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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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Related in: MedlinePlus

Preventing KAHRP PEXEL processing by mutation affects signal sequence processingImmunoblot(A) and coomassie gel (B) of KAHRPR>A chimeric proteins after immunoaffinity purification from the saponin pellet. The bands indicated by arrows in (B) were excised and subjected to MS. C) Mass spectra of the most N-terminal peptide from the second upper band (▪) in (B) showing that the chimaera is processed at the site predicted by SignalP (32LKC-SNN36) and N acetylated. D) Mass spectra of the most N-terminal peptide from the uppermost band (♦) in (B) showing that the chimaera contains residues present in the signal sequence (i.e. N-terminal to the SignalP processing site; 31VLKC34) and has retained the signal sequence.
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fig05: Preventing KAHRP PEXEL processing by mutation affects signal sequence processingImmunoblot(A) and coomassie gel (B) of KAHRPR>A chimeric proteins after immunoaffinity purification from the saponin pellet. The bands indicated by arrows in (B) were excised and subjected to MS. C) Mass spectra of the most N-terminal peptide from the second upper band (▪) in (B) showing that the chimaera is processed at the site predicted by SignalP (32LKC-SNN36) and N acetylated. D) Mass spectra of the most N-terminal peptide from the uppermost band (♦) in (B) showing that the chimaera contains residues present in the signal sequence (i.e. N-terminal to the SignalP processing site; 31VLKC34) and has retained the signal sequence.

Mentions: We sought to characterise the N-termini of the different protein populations present with respect to mutation of the KAHRP PEXEL (Figure 2A). The KAHRPR>A chimeric proteins were purified by immunoaffinity chromatography from the saponin pellet (parasite located) and four GFP-containing bands were observed (Figure 5A). The upper two bands of 31 and 35 kDa were the same larger bands observed previously (Figure 2A), while the lower two bands represent some processing in the PEXEL of the large-scale sample (29 kDa; also faintly present in Figure 2A) and degradation to GFP (26.5 kDa; also present in Figure 2A), respectively, The upper two bands were excised from a gel (arrows in Figure 5B) and subjected to LC-MS/MS. From the 31-kDa protein species (▪ in Figure 5B), we identified the KAHRPR>A chimaera with 83% coverage and the most N-terminal peptide had a mass of m/z 1903.70 with the sequence Ac-35SNNCNNGNGSGDSFDFR51 (Figure 5C), indicating that this species was processed, not within the PEXEL, but at the site predicted by SignalP (32LKC-SN36) and, notably, was N acetylated.


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

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

Preventing KAHRP PEXEL processing by mutation affects signal sequence processingImmunoblot(A) and coomassie gel (B) of KAHRPR>A chimeric proteins after immunoaffinity purification from the saponin pellet. The bands indicated by arrows in (B) were excised and subjected to MS. C) Mass spectra of the most N-terminal peptide from the second upper band (▪) in (B) showing that the chimaera is processed at the site predicted by SignalP (32LKC-SNN36) and N acetylated. D) Mass spectra of the most N-terminal peptide from the uppermost band (♦) in (B) showing that the chimaera contains residues present in the signal sequence (i.e. N-terminal to the SignalP processing site; 31VLKC34) and has retained the signal sequence.
© Copyright Policy
Related In: Results  -  Collection

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

fig05: Preventing KAHRP PEXEL processing by mutation affects signal sequence processingImmunoblot(A) and coomassie gel (B) of KAHRPR>A chimeric proteins after immunoaffinity purification from the saponin pellet. The bands indicated by arrows in (B) were excised and subjected to MS. C) Mass spectra of the most N-terminal peptide from the second upper band (▪) in (B) showing that the chimaera is processed at the site predicted by SignalP (32LKC-SNN36) and N acetylated. D) Mass spectra of the most N-terminal peptide from the uppermost band (♦) in (B) showing that the chimaera contains residues present in the signal sequence (i.e. N-terminal to the SignalP processing site; 31VLKC34) and has retained the signal sequence.
Mentions: We sought to characterise the N-termini of the different protein populations present with respect to mutation of the KAHRP PEXEL (Figure 2A). The KAHRPR>A chimeric proteins were purified by immunoaffinity chromatography from the saponin pellet (parasite located) and four GFP-containing bands were observed (Figure 5A). The upper two bands of 31 and 35 kDa were the same larger bands observed previously (Figure 2A), while the lower two bands represent some processing in the PEXEL of the large-scale sample (29 kDa; also faintly present in Figure 2A) and degradation to GFP (26.5 kDa; also present in Figure 2A), respectively, The upper two bands were excised from a gel (arrows in Figure 5B) and subjected to LC-MS/MS. From the 31-kDa protein species (▪ in Figure 5B), we identified the KAHRPR>A chimaera with 83% coverage and the most N-terminal peptide had a mass of m/z 1903.70 with the sequence Ac-35SNNCNNGNGSGDSFDFR51 (Figure 5C), indicating that this species was processed, not within the PEXEL, but at the site predicted by SignalP (32LKC-SN36) and, notably, was N acetylated.

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