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Evidence for a role for a Plasmodium falciparum homologue of Sec31p in the export of proteins to the surface of malaria parasite-infected erythrocytes

Akinola Adisa1, Frank R. Albano, John Reeder2, Michael Foley1 and Leann Tilley1,*

1 Department of Biochemistry, La Trobe University, Melbourne, Victoria, Australia
2 Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea



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  		Fig. 1. Analysis of the sequence of Sec31p from P. falciparum. (A) Alignment of the amino acid sequences of the WD-40 domains of P. falciparum Sec31p and human Sec31A. Identical amino acids are repeated in the central line. Similar amino acids are denoted with a +. The residues that form the four WD-40 motifs of P. falciparum Sec31p and five of the six human WD-40 motifs are underlined. (B) Domain structure of PfSec31p. Approximate boundaries between the WD40 domain, the intervening domain, the proline-rich domain and the C-terminal domain are indicated. Regions produced as recombinant proteins for the generation of antibodies are indicated by solid lines.

 


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  		Fig. 2. Western blot and immunoprecipitation analysis of PfSec31p. (A) Lysates of E. coli expressing PfSec31(WD) (lanes a,b) and recombinant PfSec31(WD) isolated by affinity chromatography on GSH-Sepharose (lanes c,d) were subjected to SDS-PAGE (10% acrylamide) and visualised by Coomassie blue staining (lanes a,c) or transferred to nitrocellulose filters and incubated with rabbit antiserum against PfSec31(WD) followed by horse radish peroxidase (HRP)-conjugated anti-rabbit IgG and visualised using ECL (lanes b,d). (B) Saponin-lysed uninfected red blood cells (lanes a,b) or harvested saponin-lysed parasitised erythrocytes (lanes c,d) were subjected to SDS-PAGE (10% acrylamide) and stained with Coomassie blue (lane a,c) or transferred to nitrocellulose and probed with rabbit antiserum against PfSec31(WD) (lanes b,d). (C) [35S]-methionine/cysteine-labelled infected erythrocytes were solubilised in detergent and the sample was subjected to an immunoprecipitation protocol using either pre-bleed rabbit serum (lane a) or rabbit anti-PfSec31(WD) antiserum (lane b). (D) Harvested saponin-lysed parasitised erythrocytes (lanes a,b) were subjected to SDS-PAGE (10% acrylamide) and transferred to nitrocellulose and probed with either pre-bleed rabbit serum (lane a) or rabbit antiserum against PfSec31(int) (lane b). An ~160 kDa band that might correspond to a full-length monomeric form of PfSec31p in the parasite samples is marked with an arrow. Higher and lower molecular mass species are indicated with asterisks.

 


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  		Fig. 3. Solubility studies and time-course of expression of PfSec31p. (A) Mature-stage-parasitised erythrocytes (FAC8 strain, ~106 cells) were either sonicated in PBS (lanes a,b), or agitated in 2% Triton X-100 (lanes c,d) for 20 minutes at 4°C or sonicated in 50 mM NaHCO3, pH 10 (lanes e,f) and the samples kept for 30 minutes at 4°C. The soluble and particulate fractions were separated by centrifugation and the supernatant (lanes b,d,f) and pellet (lanes a,c,e) fractions were subjected to SDS-PAGE (7.5% acrylamide) and transferred to a nitrocellulose membrane. The filters were incubated with antiserum against recombinant PfSec31(WD) followed by HRP-conjugated anti-rabbit IgG and visualised using ECL. (B) Aliquots of a synchronous culture of [35S]-methionine/cysteine-labelled parasitised erythrocytes (FAC8 strain, 10% parasitemia) were harvested at the ring stage (lane a), the trophozoite stage (lane b), the schizont stage (lane c) or the ring stage of the second cycle (lane d). The samples were solubilised, immunoprecipitated with anti-PfSec31(WD) antiserum, subjected to SDS-PAGE (7.5% acrylamide) and visualised by phosphorimage analysis. A ~160 kDa band that might correspond to a full-length monomeric form of PfSec31p is marked with an arrow.

