Fig 1. Subcellular location of PfAK2/GFP and the non-myristoylated G2A variant, as determined by live fluorescence microscopy and cell fractionation using SLO and saponin lysis together with a protease protection assay.
(A) The AK2/GFP and the AK2G2A/GFP fusion proteins were expressed using the CRT promoter from an episomal plasmid (constructs indicated above the images). AK2/GFP is located at the periphery of the intracellular parasite as judged by epifluorescence microscopy, and is associated with one or two protuberances towards the host cell cytoplasm present on each parasite (indicated by white arrow). In contrast, the AK2G2A/GFP chimera is located within the parasite cytosol. The infected cell was visualised by differential interference contrast (DIC), intrinsic fluorescence of the GFP identified the location of the AK2/GFP fusion protein, and parasite nuclei were detected by Hoechst staining. Overlay: green (GFP), blue (DNA). Scale bar—3 μm. (B) The PfAK2/GFP transgenic parasite and (C) the G2A variant parasite were both subjected to SLO and saponin lysis, separated into soluble supernatant (SN) and pellet (P) fractions, and then part of the pellet fraction was treated with Proteinase K (PrK). Western blot analysis with equal cell equivalents was performed using anti-GFP, anti-SERP (a soluble PV protein), and anti-aldolase (a parasite cytoplasm protein) antibodies. Size markers are in kDa. The absence of the protein band corresponding to the PfAK2/GFP fusion protein in the saponin pellet fraction treated with Proteinase K indicates secretion of the protein beyond the PPM.