplasmic reticulum in addition to the parasitophorous vacuole or erythrocyte cytosolImages captured by immunofluorescence microscopy show substantial colocalisation of GBP130 chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild-type PEXEL chimaera is efficiently exported to the erythrocyte cytosol (uppermost panels). All mutants show some accumulation within the parasitophorous vacuole and low-level fluorescence in the erythrocyte cytosol (lower panels), and this is evident, upon careful examination, in previously published images of these chimaeras (19). The presence of GBP130RILE>A in the saponin supernatant in Figure 4 suggests localisation predominantly in the parasitophorous vacuole (signal sequence processed). This is confirmed by immunofluorescence microscopy (compare GBP130RILE>A in this figure to KAHRPRLQ>A in Figure 6, the latter of which retains the signal sequence and localises more in the ER as a result). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.
Mentions: To confirm the effect of PEXEL mutations on subcellular localisation of the KAHRP and GBP130 chimaerae, we used immunofluorescence analysis (IFA) to colocalise them with known markers of the ER and parasitophorous vacuole (Figures 6 and 7). Consistent with previous results (19), we observed accumulation of each PEXEL mutant chimaera in the parasitophorous vacuole, as shown by colocalisation with the parasitophorous vacuole membrane protein EXP-2 (Figures 6 and 7) (26). We also observed colocalisation with the endoplasmic reticulum calcium-binding protein ERC (Figures 6 and 7) (27) consistent with the ER as a transit organelle during export. We observed strong fluorescence of the KAHRPR>A, KAHRPL>A and KAHRPRLQ>A GFP chimaeras in the ER but decreased levels for the KAHRPQ>A GFP chimaera (Figure 6). This may be attributed to accumulation at the ER membrane of the subspecies of each mutant chimaera that retained the signal peptide. If so, this would suggest that the proteins were retained in a folded state.

Bottom Line: The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner.Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell.Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Melbourne, Australia.

ABSTRACT
The intracellular survival of Plasmodium falciparum within human erythrocytes is dependent on export of parasite proteins that remodel the host cell. Most exported proteins require a conserved motif (RxLxE/Q/D), termed the Plasmodium export element (PEXEL) or vacuolar targeting sequence (VTS), for targeting beyond the parasitophorous vacuole membrane and into the host cell; however, the precise role of this motif in export is poorly defined. We used transgenic P. falciparum expressing chimeric proteins to investigate the function of the PEXEL motif for export. The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner. Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell. Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell. This work defines the role of each residue in the PEXEL for export into the P. falciparum-infected erythrocyte.

Show MeSH
Related in: MedlinePlus