 


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  		Fig. 4. Intracellular location of PfSec31p and effect of BFA treatment on export of PfSec31p. Erythrocytes infected with ring stage (a,b,i) or mature (c,d,j-l) malaria parasites (K1 strain) were labelled with affinity purified rabbit anti-PfSec31(WD) antiserum (a-d) or affinity-purified rabbit anti-PfSec31(int) antiserum (i-l) followed by a fluorescein-conjugated anti-rabbit IgG. Transmission images of a-d are shown in e-h. Transmission images of i-l are shown in m-p. Optical slices were collected by confocal microscopy through the centre of the parasite (a,i,j) or near the surface of the parasitised erythrocyte (b-d,k,l). In (a) and (i), some PfSec31p-containing structures in the erythrocyte cytosol are marked with white arrowheads. A PfSec31p-containing structure in the parasite cytosol in (a) is marked with a blue arrowhead. Erythrocytes infected with synchronised ring stage parasites (FAC8 strain, 2-10 hour rings) were incubated for 18 hours in the presence of 5 µg ml-1 BFA (q,r) or an equivalent volume of methanol (s,t). Smears were prepared for immunofluorescence microscopy and visualised using affinity-purified rabbit anti-PfSec31(WD) antiserum. Bar in (a), 5 µm.

 


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  		Fig. 5. Comparison of the location of PfSec31p with the ER-located protein PfERC in P.-falciparum-infected erythrocytes and semiquantitative analysis of the amount of PfSec31p in the erythrocyte cytosol. (a-h) Asynchronous infected erythrocytes (K1 strain) were probed with affinity-purified rabbit anti-PfSec31(WD) antiserum followed by a fluorescein-conjugated anti-rabbit IgG (green fluorescence) and a murine antiserum recognising the ER-located protein PfERC followed by Alexa-Fluor-568-conjugated anti-mouse IgG (red fluorescence). A region of partial overlap of PfSec31p and PfERC in the parasite cytosol in (d) is marked with an arrowhead. (i-p) Asynchronous infected erythrocytes (K1 strain) were probed with rabbit anti-PfSec31(WD) antiserum (green fluorescence; i-l) or a murine anti-PfERC (red fluorescence; m-p). Four optical slices (0.6 µm) were collected by confocal microscopy from the bottom to the top of the parasitised erythrocytes.

 


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  		Fig. 6. Comparison of the location of PfSec31p with that of other parasite antigens in P.-falciparum-infected erythrocytes. (a-d) Asynchronous infected erythrocytes (K1 strain) were probed with affinity-purified rabbit anti-PfSec31(WD) antiserum followed by a fluorescein-conjugated anti-rabbit IgG (b; green fluorescence) and a murine monoclonal antibody recognising the PV-located protein Exp1 followed by rhodamine-conjugated anti-mouse IgG (c; red fluorescence). An optical slice was collected through the centre of the parasite by confocal microscopy. Some PfSec31p-containing structures in the erythrocyte cytosol are marked with white arrowheads and a PfSec31p-containing structure in the parasite cytosol is marked with a blue arrowhead (d). (a) A transmission image of (b-d). (e-l) Asynchronous infected erythrocytes (K1 strain) were probed with mouse anti-PfSec31(WD) antiserum followed by a fluorescein-conjugated anti-mouse IgG (green fluorescence) and a rabbit anti-PfSar1p antiserum followed by Alexa-Fluor-568-conjugated anti-rabbit IgG (red fluorescence). Optical slices were collected near the surface of the parasitised erythrocytes. Some PfSec31p- and PfSar1p-containing structures are marked with arrowheads. (m-p) Asynchronous infected erythrocytes were probed with affinity-purified rabbit anti-PfSec31(WD) antiserum followed by fluorescein-conjugated anti-rabbit IgG (green fluorescence) and a murine monoclonal antibody recognising PfEMP3, followed by Alexa-Fluor-568-conjugated anti-mouse IgG (red florescence). An optical slice was collected near the surface of the parasitised erythrocyte. Two structures in which there is partial overlap of PfSec31p and PfEMP3 in the erythrocyte cytosol are marked with arrowheads. (q-x) Asynchronous infected erythrocytes were probed with a mouse anti-PfSec31(WD) antiserum followed by Alexa-Fluor-568-conjugated anti-mouse IgG (red fluorescence) and an affinity-purified rabbit anti-PfEMP1 antibody followed by fluorescein-conjugated anti-rabbit IgG (green florescence). Optical slices were collected near the surface of the parasitised erythrocytes. Some PfSec31p- and PfEMP1-containing structures in the erythrocyte cytosol are marked with arrowheads. Bar in (a), 5 µm.

 

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