Condensation of Ede1 promotes the initiation of endocytosis.

Clathrin-mediated endocytosis is initiated by a network of weakly interacting proteins through a poorly understood mechanism. Ede1, the yeast homolog of mammalian Eps15, is an early-arriving endocytic protein and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and endocytic uptake is decreased. We show that in yeast cells, cytosolic concentration of Ede1 is buffered at a critical level. Excess amounts of Ede1 form large condensates which recruit other endocytic proteins and exhibit properties of phase-separated liquid droplets. We demonstrate that the central region of Ede1, containing a coiled-coil and a prion-like region, is essential for both the condensate formation and the function of Ede1 in endocytosis. The functionality of Ede1 mutants lacking the central region can be partially rescued by an insertion of heterologous prion-like domains. Conversely, fusion of a heterologous lipid-binding domain with the central region of Ede1 can promote clustering into stable plasma membrane domains. We propose that the ability of Ede1 to form condensed networks supports the clustering of early endocytic proteins and promotes the initiation of endocytosis.

Computational and structural evidence for neurotransmitter-mediated modulation of the oligomeric states of human insulin in storage granules.

Human insulin is a pivotal protein hormone controlling metabolism, growth, and aging and whose malfunctioning underlies diabetes, some cancers, and neurodegeneration. Despite its central position in human physiology, the in vivo oligomeric state and conformation of insulin in its storage granules in the pancreas are not known. In contrast, many in vitro structures of hexamers of this hormone are available and fall into three conformational states: T6, T3Rf3, and R6 As there is strong evidence for accumulation of neurotransmitters, such as serotonin and dopamine, in insulin storage granules in pancreatic ß-cells, we probed by molecular dynamics (MD) and protein crystallography (PC) if these endogenous ligands affect and stabilize insulin oligomers. Parallel studies independently converged on the observation that serotonin binds well within the insulin hexamer (site I), stabilizing it in the T3R3 conformation. Both methods indicated serotonin binding on the hexamer surface (site III) as well. MD, but not PC, indicated that dopamine was also a good site III ligand. Some of the PC studies also included arginine, which may be abundant in insulin granules upon processing of pro-insulin, and stable T3R3 hexamers loaded with both serotonin and arginine were obtained. The MD and PC results were supported further by in solution spectroscopic studies with R-state-specific chromophore. Our results indicate that the T3R3 oligomer is a plausible insulin pancreatic storage form, resulting from its complex interplay with neurotransmitters, and pro-insulin processing products. These findings may have implications for clinical insulin formulations.