The ESCRT machinery colocalizes with ubiquitinated Fth1-GFP during RapID degradation. (A) Design of the RapIDeg system for Fth1 degradation. (B) Western blots showing the fast degradation of Fth1-GFP-2xFKBP. (C) Quantification (±SD, n = 3) of protein levels in B. (D) Snapshots showing the colocalization of Fth1-GFP-2xFKBP with Vps4-3HA-mCherry during degradation. Dashed lines indicate the periphery of yeast cells. (E and F) Quantification of the number of Fth1 punctae per cell and their colocalization with Vps4-3HA-mCherry in D. Error bars represent SD. Numbers on each column indicate the total number of cells counted. (G) Snapshot Imaging showing the colocalization of Fth1-GFP-2xFKBP with Hse1-mCherry during degradation. (H and I) Quantification of the number of Fth1 punctae per cell and their colocalization with Hse1-mCherry in D. (J) Time-lapse imaging to show the colocalization (arrows) of Fth1-GFP-2xFKBP punctae with Hse1-mCherry during degradation. Ub, ubiquitin. Scale bars, 3 µm.