PMIX localizes to rhoptries, whereas PMX localizes to exonemes. (A and B) The PMIX gene was tagged with 3X hemagglutinin (HA) sequence at the 3′ end of the endogenous open reading frame. Tagged parasites grew normally. Segmented schizonts were prepared for immuno–electron microscopy, and PMIX was visualized with an anti-HA antibody and an 18-nm colloidal gold-labeled secondary antibody. Colocalization with an anti-RAP1 antibody, a marker for rhoptries, was performed using a 12-nm colloidal gold-labeled secondary antibody. The arrowhead points to an 18-nm particle; the arrow points to a 12-nm particle. (A) schizont; (B) merozoite. (C and D) The PMX gene was tagged with HA sequence. (C) Schizont; (D) enlarged view of the boxed area in (C). (E) The PMX gene was tagged with green fluorescent protein (GFP) sequence, and the exoneme marker SUB1 was tagged with 3X HA. Segmented schizonts were prepared for immuno–electron microscopy, PMX was visualized with an anti-GFP antibody and an 18-nm colloidal gold-labeled secondary antibody, and SUB1 was visualized with an anti-HA antibody and a 12-nm colloidal gold-labeled secondary antibody. Two examples are shown. Controls omitting the primary antibody were negative in all cases. (F and G) Electron micrographs of PMIXapt cultured with (F) or without (G) aTc. Note the apical granularity and discoid morphology (G). See fig. S1 for statistics. Scale bars, 500 nm [(A) to (C), (F), and (G)]; 100 nm (D); 200 nm (E). R, rhoptry; N, nucleus.

Nasamu AS, Glushakova S, Russo I, Vaupel B, Oksman A, Kim AS, Fremont DH, Tolia N, Beck JR, Meyers MJ, Niles JC, Zimmerberg J, Goldberg DE. Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Science. 2017 358(6362):518-522. PMID: 29074774.