Endocytosis of a zinc transceptor ZIP4 is mediated by AP2 through an atypical dileucine motif

Human ZIP4 is an essential zinc transporter for dietary zinc absorption and also a transceptor that undergoes zinc-dependent endocytosis to regulate cellular zinc uptake capacity in response to changes in zinc availability. However, the detailed molecular mechanism of this post-translational regulation remains elusive. In this work, we focus on the LxL motif (formerly LQL motif), which is located in the longest cytosolic loop and indispensable for ZIP4 endocytosis, to elucidate how it is involved in ZIP4 endocytosis in a zinc-responsive manner. Integrating a combination of biochemical, cell/molecular biology and modelling approaches, our data collectively support a working model that the LxL motif, which is found to be more exposed and/or less ordered upon increased zinc availability, acts as an atypical dileucine motif to interact with the adaptor protein complex 2 for clathrin-mediated endocytosis. These findings provide insights into the molecular basis of zinc-dependent ZIP4 endocytosis, advancing the understanding of cellular zinc homeostasis and providing a paradigm for mechanistic studies of substrate-induced endocytosis of other nutrient transceptors. HighlightsO_LIHydrophobicity of the LxL motif is essential for ZIP4 endocytosis C_LIO_LIEndocytosis of endogenously expressed ZIP4 depends on Clathrin and AP2 C_LIO_LIThe LxL motif binds to the {sigma}2 subunit of AP2 C_LIO_LICellular zinc levels regulate the structure of the LxL motif C_LI