Eukaryotic cells maintain homeostasis of their outer membrane by controlled internalization of lipid and protein constituents via endocytosis. Endocytosis is evolutionary conserved and uses similarly folded domains. How these structural folds are combined into proteins and protein complexes, however, differs between eukaryotic kingdoms. The TPLATE complex (TPC) in plants is an evolutionary ancient protein module that combines several protein domains with a conserved role in endocytosis into a single octameric protein complex. Its molecular architecture, lipid-nucleated condensate formation and requirement for clathrin cage curvature revealed its function in endocytosis initiation in plants. Mechanistic understanding of how this complex drives membrane deformation during plant endocytosis is, however, lacking. Here we used an integrative structural approach to obtain a precise molecular structure of the TPC of Arabidopsis thaliana. In addition, our approach allowed visualizing the structural flexibility that hallmarks this enigmatic complex. We prove that the intrinsic structural flexibility is required for its functionality and membrane recruitment. The membrane-binding interface consists of several domains with differential lipid preferences. Finally, we demonstrate via molecular dynamics simulations that the crescent shape of the structured part of the complex is sufficient for membrane curvature generation. Our mechanistic insight, obtained by a combined biochemical and computational approach, shows that the structured part of the TPC likely contributes to the execution of plant endocytosis, which does not depend on cytoskeletal-based force generation.

Department of Experimental Plant Biology Faculty of Science Charles University Prague Prague Czech Republic

Department of Plant Biotechnology and Bioinformatics Ghent University Ghent Belgium

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

Structural Biology Brussels Vrije Universiteit Brussel Brussels Belgium

VIB Center for Plant Systems Biology Ghent Belgium

VIB VUB Center for Structural Biology Brussels Belgium

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