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. 2017 Sep 12;6:e27480. doi: 10.7554/eLife.27480

Figure 3. ASP3 is a maturase for microneme proteins and impacts microneme processing post-exocytosis via SUB1.

(A) Schematic representation of MICs and their processing pre-exocytosis (red) and post-exocytosis by ROM4/5 (light blue). (B) Immunoblots assessing the processing of the microneme proteins M2AP, MIC3, MIC6, AMA1 upon ASP3 knockdown. Catalase was used as loading control. (C) ASP3ty but not asp3ty-D299A can rescue the processing of MIC6 in absence of ASP3myc. (D), (E), (F) Immunoblots assessing the processing of the microneme proteins MIC5, TLN4 and PLP1 upon ASP3 knockdown. MIC5 was Ty-tagged at the endogenous locus in ASP3-iKD and transient plasmid transfection of epitope tagged TLN4 was used. (G) IFAs evaluating the localization of microneme proteins upon ASP3 depletion – M2AP, MIC6 and MIC3, co-localized with either MIC2 or AMA1. White arrowhead points to the pro form of M2AP. (H) Immunoblot analyses of ESA from ASP3ty-iKD parasites, +/−ATc, after 2% EtOH stimulation show impaired post-exocytosis cleavage of MIC2, M2AP and MIC4. (I) Schematic representation of microneme proteins and their putative processing post-exocytosis by SUB1. (J) Endogenously tagged SUB1 was localized in the microneme independently of ASP3. (K) Depletion of ASP3 showed impaired processing of SUB1in ESA analyses. TgSUB1 was detected using an antibody raised against PfSUB1. Black arrowheads represent pro and mature forms of the proteins.

Figure 3.

Figure 3—figure supplement 1. Effect of ASP3 depletion on MICs.

Figure 3—figure supplement 1.

(A) In parasites lacking ASP3, MIC2, MIC4, CPL, MIC8 processing is unchanged. Catalase was used as loading control. Arrowheads represent pro and mature forms of the proteins. (B) Microneme protein localizations were not affected by ASP3 depletion (48 hr +/−ATc). (C) Secretion of MIC8 was not affected with ASP3 depletion, as assessed by western blot analyses of excreted-secreted antigens (ESA) and pellets from ASP3ty-iKD parasites, +/−ATc, after stimulation with 2% Ethanol (EtOH). MIC2 was used as positive control for secretion, while Catalase was used as cytosolic control and GRA1 for constitutive secretion. Full length and secreted fragments are showed with arrowheads.