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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

Role of the PEXEL residues in processing and export to the erythrocyteImmunoblots with α-GFP antibodies against KAHRP chimaeras from tetanolysin pellets (A) and supernatant (B) or GBP130 chimaeras from tetanolysin pellets (C) and supernatant (D) are shown. Antibodies to aldolase (48) were used as a permeabilisation control, as described previously (49) and reflects the quantity of protein loaded in each lane. While equal proportions of pellet and supernatant were loaded for each chimaera, equal loadings could not be achieved between different chimaeras because of differences in episomal expression. The densitometric analyses below (see G and H) were thus limited to comparing only between pellet and supernatant fractions of the same chimaera. Upper bands in (D, lanes 2 and 3) represent slight vacuolar leakage. E) Predicted protein sizes of KAHRP chimaeras after differential N-terminal processing; ♦ represents full-length chimaeras with signal sequence; ▪ represents processing at/near the site predicted by SignalP; H represents chimaeras processed downstream of prediction by SignalP (i.e. within the PEXEL); ▴ represents degradation to GFP/YFP only (confirmed by MS; Figure S2). The linker upstream of YFP in KAHRPWT (Figure 1) is not depicted here. While the N-termini of each KAHRP chimaera are the same, except for the mutations shown, the C-terminal YFP linker in KAHRPWT explains the minor size shift in (A) and (B) between WT and other chimaeras processed at the PEXEL (i.e. those depicted with *). F) Predicted protein sizes of GBP130 chimaeras after differential N-terminal processing. Varying exposures within the linear range of the blots represented in (A–D) were scanned at high resolution and densitometry was undertaken to approximately quantify the differential N-terminal processing and cellular localisation of KAHRP (G) and GBP130 (H) chimaeras. For each chimaera, percentages were calculated by dividing the intensity of each band in the supernatant (exported to host) or pellet (ER or parasitophorous vacuole) by the sum of the band intensities for that chimaera (total tagged chimaera) and multiplying by 100. Percentages between chimaerae are directly comparable. WT, wild type; SS, signal sequence; PV, parasitophorous vacuole.
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fig02: Role of the PEXEL residues in processing and export to the erythrocyteImmunoblots with α-GFP antibodies against KAHRP chimaeras from tetanolysin pellets (A) and supernatant (B) or GBP130 chimaeras from tetanolysin pellets (C) and supernatant (D) are shown. Antibodies to aldolase (48) were used as a permeabilisation control, as described previously (49) and reflects the quantity of protein loaded in each lane. While equal proportions of pellet and supernatant were loaded for each chimaera, equal loadings could not be achieved between different chimaeras because of differences in episomal expression. The densitometric analyses below (see G and H) were thus limited to comparing only between pellet and supernatant fractions of the same chimaera. Upper bands in (D, lanes 2 and 3) represent slight vacuolar leakage. E) Predicted protein sizes of KAHRP chimaeras after differential N-terminal processing; ♦ represents full-length chimaeras with signal sequence; ▪ represents processing at/near the site predicted by SignalP; H represents chimaeras processed downstream of prediction by SignalP (i.e. within the PEXEL); ▴ represents degradation to GFP/YFP only (confirmed by MS; Figure S2). The linker upstream of YFP in KAHRPWT (Figure 1) is not depicted here. While the N-termini of each KAHRP chimaera are the same, except for the mutations shown, the C-terminal YFP linker in KAHRPWT explains the minor size shift in (A) and (B) between WT and other chimaeras processed at the PEXEL (i.e. those depicted with *). F) Predicted protein sizes of GBP130 chimaeras after differential N-terminal processing. Varying exposures within the linear range of the blots represented in (A–D) were scanned at high resolution and densitometry was undertaken to approximately quantify the differential N-terminal processing and cellular localisation of KAHRP (G) and GBP130 (H) chimaeras. For each chimaera, percentages were calculated by dividing the intensity of each band in the supernatant (exported to host) or pellet (ER or parasitophorous vacuole) by the sum of the band intensities for that chimaera (total tagged chimaera) and multiplying by 100. Percentages between chimaerae are directly comparable. WT, wild type; SS, signal sequence; PV, parasitophorous vacuole.

Mentions: To separate exported chimaeras (erythrocyte cytosol) from nonexported chimaeras (parasite and parasitophorous vacuole) and enable visualisation of potential processing differences, we used the selective pore-forming toxin tetanolysin (25) followed by analysis of the fractions by immunoblot with α-GFP antibodies. A number of different sized GFP chimaerae was observed in the tetanolysin pellets, suggesting PEXEL-dependent N-terminal processing (Figure 2A,C). No size difference was observed for chimaeras with a wild-type PEXEL between tetanolysin supernatants (exported) and pellets (not exported) (Figure 2B,D), suggesting PEXEL-dependent processing occurred before translocation to the host cell. This agrees with previous work that suggested PEXEL processing occurred in the parasite ER before export (24).


