<p><i>Pf</i>ATG18 marks the DVM without junctional enrichment.</p>

RAP-inducible DiCre excises the <i>loxP</i>-flanked sequence. Shown are the endogenous wild-type locus, the repair template, and the modified locus before and after excision. Arrowheads and dotted lines denote the positions of the primers and the expected PCR products, with amplicon sizes indicated.

A second independent line (<i>atg18:loxP</i>) lacks the mNG tag. <b>(C)</b> <i>Pf</i>ATG18 is expressed throughout the entire asexual intraerythrocytic cycle. Representative parasite stages are shown. DNA: Hoechst 33342. <b>(D)</b> <i>Pf</i>ATG18 localizes around hemozoin, visualized using two crossed polarizers. <b>(E)</b> <i>Pf</i>ATG18-mNG staining delineates the DV, as demonstrated by co-localization with PM2-mCh. <b>(F,G)</b> <i>Pf</i>ATG18 colocalizes with CRT at the DVM (F) and is occasionally observed at DV-adjacent compartments (G, arrowhead). <b>(H-J)</b> <i>Pf</i>ATG18 is not enriched at DV-lobe junctions. <b>(H)</b> Normalized CRT-mCh and <i>Pf</i>ATG18-mNG intensities were subtracted to reveal signals exclusive to each protein.

The arrowhead marks the membrane junction. Maximum ATG18-mNG intensities at DV, lobe, and junction (I) showed no junctional accumulation beyond the sum of both membranes <b>(J)</b>. Individual and mean values; one-way ANOVA with Tukey’s test; n = 30 parasites, 3 experiments. n.s., <i>p</i> > 0.05; ***, <i>p</i> < 0.001. <b>(K-O)</b> <i>Pf</i>ATG18 shows no overlap with fluorescently tagged early (K) or late (L) endosomal markers, retromer components (M,N) or apicoplast-resident folate transporter 2 (FT2, <b>O)</b> [<a href="plospathogens.org/article/info:doi/10.1371/journal.ppat.1014608#ppat.1014608.ref045" target="_blank">45</a>].

Scale bars: 5 µm. Source data are provided as a <a href="plospathogens.org/article/info:doi/10.1371/journal.ppat.1014608#ppat.1014608.s015" target="_blank">S3 File</a>.</p>