Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding

Klaas Yperman,Anna C Papageorgiou,Martin Potocký,Roman Pleskot,Yehudi Bloch,Savvas N Savvides,Dominique Eeckhout,Peter Vandenabeele,Qihang Jiang,Frank Vanhaecke,Romain Merceron,R A Grigoryan ,Jelle Van Leene,Konstantinos Tripsianes,Thomas Evangelidis,Geert De Jaeger,Steven De Munck,Pieter Tack,László Vincze ,Daniël Van Damme

doi:10.1038/s41467-021-23314-6

Abstract

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Highlights

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane
The residence time of integral membrane proteins at the plasma membrane is controlled by internalization signals that are recognized by adaptor protein complexes, which mediate a process named clathrin-mediated endocytosis (CME)
Adaptor Protein-2 complex (AP-2) and TPLATE complex (TPC) likely have independent and complementary roles in CME3. Both protein complexes have a core-complex of four subunit[5], which in the case of TPC is associated with four additional subunits (TWD40-1, TWD40-2, AtEH1/Pan[1], and AtEH2/Pan1)[6]

Summary

Introduction

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. Set out to characterize the function of the EH domains of the AtEH/Pan[1] proteins in Arabidopsis. To structurally characterize both EH domains of AtEH1/Pan[1], we expressed recombinant proteins in E.coli and purified highly monodisperse samples for X-ray crystallography and NMR (Supplementary Fig. 1).

Results

Conclusion