The N-terminus of Plasmodium falciparum (Pf)ThrRS, PfGlyRS and PfAlaRS directs apicoplast targeting. (A) PfThrRS1–60-GFP, PfAlaRS1–70-GFP and PfGlyRS1–68-GFP transfected parasites were labelled with anti-GFP and anti-acyl carrier protein antibodies and DAPI. The aminoacyl-tRNA synthetase–GFP fusions overlap with the apicoplast marker ACP, demonstrating that the N-terminus of these gene products is sufficient for apicoplast targeting. Bright field panels show the parasite (P) residing in its host erythrocyte (E). (B) Live cell microscopy shows that the N-terminal fragments of the nuclear encoded organellar tRNAs (aaRS) do not direct trafficking to mitochondria. Live parasites were stained using Mitotracker to highlight the mitochondria and this was compared with the GFP signal for each of the aaRS–GFP parasites. In all cases the mitochondria labelled a distinct and separate organelle from the GFP, indicating that these N-terminal leaders do not direct mitochondrial trafficking.Jackson KE, Pham JS, Kwek M, De Silva NS, Allen SM, Goodman CD, McFadden GI, Ribas de Pouplana L, Ralph SA. Dual targeting of aminoacyl-tRNA synthetases to the apicoplast and cytosol in Plasmodium falciparum. Int J Parasitol. 2012 42:177-86. PMID:

Epitope-tagging of Plasmodium falciparum tRNAThr, tRNAGly and tRNAAla. The single, chromosomal copies of PfThrRS, PfGlyRS and PfAlaRS were tagged with a triple hemagglutinin through transfection and 3’ replacement. Immunfluorescence assays show that the majority of each of these epitope tagged proteins is localized to the parasite cytosol. An early trophozoite and a schizont stage parasite are shown for each parasite line. Parasites were labelled with anti-HA and anti-acyl carrier protein antibodies and DAPI. Bright field panels show the parasite (P) residing in its host erythrocyte (E). Some epitope tagged protein colocalises with the apicoplast marker ACP, but the dominant signal from the cytosolic HA-tagged protein in these parasites makes it unclear whether this is specific colocalisation or signal from adjacent protein. The very small size of the apicoplast (~200 nm diameter) makes this question difficult to resolve by light microscopy under these conditions. Jackson KE, Pham JS, Kwek M, De Silva NS, Allen SM, Goodman CD, McFadden GI, Ribas de Pouplana L, Ralph SA. Dual targeting of aminoacyl-tRNA synthetases to the apicoplast and cytosol in Plasmodium falciparum. Int J Parasitol. 2012 42:177-86.