Condensation of Ede1 promotes the initiation of endocytosis

Abstract

Clathrin-mediated endocytosis is initiated by a network of weakly interacting proteins through a poorly understood mechanism. Ede1, the yeast homologue 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.

The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance

“…5d). These results suggest that IR puncta are not simply concentrations of IR constrained as a consequence of being fully enveloped by membranes, but instead can sometimes be partially associated with membranes, consistent with previously published results for IR 52 and other protein assemblies associated with plasma membranes and endosomes such as those formed by other signaling factors and neuronal post-synaptic densities 27,30,[53][54][55][56] . In the nucleus, IR puncta were found colocalized with markers of the transcriptional machinery (MED1 and RNA Polymerase II) at the insulin-responsive genes FASN, SREBF1, and TIMM22 (Supplementary Fig.…”

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast

“…Further, we observed here and previously ( Roberts-Galbraith and Gould, 2010 ) that Cdc15 cortical condensates recruit other CR components, and within the CR, Cdc15 has been observed in node-like structures by super-resolution imaging ( Laplante et al, 2016 ; McDonald et al, 2017 ). Taken together, these observations suggest that CR assembly involves condensation of a key membrane-bound scaffold, analogous to what occurs at other actin-based structures such as sites of endocytosis in yeast and focal adhesions in higher eukaryotes ( Day et al, 2021 ; Case et al, 2022 ; Kozak and Kaksonen, 2022 ).…”

Quantitative mapping of synaptic periactive zone architecture and organization

“…Pre-deployment could provide an essential mechanism to allow for low latency vesicle recycling or release site clearance, to build a large pool of molecules for high-capacity cargo binding and internalization, or for other emergent requirements such as limiting cargo diffusion (Azarnia Tehran and Maritzen, 2022). This pre-deployment suggests that different regulatory mechanisms may control the molecular dynamics and activation of membrane remodeling at synapses compared to non-neuronal cells, and could result from multiple mechanisms including specialized lipid domains (Khuong et al, 2010;Li et al, 2020;Wahl et al, 2016) or protein phase separation (Imoto et al, 2021;Kozak and Kaksonen, 2022). Yet to date most studies of synaptic endocytic protein dynamics have focused on global transitions between membrane compartments such as the synaptic vesicle pool and the plasma membrane (Bai et al, 2010;Denker et al, 2011;Li et al, 2020;Winther et al, 2015Winther et al, , 2013 or general diffusion (Reshetniak et al, 2020;Wilhelm et al, 2014), and there remains no data that show how the spatio-temporal organization of endocytic proteins might drive synaptic vesicle recycling.…”