The ER–endosome contact site localization of Vps13 is critical for ESCRT-mediated sorting. (A) Schematic of Vps13 mutants. (B) The ribbon cartoon of Vps13 was generated using AlphaFold2. (C) The surface model of the alphafold predicted structure of Vps13. Blue and yellow indicate hydrophilic and hydrophobic residues, respectively. (D) Vps13–GFP localization at the ER–endosome contact site. Vps13–GFP, DsRed–HDEL (ER), and Mup1–BFP (endosome) expressing WT and vps4Δ cells were stimulated with methionine for 30 min. Scale bar: 1 µm. (E) Quantification of Mup1-BFP puncta associated with the ER (DsRed-HDEL) from D. (F) Quantification of Vps13–GFP localization at the ER–endosome contact site from D. (G) Live-cell imaging analysis of Vps13–GFP at the ER-endosome contact site. Vps13–GFP, DsRed–HDEL (ER), and Mup1–BFP (endosome) expressing WT cells were stimulated with methionine for 30 min. Scale bar: 500 nm. (H) Two-dimensional cross-sections and three-dimensional models of vps4Δ cells. ER is traced in green. Ribosomes are indicated as green dots. The endosome stacks are shown in different colors to differentiate individual membranes. Round endosomes are traced in yellow. Larger tubular and cisternal structures are in various shades. Scale bars: 100 nm. (I and K) Quantification of Mup1 sorting in vps13 mutants from Fig. S2, J and K. (J) Mup1–GFP localization after methionine stimulation in vps13 mutants. Scale bar: 1 µm.