Dominant negative modulation of plant endocytosis to identify fast-cycling cargoes of the TPLATE and AP-2 adaptor complexes

The plant plasma membrane (PM) contains a wide range of receptors, channels and other membrane proteins that control nutrient uptake and mediate communication of the cell with the outside world. The PM proteins, upon perception of hormonal stimuli, ligands or  pathogen presence, transfer these signals over the PM via signaling cascades that ultimately control plant growth and development. Removal of PM material and extracellular ligands (known as cargo), depends on retrograde transport by endocytosis, in which the cargo is predominantly internalized using coated vesicles. The main endocytic pathway in plants and animals is clathrin-mediated endocytosis (CME) defined by the involvement of the scaffold protein clathrin guiding the formation of a cage around the invaginating membrane. Up to date there are two known adaptor complex being involved in CME: well-studied Adaptor Protein Complex 2 (AP-2) and the rather unknown TPLATE complex (TPC). What is the exact role of TPC during endocytosis? What caused its evolutionary retention in plants compared to other Kingdoms? Can TPC-dependent, yet AP-2 independent endocytosis be attributed to cargo specificity? To answer this questions, I will develop an inducible tools to block endocytosis in plants at the protein level. Moreover, comparative PM proteome analysis will help me to unravel cargo proteins specifically recognized by TPC or AP-2 transport machinery. This project builds on preliminary work and comprises multiple approaches.