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.

Archaeometry Research Group Department of Archaeology Ghent University Ghent Belgium

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biochemistry and Microbiology Ghent University Ghent Belgium

Department of Chemistry 10 ray Microspectroscopy and Imaging XMI Research Unit Ghent University Ghent Belgium

Department of Chemistry Atomic and Mass Spectrometry A and MS Research Unit Ghent University Ghent Belgium

Department of Plant Biotechnology and Bioinformatics Ghent University Ghent Belgium

Institute of Experimental Botany Academy of Sciences of the Czech Republic Prague 6 Czech Republic

VIB Center for Inflammation Research Ghent Belgium

VIB Center for Plant Systems Biology Ghent Belgium

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