Lipid-binding features and phosphoinositide-dependent function of the endocytic adaptor protein 2 (AP2)-complex from Arabidopsis thaliana

Abstract

Regulation of plasma membrane (PM) composition is crucial for plant cell function. Clathrin-mediated endocytosis (CME) is a key pathway for protein internalization from the PM. This study explores how anionic phospholipids, particularly phosphoinositides (PIs) regulate CME in Arabidopsis. Mutants with disrupted PI metabolism showed altered membrane order and impaired endocytosis. The AP2-complex, crucial for CME initiation, was found to directly bind PIs, especially PtdIns(4,5)P2, via polybasic regions. Mutated AP2-subunits with reduced lipid binding led to defective CME, as shown by reduced FM4-64 uptake and altered cargo internalization. AP2-subunits interacted with various lipid kinases important for PI biosynthesis, including PIP5K1, PIP5K2 and PIP5K6, which have been functionally implicated in CME. These findings suggest that PtdIns(4,5)P2 modulates CME through direct interaction with AP2, highlighting a molecular mechanism linking PI-metabolism to membrane trafficking in plants.