GFP targeting by various P. falciparum redox proteins. (A) Apicoplast targeting of the AOP N-terminus. (B) Dual localization (cytosol and apicoplast) of the TPxGl N-terminal amino acid sequence. (C) ER-targeting of Trx3. (D) Mitochondrial targeting of Tlp2. (E) Mitochondrial targeting of GLP3. (F) Apicoplast targeting of the GILP N-terminal sequence. Colocalization of GFP with the mitochondrial dye MitoTrackerOrange in fixed cells. Colocalization of GFP and the apicoplast marker ACP PFB0385w or the ER marker BiP PF11_0098 in fixed, immunodecorated cells. Kehr S, Sturm N, Rahlfs S, Przyborski JM, Becker K. Compartmentation of redox metabolism in malaria parasites. PLoS Pathog. 2010 6:e1001242.

The modular gene architecture reflects the dual localization of PfAOP. Confocal live cell imaging of blood stage parasites expressing the indicated PfAOP-GFP chimera. The BTS is necessary and sufficient to target PfAOP to the apicoplast, as revealed by fluorescence microscopy and subcellular fractionation assays with transgenic parasites expressing different GFP-tagged PfAOP constructs. Removal of the N-terminus in PfAOPΔN-term-GFP abrogated the apicoplast targeting, whereas constructs with mutated methionine residues at the start of the Prx5 domain (PfAOPM71A/M77A-GFP) or without the C-terminal Prx5 domain (PfAOPBTS-GFP) were found exclusively in the apicoplast.Djuika CF, Huerta-Cepas J, Przyborski JM, Deil S, Sanchez CP, Doerks T, Bork P, Lanzer M, Deponte M. Prokaryotic ancestry and gene fusion of a dual localized peroxiredoxin in malaria parasites. Microb Cell. 2015 2(1):5-13.

Dual localization of PfAOP. (A) Immunofluorescence localization of PfAOP in blood stage parasites. (C) Co-localization analysis of PfAOP and a chimera of acyl-carrier protein and GFP (ACP-GFP) as an apicoplast-localized marker protein. Left, upper panel: a rather strong fluorescence was detected in the parasite cytosol. Left: Cytosolic or apicoplast localization of mutated and truncated GFP-tagged PfAOP. Additional confocal live cell images of blood stage parasites expressing the indicated PfAOP-GFP chimera. Early trophozoite to late schizont stage parasites are shown from top to bottom for each construct. Scale bar, 5 μm. Unprocessed full length PfAOP was not observed.Djuika CF, Huerta-Cepas J, Przyborski JM, Deil S, Sanchez CP, Doerks T, Bork P, Lanzer M, Deponte M. Prokaryotic ancestry and gene fusion of a dual localized peroxiredoxin in malaria parasites. Microb Cell. 2015 2(1):5-13.