Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

Javier Lizarrondo,Martin A Schroer,Katharina Veith,Roland Thuenauer,Maria M Garcia-Alai,Dmitri I Svergun,Haydyn D T Mertens,Stephen P Muench,Stephan Niebling,Michal Skruzny,Marc Abella,Frank Sobott,David P Klebl

doi:10.1038/s41467-021-23151-7

Abstract

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.

Highlights

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination
Clathrin-mediated endocytosis requires the concerted action of many proteins to coordinate cargo recruitment, endocytic coat formation, and membrane bending
We determined the structure of a 16-mer assembly of the membrane-binding domains of Hip1R homolog Sla[2] and epsin Ent[1], AP180 Nterminal homology (ANTH) and epsin N-terminal homology (ENTH) domains, respectively, with PIP2 (8 ANTH:[8] ENTH:[20] PIP2) using cryoEM

Summary

Introduction

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. A time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites. Another question involving endocytic adaptors is whether this remodeling is stabilized by multimeric complex protein scaffolds in addition to hydrophobic insertions of epsin and AP180 amphipathic α0 helices[5,31,32,33,34,35] For all these questions the knowledge of the mechanism of ANTH and ENTH recruitment to the PIP2-enriched membrane and the detailed structure of AENTH assembly is of high importance. Our findings provide a molecular understanding of key adaptor protein structure during endocytic vesicle formation and demonstrate that the mechanism of assembly is not based on weak lipidprotein interactions, but on lipid-mediated oligomeric states

Methods

Results

Conclusion