Plasmodium falciparum-encoded exported hsp70/hsp40 chaperone/co-chaperone complexes within the host erythrocyte
Article first published online: 24 AUG 2012
© 2012 Blackwell Publishing Ltd
Volume 14, Issue 11, pages 1784–1795, November 2012
How to Cite
Külzer, S., Charnaud, S., Dagan, T., Riedel, J., Mandal, P., Pesce, E. R., Blatch, G. L., Crabb, B. S., Gilson, P. R. and Przyborski, J. M. (2012), Plasmodium falciparum-encoded exported hsp70/hsp40 chaperone/co-chaperone complexes within the host erythrocyte. Cellular Microbiology, 14: 1784–1795. doi: 10.1111/j.1462-5822.2012.01840.x
- Issue published online: 16 OCT 2012
- Article first published online: 24 AUG 2012
- Accepted manuscript online: 1 AUG 2012 07:03AM EST
- Manuscript Revised: 17 JUL 2012
- Manuscript Accepted: 17 JUL 2012
- Manuscript Received: 22 MAY 2012
- DFG. Grant Number: SPP1580
- Australian NHMRC
Fig. S1. Sequencing of cDNA coding for PfHsp70-x reveals a putative signal peptide. In the original annotation (MAL7P1.228), the AUG initiation codon was missing due to an a > t point mutation (box), and included a TAA stop codon (bold italic). The new gene model (JN900252) now includes the correct start codon (bold). Putative signal sequence (SignalP 4.0) is underlined. The ATPase domain typical for Hsp70s is shown shaded.
Fig. S2. Alignment of PfHsp70-x with PfHsp70-1 (cytosolic), PfHsp70-2 (ER resident), and PfHsp70-3 (mitochondrial). Red, predicted signal peptide (SignalP 4.0); light blue, putative export motif of PfHsp70-x; dark blue, EEVD/EEVN motif; green, ER retrieval signal SDEL; bold, peptide used to generate antisera MRaX; underlined, recombinant protein used to generate BIaX antisera.
Fig. S3. Hsp70 homologues used for phylogenetic analysis. For P. reichenowi multiple accession numbers are included, as no full-length clones could be identified.
Fig. S4. Full phylogenetic tree of sequences analysed in Fig. 1.
Fig. S5. Identification of a P. reichenowi Hsp70-x and alignment with PfHsp70-x. Three individual sequenced Sanger clones cover the majority of the Hsp70-x coding sequence. Red box, export sequence; blue box, missing sequence information.
Fig. S6. Specificity test of PfHsp70-x antisera.
A. MRaX. 1 × 107 infected/non-infected erythrocytes per lane. INF, infected erythrocytes; NI, erythrocyte only control; r70x, recombinantly produced PfHsp70-x; r70-1, recombinantly produced PfHsp70-1; 3D770x:GFP, protein sample from transfectant line 3D770x:G; PI, pre-immune serum. A band of the predicted molecular mass is only detected when using the antibody specific for the respective recombinant protein. r70x and r70-1 run slightly higher due to the presence of a polyhistidine tag.
B. BIaX. α-Hsp70x was purified on a recombinant fragment of PfHsp70-x (r70-x). α-Hsp70-1 was purified on a recombinant fragment of PfHsp70-1 (r70-1). Following purification and cross absorption against the respective protein, each antisera specifically recognizes only the target protein.
C. Indirect immunofluorescence localization of PfHsp70-x using BIaX. Green, α-70x; Blue, Hoechst.
D. Indirect immunofluorescence localization of PfHsp70-x using MRaX in cells infected with 3D7GFP:70x. A high degree of signal localization can be observed.
E. Indirect immunofluorescence localization of PfHsp70-1. In merge and overlay images: Green, α-PfHsp70-1; Blue, Hoechst.
Fig. S7. A. Stage dependent colocalization of PfHsp70-x and PfEMP1. Upper infected cell (ring stage) shows a higher degree of signal colocalization than lower infected cell (trophozoite).
B. Indirect immunofluorescence of PfHsp70-x and PfEMP1 using confocal microscopy. In merge and overlay: Green, α-70x; Red, α-ATS; Blue, Hoechst.
C. Indirect immunofluorescence localization of PfHsp70-x using anti-SBP1, anti-MAHRP2 and anti-STEVOR (STV) antisera in cells infected with 3D7GFP:70x. In merge and overlay images: Green, GFP; red, specific antisera; Blue, Hoechst. No colocalization can be observed between the GFP and antibody-specific signals.
Fig. S8. Gel filtration. Infected cells were fractionated as described in materials and methods. Size markers in kDa. RBC, red blood cell; IRBC, infected red blood cell.
Fig. S9. Parasite lines with hrpii deletion still express PfHsp70-x. Western blot analysis of protein samples derived from D10 and 3D7 parasite lines. Both parasite lines express Hsp70-x (upper panel) whereas only 3D7 still expresses HRPII (lower panel). 1 × 107 parasites/lane. Size markers in kDa.
Movie S1. Time lapse live cell imaging 3D7G:70x infected erythrocytes. Note: Signal strength decreases during imaging due to GFP bleaching.
Appendix S1. Supplementary materials and methods.
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