Fig. 1: PfVAP is an essential ER protein and binds FFAT motifs.

a Domain cartoon of PfVAP. MSP, Major Sperm Protein domain; CC, coiled coil; TM, transmembrane. b Fluorescence microscopy images of Halo-PfVAP^endo parasites (Supplementary Fig. 1b–c) episomally co-expressing the TM domain of Sec61β fused to mScarlet (mSc-TM^Sec61β). Arrowheads, PfVAP enriched in hotspots at ER (also in Supplementary Fig. 1e). c Schematic and quantification of PfVAP loss in GFP-2xFKBP-PfVAP^endo parasites episomally co-expressing mSc-TM^Sec61β after diCre-mediated gene excision induced using rapalog (+Rapa, PfVAP cKO) [left panel, 3-4 experimental replicates, with 36, 17, 62 and 38 parasites scored for the first timepoint (cycle 1, day 2); 53, 20, 68 parasites for the second timepoint (cycle 2, day 3); 61, 33, 41, 88 parasites for the third timepoint (cycle 2, day 4)] and representative fluorescence microscopy images from both cycles parasites (right panel). d Growth of GFP-2xFKBP-PfVAP^endo parasites with rapalog (PfVAPcKO) or without (control), measured by flow cytometry over 7 days (left panel) and growth relative to control (Cntrl) on day 7 in 3 experimental replicates (right panel; bars, average and SD; p-value, two-tailed paired t test). e Schematic of the experimental design to test PfVAP and FFAT motif binding. The motif is anchored to the PM via Lyn, promoting ER-PM MCS formation if bound to PfVAP. f Conventional and mutated FFAT sequences used, based on the opisthokont consensus. g Representative confocal microscopy images of Halo-PfVAP^endo parasites episomally expressing the indicated FFAT motif (left panel) and quantification of ER-PM MCSs distance (percentage of total PM circumference, right panel), shown as superplot¹⁰³, from n = 3 independent experiments (right panel) with a total of 28 and 29 parasites (1- or 2-nuclei trophozoite stage) quantified for the conventional (Conv.) and mutated (Mut.) motif, respectively; colors indicate independent experiments (small dots, individual parasites; large dots, average of each experiment; black lines, mean and SD; p-value, two-tailed unpaired t test of the means). DIC, differential interference contrast; Nuclei, Hoechst 33342; scale bars, 2 µm.