SH3Ps recruit auxilin-like vesicle uncoating factors into clathrin-mediated endocytosis

Abstract

Clathrin-mediated endocytosis (CME) is an essential process of cellular cargo uptake operating in all eukaryotes. In animal and yeast, CME involves BAR-SH3 domain proteins, endophilins and amphiphysins, which function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains BAR-SH3 domain proteins SH3P1-3, but their role is poorly understood. We identify SH3P1-3 as functional homologues of endophilin/amphiphysin. SH3P1-3 bind to discrete foci at the plasma membrane (PM), and colocalization indicates late recruitment of SH3P2 to a subset of clathrin-coated pits. PM recruitment pattern of SH3P2 is nearly identical to its interactor, a putative vesicle uncoating factor AUXILIN-LIKE1, and SH3P1-3 are required for most of AUXILIN-LIKE1 PM binding. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors, rather than the uncoating phosphatases synaptojanins. Furthermore, we identify an unexpected redundancy between SH3P1-3 and a plant-specific endocytic adaptor, TPLATE complex, showing a contribution of SH3P1-3 to gross CME.

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo

“…Proteins associated with the endocytic machinery such as TARGET OF MYB1 (TOM1)-LIKE (TOL) or SH3P2 are also capable of binding ubiquitin 23,32,33 . A role of SH3P in recognizing ubiquitinated cargo would be in agreement with the recent finding that nosh enhanced the plant developmental defects of a triple sh3p mutant, indicating that these proteins have a non-redundant role in plant development 72 . In mammalian cells, WD40-repeat βpropellers also bind ubiquitin 73 , which suggests that two TWD40 subunits of TPC might potentially also play a role in this.…”

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

“…Subcellular fractionations and quantitative colocalization analyses between SH3P2-GFP and compartment markers in the sac9 mutant background will be needed to fully understand how SAC9 impacts SH3P2 localization. In addition, the exact function of SH3P2 in endocytosis is also elusive and it will be important to analyze it in detail in the future ( Adamowski et al, 2022 ). Finally, one of the clear limitations in interpreting the sac9 phenotype, which is common to most genetic approaches, is the accumulation of defects over a long-time period in the mutant.…”

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

“…Subcellular fractionations and quantitative colocalization analyses between SH3P2-GFP and compartment markers in the sac9 mutant background will be needed to fully understand how SAC9 impacts SH3P2 localization. In addition, the exact function of SH3P2 in endocytosis is also elusive and it will be important to analyze it in details in the future (Adamowski et al, 2022). Finally, one of the clear limitations in interpreting the sac9 phenotype, which is common to most genetic approaches, is the accumulation of defects over a long-time period in the mutant.…”