Gene expression constructs not targeted to the apicoplast now co-localised with the plasma membrane.

TMDs are represented by boxes and are joined by loops. All PfoTPT constructs are episomally expressed under the PfCRT promoter and tagged with triple HA at the C-terminus detected with anti-HA and secondary antibody conjugated to FITC (green). The parasite plasma membrane is detected with anti-PfNTP1 (red). Nuclei are stained with Hoechst (blue), and transmitted light images of the parasites within their host red blood cell are shown at right. Scale bars = 2μm. A. Point mutation of tyrosine residue at position 10 (✪) in the synthetic PfoTPT (Y10A) relocates protein from the apicoplast (B) to the plasma membrane. B. Removal of TMD 10 (TMD1to9), which completely abrogates targeting to the apicoplast (C), does not result in plasma membrane localisation. C. Removal of TMDs 9 and 10 (TMD1to8), which completely abrogates targeting to the apicoplast (D), results in some protein localisation to the plasma membrane. D. Removal of TMDs 7, 8, 9 and 10 (TMD1to6), which completely abrogates targeting to the apicoplast (E), results in localisation of the protein that mostly overlaps with the parasite plasma membrane marker, PfNT1. Note that the erythrocyte is infected with three separate parasites. E. Removal of TMDs 6, 7, 8, 9 and 10 (TMD1to5), which completely abrogates targeting to the apicoplast (F), results in localisation of the protein that mostly overlaps with the parasite plasma membrane marker, PfNT1. Note that the erythrocyte is infected with two separate parasites. F. Removal of TMDs 3, 4, 5, 6, 7, 8, 9 and 10 (TMD1to2), which completely abrogates targeting to the apicoplast (G), results in some re-localisation of the protein to the plasma membrane. G. Removal of TMDs 2, 3, 4, 5, 6, 7, 8, 9 and 10 (TMD1), which completely abrogates targeting to the apicoplast (H), results in some localisation of the protein to the plasma membrane. H. Recombining TMD1 with TMD10 (TMD1+10) was unable to reconstitute apicoplast targeting (I) and resulted in some plasma membrane targeting.

Lim L, Sayers CP, Goodman CD, McFadden GI. Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum. PLoS One. 2016 11(7):e0159603.