The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo

Abstract

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive.Here, we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this 2 evolutionary specific appendage modification allows TPC formation, but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

Protein Phosphorylation Orchestrates Acclimations ofArabidopsisPlants to Environmental pH

“…TPC may also be involved in the regulation of H + -fluxes across the plasma membrane. A mutant harboring defects in the large TPLATE subunit TASH3 – referred to as nosh (no SH3) - showed reduced internalization of TRANSMEMBRANE KINASE 1 (TMK1) (81). TMK1 is required for auxin-induced activation of plasma membrane H + -ATPases via phosphorylation, promoting cell wall acidification and cell elongation (82).…”

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants

“…We also identified a link between AtEH1 and TOM1-LIKE (TOL) proteins, which function in plants as ubiquitin receptors, analogous to the ESCRT-0 complex (Korbei et al, 2013; Mosesso et al, 2019; Moulinier-Anzola et al, 2020). We speculate that recognition of TOL proteins by AtEH subunits promote condensation and endocytosis initiation at membrane regions enriched for ubiquitinated cargo, possibly in co-operation with the TPC subunit TASH3 (Grones et al, 2022). This coupling of TPC recruitment and condensation to the co-operative recognition of lipids and ubiquitinated cargo would ensure the highly regulated assembly of clathrin-coated vesicles on the plasma membrane.…”

“…Images were acquired using the Leica SP8X system using a ×40 W 1.2 NA objective using 488 nm laser excitation, and collection from 650nm - 750nm. The ratio of plasma membrane signal and cytosolic signal were calculated using an automated detection macro in ImageJ ‘detect_membrane_and_cytoplasm_from_a_roi.ijm’ (Grones et al, 2022), using a threshold set to 60.…”

“…Samples were loaded and separated on a 4–20% SDS–PAGE TGX gel (BioRad) and subsequently blotted on polyvinylidenedifluoride (PVDF; BioRad). Membranes were blocked overnight at 4C in 5% skimmed milk in PBS-T. Next, the blots were incubated with the primary antibodies (α-TPLATE (Yperman et al, 2021b), 1:1000], α-AtEH1/Pan1 (Grones et al, 2022), 1:1000], α-CHC [Agrisera, AS10690, 1:2000], α-Tubulin [Sigma-Aldrich, T5168, 1:5000] and secondary antibodies (α-rabbit-HRP [Cytiva, NA934, 1:10000], α-mouse-HRP [Cytiva, NA931, 1:10000]) in 3% skimmed milk in PBST for 1 h. HRP-conjugated antibodies were detected using western lighting plus-ECL reagent (Perkin elmer).…”

“…TPC is essential for plant life, as viable knock-out mutants have not been identified (Gadeyne et al, 2014; Van Damme et al, 2006; Wang et al, 2019). Recent studies have established the critical role of TPC during endocytosis by demonstrating its interaction with many essential endocytic components, including anionic phospholipids (Yperman et al, 2021a; Yperman et al, 2021b), the AP-2 complex (Gadeyne et al, 2014; Wang et al, 2023), clathrin (Van Damme et al, 2011), and ubiquitinated cargo (Grones et al, 2022). Furthermore, destabilisation of TPC leads to impaired endocytic internalisation, and disrupts membrane bending during clathrin-coated pit formation (Johnson et al, 2021; Wang et al, 2021).…”