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Apicoplast trafficking signals usually consist of N\u2010terminal signal and transit peptides, but the trafficking signal of Pf<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>x<jats:sub>Gl<\/jats:sub> appears to exhibit important differences. As transfection is a protracted process in <jats:italic>P.\u00a0falciparum<\/jats:italic>, we expressed the N terminus of Pf<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>x<jats:sub>Gl<\/jats:sub> as a <jats:styled-content style=\"fixed-case\">GFP<\/jats:styled-content> fusion protein in a related apicomplexan, <jats:italic>Toxoplasma gondii<\/jats:italic>, in order to dissect its trafficking signals. We show that Pf<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>x<jats:sub>Gl<\/jats:sub> possesses an N\u2010terminal signal anchor that takes the protein to the endoplasmic reticulum in <jats:italic>Toxoplasma<\/jats:italic>\u2014this 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 <jats:styled-content style=\"fixed-case\">ER<\/jats:styled-content> lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered Pf<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>x<jats:sub>Gl<\/jats:sub> targeting in <jats:italic>Toxoplasma<\/jats:italic>, in <jats:italic>Plasmodium<\/jats:italic>. This study has demonstrated that Pf<jats:styled-content style=\"fixed-case\">TP<\/jats:styled-content>x<jats:sub>Gl<\/jats:sub> 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 <jats:italic>T.\u00a0gondii<\/jats:italic> as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.<\/jats:p>","DOI":"10.1002\/2211-5463.12527","type":"journal-article","created":{"date-parts":[[2018,9,22]],"date-time":"2018-09-22T13:15:18Z","timestamp":1537622118000},"page":"1746-1762","update-policy":"http:\/\/dx.doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Heterologous expression in <i>Toxoplasma gondii<\/i> reveals a topogenic signal anchor in a <i>Plasmodium<\/i> apicoplast protein"],"prefix":"10.1002","volume":"8","author":[{"given":"Aishwarya","family":"Narayan","sequence":"first","affiliation":[{"name":"Department of Biosciences and 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