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.

• MeSH
• adaptorové proteiny signální transdukční chemie   genetika   MeSH
• buněčná membrána metabolismus   MeSH
• endocytóza * MeSH
• geneticky modifikované rostliny MeSH
• krystalografie rentgenová MeSH
• membránové proteiny chemie   MeSH
• proteinové domény MeSH
• proteiny huseníčku MeSH
• proteiny vázající vápník chemie   genetika   MeSH
• rostlinné proteiny chemie   genetika   MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky MeSH
• tabák genetika   MeSH
• transport proteinů MeSH
• vazba proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH
• proteiny huseníčku MeSH
• proteiny vázající vápník MeSH
• rostlinné proteiny MeSH
• TPLATE protein, Arabidopsis MeSH Prohlížeč