Jeudi 06 mai 2021 à 14h
Functionally disrupting the endocytic TPLATE Complex at the protein level
Daniël Van Damme
(Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium – VIB/UGent Center for Plant Systems Biology, Ghent, Belgium)
Clathrin-Mediated Endocytosis (CME) is a well-characterized eukaryotic pathway and the major mechanism by which the cell internalizes membrane proteins, along with associated lipids and ligands (together termed cargo), from the plasma membrane (PM). CME is characterized by adaptor proteins and multi-subunit adaptor complexes that bridge the gap between the cargo and the clathrin cage. Two CME-involved adaptor protein complexes function in concert at the PM in plants – the tetrameric adaptor protein complex 2 (AP-2) and the octameric TPLATE complex (TPC). In contrary to viable AP-2 single subunit mutants, TPLATE single subunit knockouts are male sterile and conditional silencing causes seedling lethality, but only upon prolonged induction. In order to address the primary effects on CME from disrupting TPC function at the PM in somatic cells, we aimed to develop fast, non-transcriptional approaches.
As TPC functions exclusively at the PM, we hypothesized that removing it from the PM should instantly abolish its’ function. In order to achieve this, we have explored three different approaches: nanobody-dependent sequestration, rapamycin-dependent delocalization and conditional complex destabilization.
Delocalization of a protein away from its site of action in a complemented mutant background will generate a functional knockout. Nanobodies recognizing a particular fluorophore allow organelle-specific delocalization of any fluorophore-coupled protein of interest. Similarly, the Knock sideways system (KS) uses rapamycin to force the heterodimerization of two protein domains: FRB and FKBP, allowing conditional delocalization of proteins to a desired subcellular location. Thirdly, a conditional functional knock-out approach was obtained by exploring specific point mutations in TPLATE, of which one of them destabilized TPC at elevated temperature. We have generated proof-of-concept for the KS system in N. benthamiana and applied all three systems in Arabidopsis seedlings.
Jie Wang, Klaas Yperman, Peter Grones, Qihang Jiang, Jonathan Dragwidge, Evelien Mylle, Eliana Mor, Jonah Nolf, Dominique Eeckhout, Geert De Jaeger, Bert De Rybel, Roman Pleskot and Daniël Van Damme (2021). Conditional destabilization of the TPLATE complex impairs endocytic internalization. PNAS 118: e2023456118.
Joanna Winkler, Andreas De Meyer, Evelien Mylle, Peter Grones, Veronique Storme, Daniël Van Damme (2021). Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity. Frontiers in Plant Science 12: 538580.
Joanna Winkler, Evelien Mylle, Andreas De Meyer, Benjamin Pavie, Julie Merchie, Peter Grones, Daniël Van Damme (2021). Rapamycin-dependent delocalization as a novel tool to reveal protein-protein interactions in plants. The Plant Cell doi: 10.1093/plcell/koab004. Online ahead of print.
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Contact : Alexandre Martinière