Skip to main content
. 2024 Aug 21;3(9):pgae342. doi: 10.1093/pnasnexus/pgae342

Fig. 5.

Fig. 5.

In condensates and at endocytic sites, loss of Eps15–ubiquitin interactions results in increased molecular exchange of Eps15. a) Representative image series of FRAP of Eps15, Eps15 + TetraUb, and Eps15ΔUIM + TetraUb droplets, respectively. Scale bar = 5 μm. b, c) Fluorescence recovery curves for Eps15 (b) or Eps15ΔUIM (c) droplets in the presence of MonoUb or TetraUb. Eps15/Eps15ΔUIM concentration was maintained at 7 μM with the addition of 1 μM MonoUb and 0.25 μM TetraUb, respectively. Droplets were made in 20 mM Tris–HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA, and 1 mM EGTA at pH 7.5 with 3% w/v PEG8000 and all droplet experiments were conducted at room temperature. Data shown as mean ± SE. n = 6 droplets under each condition. Mobile fractions and t1/2: Eps15 (85.9 ± 5.3%, 12.5 ± 0.8 s), Eps15 + MonoUb (82.7 ± 10.9%, 18.0 ± 1.3 s), Eps15 + TetraUb (79.0 ± 9.3%, 31.1 ± 1.2 s), Eps15ΔUIM (81.5 ± 12.1%, 21.4 ± 1.1 s), Eps15ΔUIM + MonoUb (75.74 ± 6.5%, 21.8 ± 1.9 s), and Eps15ΔUIM + TetraUb (76.3 ± 11.3%, 24.12 ± 1.6 s). d, e) Representative images of fluorescence recovery of Eps15-mCherry variants in clathrin-coated structures in Eps15KO cells expressing corresponding variants (d) and the average fluorescence recovery plots for each condition (e). Mobile fractions and t1/2: Eps15wt (41.3%, 19.1 s), Eps15ΔUIM (81.5%, 5.5 s), Eps15-DUB (96.7%, 8.0 s), and Eps15-DUB-dead (46.3%, 11.5 s). f, g) Representative images of fluorescence recovery of AP2-HaloTag labeled with JF646 in clathrin-coated structures in Eps15KO cells expressing corresponding Eps15 variants (f) and the average fluorescence recovery plots (g). Mobile fractions and t1/2: Eps15wt (71.9%, 29.7 s), Eps15ΔUIM (100.0%, 15.1 s), Eps15-DUB (103.9%, 6.8 s), and Eps15-DUB-dead (66.6%, 22.9 s). n = 6 pits were analyzed for each plot. Data were shown as mean ± standard error. Scale bar = 1 μm. All cell FRAP experiments were performed at 37°C. h) Schematic showing how polyubiquitin stabilizes the endocytic protein network by interacting with and cross-linking UIMs on endocytic proteins, resulting in productive clathrin-mediated endocytosis (Top). Removal of ubiquitin from the endocytic protein network using DUB decreases the network multivalency thus making the network less stable, resulting in less efficient clathrin-mediated endocytosis (Bottom).