Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae. These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein–protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein–protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.

Endocytosis in eukaryotic cells can occur via two separate mechanisms: actin-dependent and actin-independent pathways. In this study, we used the budding yeast Saccharomyces cerevisiae as a tractable model system to uncover the mechanistic basis for actin-independent endocytosis. This is directly relevant to the early stages of endocytic membrane invagination that occurs in mammalian cells through homologs of the proteins that we identify and study here in yeast (1, 2). In S. cerevisiae, membrane invagination that enables endocytosis is normally driven by growth of membrane-bound branched actin (3). A second actin-independent route to endocytosis is realized when intracellular turgor pressure is reduced. This reduction of turgor pressure alleviates the tension on plasma membranes that would normally oppose membrane invagination (1, 4). Although this actin-independent mechanism is not evident under laboratory conditions, it does occur at the hyperosmotic, high-sucrose concentrations that can be found in the wild when yeast grow on rotting fruit and under industrial fermentation conditions, particularly in the context of bioethanol production (1).In both mechanisms, endocytosis is initiated by the coordinated recruitment of a number of proteins associated with distinct stages of endocytic maturation (5). Clathrin heavy and light chains first interact with initiator proteins (Ede1 and Syp1) to form a lattice on the membrane. Subsequently, early coat proteins such as Sla1, Sla2, Ent1, Ent2, and Yap1801 (6) bind directly to the adaptor–clathrin lattice and form the cortical body (5). Electron microscopy data highlight the existence of hemispherical membraneless bodies around endocytic sites. These bodies are identifiable by following the localization of labeled endocytic coat proteins such as Sla1. The observed Sla1-labeled bodies are known to exclude ribosomes from regions that are near the cortical sites in the cytosol. Importantly, these endocytic bodies form even when actin is not polymerized, and the membrane is flat (7).Many of the coat proteins in bodies that form around endocytic sites include prion-like domains (PLDs). These are low-complexity intrinsically disordered domains that are enriched in polar amino acids such as glutamine, asparagine, glycine, and serine and are interspersed by aromatic residues (6, 8). Proteins with PLDs have the ability to drive the formation of membraneless biomolecular condensates through phase separation in cells (9) and in vitro (10). Condensates are mesoscale, nonstoichiometric macromolecular assemblies that concentrate biomolecules (11–13). Here, we show that endocytosis in S. cerevisiae involves the concentration of PLD-containing proteins, including the essential protein Sla1, within biomolecular condensates that form at cortical sites (14). Inferences from indirect measurements suggest that these condensates have viscoelastic properties and that they are scaffolded by a dense network of PLD-containing proteins. We show that condensate formation requires an intact PLD and the coat protein Sla1 is at the hub of the condensate-driving protein–protein interaction network. The distinctive compositional biases within PLDs of coat proteins contribute to condensate formation and function. We present a model, motivated by Hertz contact theory (15–17), to provide a plausible explanation for how interfacial tensions among condensates, the membrane, and the cytosol can enable membrane invagination and drive actin-independent endocytosis. This model shows that the formation of condensates and cohesiveness of molecular interactions within them are likely to be essential for mechanoactive processes associated with actin-independent endocytosis.