Dataset Information

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 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.

SUBMITTER: Grones P

PROVIDER: S-EPMC7613989 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

Grones Peter P De Meyer Andreas A Pleskot Roman R Mylle Evelien E Kraus Michael M Vandorpe Michael M Yperman Klaas K Eeckhout Dominique D Dragwidge Jonathan Michael JM Jiang Qihang Q Nolf Jonah J Pavie Benjamin B De Jaeger Geert G De Rybel Bert B Van Damme Daniël D

Nature plants 20221201 12

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 ...[more]

PMID: 36456802

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Project description:BackgroundThe regulatory mechanism underlying the interaction of the Rabex-5 MIU domain with ubiquitinated cargos remains unclear.ResultsRabex-5 guanine nucleotide exchange factor (GEF) mutants affected interactions of ubiquitinated cargos.ConclusionGDP/GTP exchange in the GEF domain controls the MIU domain interactions with the ubiquitinated cargos.SignificanceRabex-5 GEF activity acts as an intramolecular switch for spatiotemporal trafficking of the ubiquitinated cargos. Ubiquitin (Ub)-dependent endocytosis of membrane proteins requires precise molecular recognition of ubiquitinated cargo by Ub-binding proteins (UBPs). Many UBPs are often themselves monoubiquitinated, a mechanism referred to as coupled monoubiquitination, which prevents them from binding in trans to the ubiquitinated cargo. However, the spatiotemporal regulatory mechanism underlying the interaction of UBPs with the ubiquitinated cargo, via their Ub-binding domains (UBDs) remains unclear. Previously, we reported the interaction of Rabex-5, a UBP and guanine nucleotide exchange factor (GEF) for Rab5, with ubiquitinated neural cell adhesion molecule L1, via its motif interacting with Ub (MIU) domain. This interaction is critical for the internalization and sorting of the ubiquitinated L1 into endosomal/lysosomal compartments. The present study demonstrated that the interaction of Rabex-5 with Rab5 depends specifically on interaction of the MIU domain with the ubiquitinated L1 to drive its internalization. Notably, impaired GEF mutants and the Rabex-5(E213A) mutant increased the flexibility of the hinge region in the HB-VPS9 tandem domain, which significantly affected their interactions with the ubiquitinated L1. In addition, GEF mutants increased the catalytic efficiency, which resulted in a reduced interaction with the ubiquitinated L1. Furthermore, the coupled monoubiquitination status of Rabex-5 was found to be significantly associated with interaction of Rabex-5 and the ubiquitinated L1. Collectively, our study reveals a novel mechanism, wherein the GEF activity of Rabex-5 acts as an intramolecular switch orchestrating ubiquitinated cargo-binding activity and coupled monoubiquitination to permit the spatiotemporal dynamic exchange of the ubiquitinated cargos.

Dataset Information

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

Publications

The endocytic TPLATE complex internalizes ubiquitinated plasma membrane cargo.

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