Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

Abstract

Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane-actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2's ANTH and Ent1's ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a liganddependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.ANTH domain | membrane remodeling | PIP 2

Helping Hands for Budding Prospects: ENTH/ANTH/VHS Accessory Proteins in Endocytosis, Vacuolar Transport, and Secretion

“…Recent reports suggest that Epsins link vesicle budding sites with the cytoskeleton. The yeast epsin Ent1 directly interacts with actin via a phospho-regulated binding domain (Skruzny et al, 2012), and a similar acting binding activity has been found in mammalian Epsin1 (Messa et al, 2014). Thus, the membrane destabilizing properties of the ENTH domain, combined with mechanical force introduced by the actin cytoskeleton might lead to membrane bulging and, eventually, vesicle fission.…”

Dual Role of BAR Domain-containing Proteins in Regulating Vesicle Release Catalyzed by the GTPase, Dynamin-2

“…The Drosophila homologue of epsin, Liquid facets (Lqf), is essential for endocytosis, but its role is complex as transgenic expression of either the ENTH domain-encoding N terminus or the remaining cargo and coat-interacting C-terminal regions could rescue endocytosis in Lqf-deficient flies (57). Genetic studies in yeast have revealed roles for epsins in recognition of ubiquitinated cargo molecules (58), as a scaffold for assembly and organization of components of the early endocytic machinery (59) and as a platform for actin assembly (60). Studies in mammalian cells have suggested a role for epsin as an adaptor for ubiquitinated cargo (61,62) and shown that it is recruited early to CCPs along with other adaptor and scaffolding molecules (63), where it potentially regulates early stages of CCP maturation (62).…”

Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways

“…TORC2 inhibition also affects phosphorylation of Ark consensus motifs in Ent1. It has been proposed that phosphorylation at these sites alters the actin-binding properties of Ent1 (67). It is possible that TORC2 regulates actin organization and endocytosis via regulation of Ark1/Prk1 activity.…”