Characterization of MVB biogenesis in vps13 mutants. (A) Schematic of ESCRT-mediated sorting at the endosome. (B) The ubiquitination of Mup1–GFP. Mup1–GFP expressing cells were stimulated with methionine for 15 min and then immunoprecipitated under denatured conditions. The immunoprecipitated (IP) products were analyzed by immunoblotting using anti-GFP and anti-ubiquitin antibodies. (C) GFP–Vps27 (ESCRT-0) localization. Scale bar: 1 µm. (D) Quantification of GFP–Vps27 localization from C. (E) Snf7–GFP (ESCRT-III) localization. Scale bar: 1 µm. (F) Quantification of Snf7–GFP localization from E. (G) Thin section electron miscopy images of WT and vps13Δ yeast cells. Scale bars: 100 nm. (H) The value of quantification data of electron tomography analysis. (I) Quantification of endosome clustering. (J) Quantification of the chance of MVBs (endosomes) in WT and vps13Δ cells. (K) Fluorescence intensity of the Vps55–mNeonGreen punctate structures from Fig. 4 D. Source data are available for this figure: SourceData FS3.