FP2a trappable reporters were generated to study the relationship of FP2a with PTEX. (A) Three FP2a reporters were generated. 120 aa and 190 aa containing the first 120 aa and 190 aa of FP2a respectively. The NT reporter refers to the 190 aa reporter without the N-terminal TM domain and served as a negative control. All FP2a sequences were appended with nanoluciferase (Nluc), murine dihydrofolate reductase (DH) and a 3x FLAG (FL) epitope tag. The complete protein map for FP2a is indicated above for reference, where the reporter only contains the N-terminus sufficient for food vacuole delivery and not the C-terminus necessary for protease activity. The reporters were episomally expressed from a plasmid under the bidirectional EF1a promoter in the HSP101-HAglmS parental line and maintained under Blasticidin-S selection via the Blasticidin-S Deaminase (BSD) cassette. (B) Western blot analysis demonstrated that the 120 aa, 190 aa and NT FP2a reporters are expressed and migrate at the correct size indicated in panel A. Rabbit anti-Nluc was used to detect the Nluc tag and chicken anti-FLAG to detect the FL tag. Mouse anti-EXP2 was used as a loading control. (C) Immunofluorescence assays show that the 120 aa and 190 aa FP2a reporters are trafficked to the food vacuole. The NT FP2a reporter displayed diffuse labelling within the parasite as expected. Rabbit anti-Nluc was used to detect the reporter, mouse anti-EXP2. was used as a PVM marker and haemozoin crystals in the DIC show food vacuole (FV) localisation (indicated with a white dotted line). (D) Late ring/early trophozoites were treated ± 18 μMBFA to inhibit ER to Golgi protein secretion. The 120 and 190 aa FP2a reporters were both trapped in the ER upon BFA treatment as expected and no changes were observed for the NT reporter. Rabbit anti-ERC was used as an ER marker and mouse anti-HA to label for HSP101-HAglmS, which also traps in the ER upon BFA treatment. DAPI was used to stain the nucleus. Scale bars = 5 μm. Jonsdottir TK, Elsworth B, Cobbold S, Gabriela M, Ploeger E, Parkyn Schneider M, Charnaud SC, Dans MG, McConville M, Bullen HE, Crabb BS, Gilson PR. PTEX helps efficiently traffic haemoglobinases to the food vacuole in Plasmodium falciparum. PLoS Pathog. 2023 19(7):e1011006.
Other associated proteins
PFID | Formal Annotation |
---|---|
PF3D7_1108600 | endoplasmic reticulum-resident calcium binding protein |
PF3D7_1115700 | cysteine proteinase falcipain 2a falcipain 2 |
PF3D7_1116800 | heat shock protein 101 chaperone protein ClpB2 |