PfGatA and PfGatB localizes to the apicoplast. Transgenic PfGatA-GFP and PfGatB-GFPparasites were generated in which an episomal construct contained the bipartite apicoplast targeting sequence from PfGatA and PfGatB cloned in front of a GFP (green fluorescent protein), and the expression was controlled by the P. falciparum calmodulin 5′ promoter. Differential interference contrast and fluorescent images were captured and processed using deconvolution microscopy; a merge of the images is presented on the far right column (overlay). A and B, PfGatA-GFP and PfGatB-GFP apicoplast localization was monitored via immunofluorescence assay using an anti-GFP antibody (green), and the apicoplast was detected by staining it with anti-ACP antibody (red). Nucleic acid was stained with DAPI (blue). PfGatA-GFP and PfGatB-GFP form characteristically small and round compartments early in the infection cycle (panels A [i] and B [i]), which then elongate and develop into complex, multiply branched forms at the trophozoite stage prior to splitting into individual spots, one for each merozoite, in the schizont stage (panels A [ii] and B [ii]). PfGatA-GFP and PfGatB-GFP co-localized with ACP (α-GFP/α-ACP overlay), confirming localization to the plastid. B, PfGatA-GFP and PfGatB-GFP do not localize to the mitochondrion in erythrocytic stages. The mitochondrion was labeled using MitoTracker Red,whereas PfGatA-GFP and PfGatB-GFP were detected via immunofluorescence assay using an anti-GFP antibody (green). Nucleic acid was stained with DAPI. In the early stages of parasite development (panels B [i] and C [i]), PfGatA-GFP, PfGatB-GFP and the mitochondria are discrete single organelles. In the late trophozoite stages, the mitochondria,Mailu BM, Li L, Arthur J, Nelson TM, Ramasamy G, Fritz-Wolf K, Becker K, Gardner MJ. Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites. J Biol Chem. 2015 Aug 28.. [Epub ahead of print]