Canonical tyrosine-based motifs are required for constitutive endocytosis and polarity of IRT1 and contribute to metal uptake.

Endocytosis regulates the localization and abundance of plasma membrane proteins. Several endocytic mechanisms were shown to operate in plants. The plant metal transporter IRT1 was previously shown to undergo ubiquitin-mediated endocytosis and degradation upon excess of some of its metal substrates. However, the contribution of other endocytic mechanisms to IRT1 internalization and plant metal nutrition remains unclear. Here, we uncovered that the core machinery of clathrin-mediated endocytosis (CME) participates in constitutive IRT1 endocytosis and plant metal nutrition. IRT1 directly interacts with AP2M through cytosolic-exposed YxxΦ motifs. Alteration of IRT1 CME using point mutation in AP2 recognition motifs or ap2m knockout leads to defects in IRT1 endocytosis, secretion/recycling and polarity, irrespective of metal nutrition. Altered IRT1 subcellular localization is associated with impaired growth responses to non-iron metals. Altogether, our work highlights the importance of AP2 complex-mediated recognition of IRT1 YxxΦ motifs for the constitutive trafficking of IRT1 and its role in plant metal uptake.