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

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. The mechanistic contribution of the individual TPC subunits to plant CME remains however 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. Whereas one EH domain binds negatively charged PI(4,5)P2 lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel 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 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

The TPLATE subunit is essential for structural assembly of the endocytic TSET complex

“…Individual TPC subunits were built based on the experimental structures determined by X-ray crystallography and NMR spectroscopy or comparative models (Table S1) (Yperman et al, 2020). Given the similar domain organization in TPC, its evolutionary relationship to other coatomer complexes, and the published interaction between LOLITA-TASH3 ( Figure S1), we performed protein-protein docking of LOLITA and the TASH3 trunk domain (amino acid residues 104-894) utilizing the ClusPro2.0 docking algorithm (Gadeyne et al, 2014;Kozakov et al, 2017).…”

“…S1, Table S1). In addition, a structure was recently solved for both EH domains of AtEH1/Pan1 by NMR/X-ray crystallography (3% of the TPC sequences) ( 12 ). Based on the published interaction between LOLITA-TASH3, we performed protein-protein docking of LOLITA and the TASH3 trunk domain (amino acid residues 104-894) utilizing the ClusPro2.0 docking algorithm ( 8, 19 ).…”

“…The additional AtEH/Pan1 subunits in Arabidopsis TPC are the most structurally characterized members. They unite accessory protein interactions and membrane targeting via their EH domains while allowing dimerization through their coiled-coil regions ( 12, 13 ).…”

“…The additional AtEH/Pan1 subunits in Arabidopsis TPC are the most structurally characterized members. They represent unique plant constituents uniting accessory protein interactions and membrane targeting via their EH domains while allowing dimerization through their coiled-coil regions (Sánchez-Rodríguez et al, 2018; Yperman et al, 2020). Originally TPC was described as a major adaptor module for clathrin-mediated endocytosis but recent insight also implicated the AtEH/Pan1 subunits as initiators of actin-mediated autophagy (Wang et al, 2019).…”

“…Integrative structure determination of TPC The integrative modeling platform (IMP) package version 2.12 was used (Russel et al, 2012) to generate the structure of TPC. Individual TPC subunits were built based on the experimental structures determined by X-ray crystallography and NMR spectroscopy (Yperman et al, 2020) or comparative models created with MODELLER 9.21 (Šali and Blundell, 1993) based on the the related structures detected by HHPred (Zimmermann et al, 2018) and RaptorX (Källberg et al, 2012). Domain boundaries, secondary structures and disordered regions were predicted using the PSIPRED server (Buchan and Jones, 2019) by DomPRED (Bryson et al, 2007), PSIPRED (Jones, 1999) and DISOPRED (Jones and Cozzetto, 2014).…”