Fig. 1: Epsin1 fails to form protein condensates but is incorporated into condensates of Eps15 at low stoichiometric ratios. | Nature Communications

Fig. 1: Epsin1 fails to form protein condensates but is incorporated into condensates of Eps15 at low stoichiometric ratios.

From: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

Fig. 1

a Schematic of Eps15 and Epsin1 functional domains. Eps15 consists of three EH domains at its N terminus followed by a coiled-coil domain and a long intrinsically disordered region followed by two ubiquitin interacting motifs (UIMs) at the C terminal end. Epsin1 consists of the N-terminus ENTH (Epsin1 N-Terminal Homology) domain followed by two UIMs. The central part includes the CLAP (Clathrin/AP2 binding) region and multiple DPW motifs for binding AP2 followed by the C-terminus containing 3 NPF (Asn-Pro-Phe) motifs that bind to EH domain-containing proteins like Eps15. The side cartoons depict domain organization of Eps15 in dimeric form and Epsin1 as monomer. b Time course of fusion and re-rounding of Eps15 (12 μM) condensates (green) on contact. n = 3 independent replicates. c Varying concentration of Epsin1 (20 μM to 80 μM) in 3 weight% PEG8K. d 40 μM Epsin1 in varying weight% of PEG8K (3 weight% to 10 weight%). n = 3 independent replicates. e Eps15 (16 μM) condensates (green) incubated with 1.6 μM of Epsin1 and Epsin1ΔNPF (red), respectively. Plots on the right depict the intensity profile of the Epsin1 / Epsin1ΔNPF channel along the white dashed line shown in the corresponding images. n = 3 independent replicates. A.U. represents arbitrary units. f The distribution of the Epsin1 and Epsin1ΔNPF intensity ratio (Kapp) between the intensity inside the condensates and the solution. Partition of Amphyphysin was used as a negative control. In total, 100 condensates were analyzed under each condition. Data are mean ±  standard deviation, calculated from n = 3 independent replicates. Statistical significance was tested using an unpaired, two-tailed student’s t test on GraphPad Prism. g Representative time course of fusion events between condensates containing Eps15 and Epsin1. n = 3 independent replicates. Inset in (h) shows the interaction between Eps15 and Epsin1 (the NPF motif of Epsin1 binds to the EH domains of Eps15). All droplet experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K, at room temperature, 24 °C. All scale bars equal 5 μm.

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