Extended Data Fig. 4: Eps15 mutants and Fcho1 assemble to varying degrees in solution.

a-c, Fcho1 is labeled with Atto-594, Eps15 mutants are labeled with CF488a. Panels on the left show 7 μM Eps15 mutant alone, set of panels on the right show 6.8 μM Eps15 mutant combined with 0.2 μM Fcho1 (34:1). Cartoons depict binding interaction between Fcho1 and Eps15 mutants. a, Eps15 lacking the EH domains (Eps15-∆3xEH) does not form droplets on its own, but when combined with Fcho1 forms small droplets. b, Eps15 lacking the C-terminal disordered domain (Eps15-∆CTD) does not form droplets on its own and addition of Fcho1 does not induce droplet formation, reinforcing that the CTD of Eps15 mediates its interaction with Fcho1. c, Eps15 containing mutated Fcho1-binding DPF motifs (amino acids 623-636; Eps15-DPF>APA) robustly assembles into droplets on its own and co-assembles into droplets with Fcho1, presumably because the disordered domains of Eps15 and Fcho1 interact even in the absence of 3 key DPF motifs. Scale bar is 10 µm.