Zrt-/Irt-like protein 4 (ZIP4) belongs to Solute Carrier Family 39 (SLC39A) which controls zinc influx from the extracellular milieu to the cytoplasm. Out of 14 members in the ZIP family, ZIP4 is particularly important for zinc homeostasis as the exclusive zinc importer in the small intestine which is responsible for zinc uptake from diet. The cell surface expression of ZIP4 is regulated by endocytosis and recycling in which the internalized ZIP4 is temporarily stored in intracellular vesicles/organelles to be recycled or degraded, representing a rapid post-translational regulation of ZIP4. ZIP4 endocytosis is conducted in a zinc-dependent manner. Previous studies showed when both extracellular and intracellular zinc pools were depleted, ZIP4 endocytosis was drastically reduced, which can be restored by zinc repletion. A sorting motif in the cytoplasmic portion of the target membrane protein is supposed to be recognized by an adaptor protein for the later engagement of the endocytic machinery, which is a well-established mechanism in cargo recognition and endocytosis. By scanning the longest cytosolic loop of ZIP4, a conserved Leu-Gln-Leu (LQL) motif has been identified as an essential motif for endocytosis. In this work, we show that ZIP4 internalization is reduced when Clathrin or Adaptor protein 2 (AP2) complex subunits were knocked down by using the RNA interference technique, suggesting that ZIP4 endocytosis is through the canonical Clathrin-mediated internalization. Mutagenesis of the LQL motif indicated that the two hydrophobic residues play more important roles in mediating potential interactions with adaptor protein(s). This work provides additional insights into the post-translational regulation of nutrient transporters.

Zinc-dependent ZIP4 endocytosis mediated by adaptor protein complexes

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