Background InformationLike other apicomplexan parasites, Toxoplasma gondii harbours a four‐membraned endosymbiotic organelle – the apicoplast. Apicoplast proteins are nuclear encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi‐independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi‐independent pathway, whereas a handful of reports show that a few proteins use the Golgi‐dependent pathway. This has led to an emphasis towards the unique Golgi‐independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known.ResultsIn this report, we systematically test eight apicoplast proteins, using a C‐terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast.Conclusions and SignificanceCollectively, with these results, for the first time, we establish that the driver of the Golgi‐dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast, and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.

The T. gondii protein superoxide dismutase 2 86 (TgSOD2) is dually targeted to the apicoplast and mitochondrion (Pino et al., 2007) and a 87 single point mutation in the N-terminus of a TgSOD2 GFP fusion protein shifts its 88 localization from the mitochondrion to the apicoplast (Brydges, 2003). Clearly, open 89 questions remain; does the dual targeting of proteins to the apicoplast and mitochondrion 90 occur due to superimposed signals at the N-terminus and if so, which signals? 91To understand mechanisms underlying dual targeting, we studied the antioxidant protein, 92 Thioredoxin peroxidase 1/2 (TgTPx1/2), an alternatively spliced form of T. gondii 93 thioredoxin peroxidase, TgTPx. TgTPx1/2 localizes to both the apicoplast and mitochondrion 94 in T. gondii parasites (Pino et al., 2007), reflecting the generation of oxidative radicals in 95 these sub-cellular compartments and their need to handle oxidative stress. Through a 96 combination of bioinformatics and mutational analyses we show that an N-terminal 97 ambiguous sequence directs the dual targeting of TgTPx1/2 to the apicoplast and 98 mitochondrion. This sequence shows an overlap between the signal peptide of the apicoplast 99 targeting signal and the predicted amphipathic helix of the mitochondrial targeting signal. 100 Interestingly, proteins with mutants in the N-terminus of TgTPx1/2 are found in either 101 organelle while a truncated N-terminus results in localization of a reporter protein to punctate 102 structures close to, but not overlapping with the apicoplast. These data suggest that 103 organellar receptors compete for the ambiguous N-terminus of TgTPx1/2 and this N-terminus 104 consists of an unusual sequence where the signals for localization to each organelle are 105 overlapping yet separable. 106 107 Materials and Methods 108 109 T. gondii culture, transfection and selection 110 T. gondii parasites (wild type RH strain) were grown in primary Human Foreskin fibroblasts 111 (HFF-1) cells. HFF-1 cells were obtained from ATCC and grown in Dulbecco's Modified 112 Eagle's Medium (Gibco TM , Mfr. No. 12100-046) containing 3.7 g/L sodium bicarbonate and 113 2.38 g/L HEPES, supplemented with 10% Cosmic calf serum (HyClone TM ) and 20 mg/L 114 Gentamicin. Transient transfections of extracellular T. gondii parasites were carried out at 115 1.5 kV, 50 Ω and 25 μF using Bio-Rad GenePulser Xcell system and subjected to 116 5 immunostaining 24 hours post transfection. Stable lines were generated by Restriction 117 Enzyme Mediated Integration (REMI) using NotI followed by selection of the parasites for 118 growth in 20 µM chloramphenicol.

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.

Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle -the apicoplast. Apicoplast proteins are nuclear-encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, while the other is a unique Golgiindependent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, while a handful of reports show that a few proteins use the Golgidependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN) are trafficked through the Golgi while proteins localised exclusively to the apicoplast are trafficked independent of the Golgi.Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN suggested that the evolutionary origins of TgSOD2, TgTPx1/2 lie in the mitochondrion while TgACN appears to have originated from the apicoplast. Collectively, with these results, for the first time, we establish that the driver of the Golgidependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.