Immunolocalization of PfGCN5 following inhibition of processing of the protein. (A) Immunofluorescence analysis of PfGCN5 in DMSO (control), MG132 (100 nM) and E64d (10 μM) treated parasites. Following inhibitor treatment, fixed parasites were subjected to immunofluorescence assay (IFA) using antibodies against PfGCN5 followed by secondary antibodies tagged with Alexa fluor 488 or Alexa fluor 568 for MG132 treated or E64d treated parasites respectively. DAPI stains the nuclei of the parasite. (B) Double immunofluorescence analysis was carried out using PfAtg18-GFPexpressing MG132 (100 nM) treated parasites in the presence of antibodies against PfGCN5, GFP and PfActin (in combination) followed by respective secondary antibodies tagged with Alexa fluor 594 (PfGCN5 and PfActin) or Alexa fluor 488 (GFP-Atg18). DAPI represents nuclear staining. Co-localization of PfGCN5 with PfAtg18 but not with control actin is indicated by white arrowhead in 2nd right subpanel. The right panels show the 3D reconstruction of the selected images from the left panel. (C) Immunoelectron microscopy images to detect the in vivo localization of PfGCN5 protein in untreated and MG132 treated parasites. Secondary antibodies tagged with 10-nm gold particle against primary antibodies (PfGCN5) were used for detection. Schizont stage parasites with nucleus and food vacuole are indicated as ‘N’ and ‘V’ respectively in DMSO and MG132 treated parasites. The selected areas (squares) are also shown at the higher magnification at the bottom (N, V and N, V1, V2 for untreated and drug treated parasites, respectively). Localization of protein is shown with the white arrowhead. The scale bars are indicated in the figure. (D) Schematic diagram shows the localization of gold particles around the food vacuole of MG132 treated parasite as shown (c).

Bhowmick K, Tehlan A, Verma S, Sudhakar R, Kaur I, Sijwali PS, Krishnamachari A, Dhar SK. Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway essential for its function. J Cell Sci. 2019 Dec 20. pii: jcs.236489.