Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

Abstract

Endocytosis is the process by which cells internalise molecules from their cell surface via plasma membrane-derived vesicles. In plants, clathrin-mediated endocytosis requires the evolutionarily ancient TSET/TPLATE complex (TPC), which was lost in metazoan and fungal lineages. TPC is required for membrane bending, but how TPC functions in the initiation of endocytosis and clathrin assembly is unclear. Here we used live-cell imaging and biochemical approaches to investigate the function of the Arabidopsis thaliana TPC subunit AtEH1/Pan1. Using in vitro and in vivo experiments we found that AtEH/Pan1 proteins can self-assemble into condensates through phase separation, which is influenced by both structured and intrinsically disordered regions. The proteome composition of these condensates revealed many key endocytic components which are selectively recruited via prion-like- and IDR-based interactions, including the ESCRT-0 TOM-Like proteins. Furthermore, AtEH/Pan1 condensates selectively nucleate on the plasma membrane by binding specific phospholipid species that are recognised by their EH domains. Visualization of the ultrastructure of the endocytic condensates via CLEM-ET revealed that the coat protein clathrin can assemble into lattices within condensates. Our results reveal that AtEH/Pan1 proteins act as scaffolds to direct endocytic machinery to specific plasma membrane regions to initiate internalisation. These findings provide new insight into the interplay between membranes and protein condensates.

The Arabidopsis SAC9 enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the plasma membrane

“…Indeed, PI4P, not PI(4,5)P 2 , is the major anionic lipids that powers the plasma membrane electrostatic field ( Simon et al, 2016 ), and is very likely key to recruit endocytic proteins in plants ( Yperman et al, 2021b ; Yperman et al, 2021a ). In addition, phosphatidic acid (PA) is also important to drive the electrostatic field of the plasma membrane ( Platre et al, 2018 ) and PA could be involved in the localization of AtEH1/Pan1, a component of the TPLATE complex ( Yperman et al, 2021a ; Dragwidge et al, 2022 ).…”

The Arabidopsis SAC9 Enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the Plasma Membrane

“…Indeed, PI4P, not PI(4,5)P 2 , is the major anionic lipids that powers the plasma membrane electrostatic field (Simon et al 2016), and is very likely key to recruit endocytic proteins in plants (Yperman et al 2021b; Yperman et al 2021a). In addition, phosphatidic acid (PA) is also important to drive the electrostatic field of the plasma membrane (Platre et al 2018) and PA could be involved in the localization of AtEH1/Pan1, a component of the T-PLATE complex (Yperman et al 2021a, Dragwidge et al, 2022).…”

The “Proxitome” of Arabidopsis Processing Bodies Reveals a Condensate-Membrane Interface Instructing Actin-Driven Directional Growth

“…Furthermore, DRPs are physically linked to the TPLATE complex 6 , and enriched under HS conditions (e.g., AtEH2, log 2 FC NS/HS =0/3.1). Recently AtEH1 was shown to phase-separate to induce endocytosis 73 . We hypothesize that during stress, DCP1 may cooperate with the endocytotic machinery.…”