Figure S3.

Role of WIPI2d binding to the E3 complex goes beyond its membrane recruitment. (A) Co-sedimentation assay using DO-SUVs [75% PC:20% PE:5% PI(3)P] and the E3-GFP complex in the presence or absence of WIPI2d or Atg21. Quantification of three independent experiments (means ± SD; n = 3) is shown on the right. P, pellet; MW, molecular weight; ns, not significant; S, supernatant. (B) GFP-Trap beads coated with E3-GFP and mCherry-WIPI2d or mCherry-Atg21 or no PROPPINs were incubated with DO-SUVs containing 72% PC:20% PE:5% PI(3)P:3% ATTO390-PE. Quantification of three independent experiments (means ± SD; n = 3) is shown. AU, arbitrary units; n.s., not significant. (C) RFP-Trap beads coated with mCherry, mCherry-WIPI2d, or mCherry-Atg21 were incubated with DO-SUVs containing 72% PC: 20% PE:5% PI(3)P:3% ATTO390-PE. Quantification of three independent experiments (means ± SD; n = 3) is shown. P values were calculated using Student’s t test: **, 0.001 < P < 0.01. AU, arbitrary units. (D) E3-GFP (0.1 µM) recruitment to DO-GUVs containing 75% PC:20% PE:5% PI(3)P in the presence of wild-type (wt) WIPI2d (0.2 µM) or R108,125E mutant WIPI2d (0.2 µM) or no PROPPINs (–). The E3-GFP signal on GUVs was quantified and plotted (means ± SD; n wt = 107, n mut = 197, n no PROPPINs = 170). A blot probed for WIPI2 shows the protein input. AU, arbitrary units. (E) Coomassie-stained gel showing equal amounts of wild-type (wt) and R108,125E mutant proteins used in the bulk lipidation assays of Fig. 3. G and H.MW, molecular weight. (F) Co-sedimentation assay using DO-SUVs [75% PC:5% PI(3)P:20% PE] and the wild-type (wt) WIPI2d or R108,125E WIPI2d. MW, molecular weight.