DPAP3 is secreted at the time of egress.

(A) C2-arrested schizonts (DPAP3-HA) were either left on C2, treated with E64 after C2 wash out, or allowed to egress for 1 h in the presence of FY01. Parasite pellets from free merozoites and schizonts (insoluble fraction obtained after saponin lysis), proteins precipitated from the culture supernatant, and PV and RBC cytosol components (soluble saponin fraction), were run on a SDS-PAGE. The presence of DPAP3-HA in each fraction was visualized as a fluorescent band at around 130kDa, which correspond to the band identified by WB using an anti-HA antibody (See also S4A Fig). Hsp70 and BiP antibodies were used as WB markers of intracellular proteins (cytosol and ER, respectively), and SERA5 as a PV marker. (B) Mature DPAP3-mCh schizonts were arrested with C2 for 3 h, and egress observed by live video microscopy after C2 wash out (S1–S3 Videos). The representative still-frame pictures show DIC and mCherry signal (red) before or after PVM breakdown, and after RBCM rupture. (C) Quantification of mCherry signal measured on consecutive frames before and after PVM breakdown. Around 20% of the signal originates from the hemozoin autofluorescence (red line). As a bleaching control, the mCherry signal of schizonts that did not egress was quantified at the corresponding time frames. (D) IEM section obtained from DPAP3-GFP parasites. Close-up images of individual intracellular merozoites on the left show immunogold staining of DPAP3-GFP in close proximity to the rhoptries (green arrows) and at the apical end of merozoites (blue arrows). Images on the right show representative sections of schizonts with an intact (black arrows) or rupture PVM. Staining of extracellular DPAP3-GFP (white arrows) was only observed in schizonts lacking a PVM. Rhoptries (r), nuclei (n), and the RBCM (red arrows) are indicated. Rabbit anti-GFP and colloidal gold-conjugated anti-rabbit antibodies were used. Bar graph = 200 nm. IEM images obtained on the 3D7 control line are shown in S3D Fig, and the uncropped IEM images for the DPAP3-GFP line in S3E Fig.