A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell
Fig 5
Intra-parasite HSP101 co-localises with P5 Lys Hyp1-Nluc-mDHFR-3xFLAG reporter construct in the endoplasmic reticulum of the parasite.
(A) Representative IFA images (n = 3 independent replicates) of parasites expressing Hyp1-Nluc-mDHFR-3xFLAG constructs with WT P5 glutamate reporter exported into the erythrocyte and the mutant P5 Lys Hyp1-Nluc-mDHFR-3xFLAG reporter trapped in the parasite ER. To visualise translocon components, cells were probed with anti-EXP2 and anti-HA (to visualise HA-tagged HSP101). Anti-PfERC was used to visualise the parasite’s ER. Hyp1-Nluc-mDHFR-3xFLAG reporter proteins were localised using either anti-Nluc or anti-FLAG antibodies. The red bar in the schematic picture of the construct indicates the PEXEL motif and variations thereof. The blue bar indicates the transmembrane signal peptide. Scale bars, 5 μm. DIC, Differential Interference Contrast. DAPI was used to stain parasite nuclei. (B) Representative Z-stacks of 3D-SIM images of the HA-tagged HSP101 parasite line expressing P5 Lys Hyp1-Nluc-mDHFR-3xFLAG and probed with either anti-HA for HSP101 (top, red) or anti-EXP2 (bottom, red), and anti-Nluc (green) to probe for the cargo. Scale bars represent 5 μm. (C) Degree of co-localisation between individual PTEX components to the P5 Lys Hyp1-Nluc-mDHFR-3xFLAG was calculated by measuring Pearson’s coefficients of the merged Z-stack images of >20 cells. Statistical significances were determined using an unpaired t-test with Welch’s correction. ****, p-value<0.0001). (D) Representative Western blot (n>3) of lysates made from mid-stage trophozoites expressing WT and P5 Lys Hyp1-Nluc-mDHFR-3xFLAG constructs showing full-length and cleaved forms of the WT Hyp1-Nluc-mDHFR-3xFLAG reporter. The P5 Lys Hyp1-Nluc-mDHFR-3xFLAG reporter appears to be miscleaved after the signal peptide (58.1 ± 1.2 kDa, n = 10). The blot was probed with anti-PTEX150 antibodies as a loading control.