Epsin and Sla2 form assemblies through phospholipid interfaces

Maria M Garcia-Alai,Rob Meijers,Marko Kaksonen,Michal Skruzny,Haydyn D T Mertens,Charlotte Uetrecht,Anna Gieras,Johannes Heidemann,Dmitri I Svergun

doi:10.1038/s41467-017-02443-x

Abstract

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. However, the precise mechanism of adapter protein assembly at the cell membrane is still unknown. Here, we show that the membrane–proximal domains ENTH of epsin and ANTH of Sla2 form complexes through phosphatidylinositol 4,5-bisphosphate (PIP2) lipid interfaces. Native mass spectrometry reveals how ENTH and ANTH domains form assemblies by sharing PIP2 molecules. Furthermore, crystal structures of epsin Ent2 ENTH domain from S. cerevisiae in complex with PIP2 and Sla2 ANTH domain from C. thermophilum illustrate how allosteric phospholipid binding occurs. A comparison with human ENTH and ANTH domains reveal only the human ENTH domain can form a stable hexameric core in presence of PIP2, which could explain functional differences between fungal and human epsins. We propose a general phospholipid-driven multifaceted assembly mechanism tolerating different adapter protein compositions to induce endocytosis.

Highlights

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane
To investigate how a lipid-dependent protein complex is formed between epsin epsin N-terminal homology (ENTH) domain and the Sla2/huntingtin interacting protein 1 related (Hip1R) AP180 N-terminal homology (ANTH) domain, we first determined the binding between the PIP2 lipid and ENTH domains from S. cerevisiae alone using native MS16,17
A crystal structure was determined of a complex between the ENTH domain of Ent[2] (ENTH2) from S. cerevisiae and the PIP2 phospholipid to a resolution of 3.4 Å (Table 1)

Summary

Introduction

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. Crystal structures of epsin Ent[2] ENTH domain from S. cerevisiae in complex with PIP2 and Sla[2] ANTH domain from C. thermophilum illustrate how allosteric phospholipid binding occurs. Clathrin does not bind to the plasma membrane directly, but to a range of adapter proteins that interact with lipids on the plasma membrane Many adapter proteins, such as epsin[5], AP1806, and AP-27, contain positively charged patches that bind to the head groups of specific phospholipids. Human epsin ENTH can autonomously form homo-oligomers, whereas fungal ENTH domains need the presence of Sla[2] ANTH domains to create stable assemblies Together, these data suggest that the ENTH/ANTH complex concentrates PIP2 locally on the plasma membrane to facilitate the formation of adapter complexes with cargo, clathrin, or other adapter proteins through PIP2-dependent interfaces

Methods

Results

Conclusion