Zinc-induced exposure of an LxL motif drives clathrin-mediated endocytosis of the zinc transceptor ZIP4

ZIP4 functions as a zinc transceptor essential for dietary zinc absorption and undergoes zinc-dependent endocytosis to dynamically regulate zinc uptake capacity in response to changes in zinc availability. While ZIP4 endocytosis is known to be regulated by zinc availability, the detailed molecular mechanism of this post-translational regulation remains elusive. In this work, we investigate the endocytosis-indispensable LxL motif to elucidate the molecular basis of ZIP4 endocytosis in HEK293T cells expressing ZIP4 exogenously and in cancer cell lines with endogenous ZIP4 expression. Integrating biochemical, cell and molecular biology, modelling, and computational approaches, our data collectively support a working model in which the LxL motif becomes more accessible at elevated cellular zinc levels and functions as a sorting signal that directly or indirectly associates with the adaptor protein complex 2 for clathrin-mediated endocytosis. These findings provide mechanistic insight into zinc-dependent ZIP4 endocytosis, advance our understanding of cellular zinc homeostasis, and establish a paradigm for studying substrate-induced endocytosis of other nutrient transceptors.