Fig. 3: PfVAP DiQ-BioID identifies FFAT-motif containing proteins.

a Schematic of hit filtering based on FFAT motifs starting with the 185 hits of the PfVAP DiQ-BioID (Fig. 2a–c) as indicated in the materials and methods and Supplementary Data 3. Scoring cut-offs are indicated; iPTM, interface predicted template modeling. b Table summarizing the 13 highest scoring FFAT motifs from (f) (Seq. Score, FFAT score based on Slee, J.A. & Levine, T.P⁴⁰. AF3 iPTM, MSP domain of PfVAP AlphaFold3 iPTM value⁴¹) and experimental data shown in (c) quantified and displayed as a superplot¹⁰³ as in Fig. 1g [3 independent replicates and a total of at least 20 parasites (1- or 2-nuclei trophozoite stage, exact number indicated below next to p-value); colors indicate independent experiments (small dots, individual parasites; large dots, average of each experiment; black lines, mean and SD); binding was considered when there was no significant difference (p ≥ 0.05) with the positive control (Fig. 1g) and indicated in green (gray, not tested; red, no binding), two-tailed unpaired t test of the means (exact n and p-values: PF3D7_0321300, 25, 0.0026; PF3D7_0726600, 31, 0.0003; PF3D7_1131800, 23, 0.0917; PF3D7_1206700, 20, 0.5591; PF3D7_1211200, 27, 0.727; PF3D7_1311600, 25, 0.8825; PF3D7_1316600, 25, 0.9431; PF3D7_1346400, 25, 0.1544; PF3D7_0604100, 27, 0.7238; PF3D7_0727100, 24, 0.0012; PF3D7_1241900, 25, 0.0015)]. Conservation of motifs (Conserv.) is indicated in green (no conservation in red); ** (yellow), motif that is conserved but part of a folded region, hence unlikely to bind PfVAP. c Representative confocal microscopy example images of Halo-PfVAP^endo parasites episomally expressing the FFAT motifs indicated and used to quantify the resulting ER-PM MCSs distance as in Fig. 1g and summarized in (b). d Alignment of the 7 binding motifs identified and a sequence logo built from their conserved homologous sequences in 10 different Plasmodium species. Amino acids are colored according to their properties: green, aromatic; red, acidic; blue, phosphorylatable. Scale bar, 2 µm.