Fig. 6: C-terminal PH domain of PfVPS13L1 interacts with PfAegerolysin at the IMC.

a Volcano plot of DiQ-BioID of the C-terminus of PfVPS13L1 showing enrichment (3 independent samples per condition; second experimental repeat in Supplementary Fig. 10c–d) of proteins in plus rapalog over control as outlined in Supplementary Fig. 10a. Enriched proteins (fold change>1.5, p < 0.05) are highlighted with a black stroke and color-coded as indicated. Moderated two-tailed t-test was applied as implemented in the limma package. IMC proximity based on previous PhIL1 BioID⁵⁸; common contaminants based on previous DiQ-BioIDs³³. b Representative fluorescence microscopy images of parasites with endogenously mNG-SW-tagged PF3D7_1364000 and episomally expressing Halo-PhIL1, during IMC formation. c–d U-ExM images of parasites in mid-stages of IMC formation endogenously expressing PfAegerolysin-smV5-SW co-immunostained for GAP45-mCh (c) or α-MORN1 (d). e Domain cartoon of PfAegerolysin. Unknown domain in turquoise. f Alphafold3-predicted interaction between HEPN-L domain of PfAegerolysin and PH domain of PfVPS13L1 (iPTM=0.84). Left panel, interacting domains shown as ribbons. Right panel, domains separated and turned to show the interaction surface, colored by conservation across apicomplexan homologs, and the residues at the interaction interface shown as sticks. Conserved residues in the interface are highlighted. g Representative confocal images of parasites episomally expressing the PH domain of PfVPS13L1 (GFP-2xPH) with or without co-expression of the HEPN-L domain of PfAegerolysin artificially localized to the PM with a Lyn-targeting sequence. h Intensity measurements across a line transversing the PM, as in (g), of the GFP-2xPH construct when expressed by itself (black), or co-expressed with the Lyn-mCh-HEPN-L construct, either WT (green) or with two point mutations in the interacting surface (E740A/Q777A; red, representative images of the mutant HEPN-L in Supplementary Fig. 10i). Graph shows mean with SD for each pixel from n = 3 independent experiments for cells co-expressing the Lyn-mCh-HEPN-L constructs (WT=10 cells; mutant=14 cells) and n = 1 for expression of the PH domain alone (5 cells). U-ExM images are maximum intensity projections of 5 (c-top), 4 (c-bottom), 9 (d-top) and 2 (d-bottom) slices. DIC, differential interference contrast; Nuclei, Hoechst 33342; scale bars, 2 µm in (b, g); 5 µm in [c-top, d-top, j, l]; 1 µm in [c-bottom, d-bottom].