Although common in plants, very few proteins are currently known to be localized to both the plastid and the mitochondrion in Plasmodium falciparum. One such protein is P. falciparum glutathione peroxidase‐like thioredoxin peroxidase (PfTPxGl) which we show, by immunofluorescence imaging and bioinformatics predictions, is localized to the apicoplast, the mitochondrion and the cytosol. The distribution of PfTPxGl was random in the population, with the protein localizing to any one organelle in some parasites and to both in others. It has been proposed that targeting to each organelle occurs via independent pathways that do not proceed via the Golgi. However, for PfTPxGl, both incubation at low temperature (15 °C) and Brefeldin A treatment reversibly blocked targeting to these organelles, suggesting the involvement of a novel trafficking route, most probably via the endoplasmic reticulum and Golgi. This idea is further supported by the lack of cleavage of the putative N‐terminal signal sequence of PfTPxGl, and this N‐terminal extension did not compromise PfTPxGl enzyme activity. In the context of evolution, a common pathway for the dual localization of a single gene product, such as the primitive endoplasmic reticulum–Golgi route, may have been retained as opposed to optimization for individual organellar import pathways.

25The secretory pathway in Plasmodium falciparum has evolved to transport proteins to 26 the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can 27 occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins

Glutathione peroxidase‐like thioredoxin peroxidase (Pf TP x Gl ) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum . Apicoplast trafficking signals usually consist of N‐terminal signal and transit peptides, but the trafficking signal of Pf TP x Gl appears to exhibit important differences. As transfection is a protracted process in P. falciparum , we expressed the N terminus of Pf TP x Gl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii , in order to dissect its trafficking signals. We show that Pf TP x Gl possesses an N‐terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma —this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered Pf TP x Gl targeting in Toxoplasma , in Plasmodium . This study has demonstrated that Pf TP x Gl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.

25The secretory pathway in Plasmodium falciparum has evolved to transport proteins to 26 the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can 27 occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins

The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.