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

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

Role of the PEXEL residues in processing and export to the erythrocyteImmunoblots with α-GFP antibodies against KAHRP chimaeras from tetanolysin pellets (A) and supernatant (B) or GBP130 chimaeras from tetanolysin pellets (C) and supernatant (D) are shown. Antibodies to aldolase (48) were used as a permeabilisation control, as described previously (49) and reflects the quantity of protein loaded in each lane. While equal proportions of pellet and supernatant were loaded for each chimaera, equal loadings could not be achieved between different chimaeras because of differences in episomal expression. The densitometric analyses below (see G and H) were thus limited to comparing only between pellet and supernatant fractions of the same chimaera. Upper bands in (D, lanes 2 and 3) represent slight vacuolar leakage. E) Predicted protein sizes of KAHRP chimaeras after differential N-terminal processing; ♦ represents full-length chimaeras with signal sequence; ▪ represents processing at/near the site predicted by SignalP; H represents chimaeras processed downstream of prediction by SignalP (i.e. within the PEXEL); ▴ represents degradation to GFP/YFP only (confirmed by MS; Figure S2). The linker upstream of YFP in KAHRPWT (Figure 1) is not depicted here. While the N-termini of each KAHRP chimaera are the same, except for the mutations shown, the C-terminal YFP linker in KAHRPWT explains the minor size shift in (A) and (B) between WT and other chimaeras processed at the PEXEL (i.e. those depicted with *). F) Predicted protein sizes of GBP130 chimaeras after differential N-terminal processing. Varying exposures within the linear range of the blots represented in (A–D) were scanned at high resolution and densitometry was undertaken to approximately quantify the differential N-terminal processing and cellular localisation of KAHRP (G) and GBP130 (H) chimaeras. For each chimaera, percentages were calculated by dividing the intensity of each band in the supernatant (exported to host) or pellet (ER or parasitophorous vacuole) by the sum of the band intensities for that chimaera (total tagged chimaera) and multiplying by 100. Percentages between chimaerae are directly comparable. WT, wild type; SS, signal sequence; PV, parasitophorous vacuole.
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Related In: Results  -  Collection

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fig02: Role of the PEXEL residues in processing and export to the erythrocyteImmunoblots with α-GFP antibodies against KAHRP chimaeras from tetanolysin pellets (A) and supernatant (B) or GBP130 chimaeras from tetanolysin pellets (C) and supernatant (D) are shown. Antibodies to aldolase (48) were used as a permeabilisation control, as described previously (49) and reflects the quantity of protein loaded in each lane. While equal proportions of pellet and supernatant were loaded for each chimaera, equal loadings could not be achieved between different chimaeras because of differences in episomal expression. The densitometric analyses below (see G and H) were thus limited to comparing only between pellet and supernatant fractions of the same chimaera. Upper bands in (D, lanes 2 and 3) represent slight vacuolar leakage. E) Predicted protein sizes of KAHRP chimaeras after differential N-terminal processing; ♦ represents full-length chimaeras with signal sequence; ▪ represents processing at/near the site predicted by SignalP; H represents chimaeras processed downstream of prediction by SignalP (i.e. within the PEXEL); ▴ represents degradation to GFP/YFP only (confirmed by MS; Figure S2). The linker upstream of YFP in KAHRPWT (Figure 1) is not depicted here. While the N-termini of each KAHRP chimaera are the same, except for the mutations shown, the C-terminal YFP linker in KAHRPWT explains the minor size shift in (A) and (B) between WT and other chimaeras processed at the PEXEL (i.e. those depicted with *). F) Predicted protein sizes of GBP130 chimaeras after differential N-terminal processing. Varying exposures within the linear range of the blots represented in (A–D) were scanned at high resolution and densitometry was undertaken to approximately quantify the differential N-terminal processing and cellular localisation of KAHRP (G) and GBP130 (H) chimaeras. For each chimaera, percentages were calculated by dividing the intensity of each band in the supernatant (exported to host) or pellet (ER or parasitophorous vacuole) by the sum of the band intensities for that chimaera (total tagged chimaera) and multiplying by 100. Percentages between chimaerae are directly comparable. WT, wild type; SS, signal sequence; PV, parasitophorous vacuole.
Mentions: To separate exported chimaeras (erythrocyte cytosol) from nonexported chimaeras (parasite and parasitophorous vacuole) and enable visualisation of potential processing differences, we used the selective pore-forming toxin tetanolysin (25) followed by analysis of the fractions by immunoblot with α-GFP antibodies. A number of different sized GFP chimaerae was observed in the tetanolysin pellets, suggesting PEXEL-dependent N-terminal processing (Figure 2A,C). No size difference was observed for chimaeras with a wild-type PEXEL between tetanolysin supernatants (exported) and pellets (not exported) (Figure 2B,D), suggesting PEXEL-dependent processing occurred before translocation to the host cell. This agrees with previous work that suggested PEXEL processing occurred in the parasite ER before export (24).

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