Model depicting newly identified steps in the FP2a trafficking pathway to the food vacuole. By integrating the data from this study, we propose a new model for the trafficking of FP2a, and potentially other Hb proteases, to the food vacuole (FV). (A) The FP2a protein enters the secretory pathway and travels from the ER to the PV with the N-terminus facing inside the lumen and C-terminus in parasite cytoplasm. (B) The protein is deposited in the PPM by vesicle fusion (TM domain shown in pink), in an N- to C-terminal direction. (C) HSP101 could help extract FP2a from the PPM and into the PV as proposed for

transmembrane domain containing exported proteins and then interact with the rest of PTEX, or (D) all the PTEX core components could participate in FP2a extraction from the PPM. FP2a is then escorted to the cytostome, which likely involves additional sorting in the PV by an unknown mechanism. (E) FP2a is taken up via the cytostome formed by invagination of the PVM and PPM. (F) FP2a is then transported to the FV together with Hb. (G) Inside the FV the Hb is digested into smaller peptides, releasing haem, which is sequestered into haemozoin crystals (Hz). (H) Reduced PTEX expression results in accumulation of Hb proteases (as shown with PM II-mScarlet) at the parasite periphery. (I) This would subsequently lead to accumulation of full-length Hb inside the FV and reduced levels of haemozoin crystal formation. 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. PMID: 37523385