Monoubiquitinated MxIRT1 acts as an iron receptor to determine MxIRT1 vacuole degradation or plasma membrane recycling via endocytosis.

IRON-REGULATED TRANSPORTER 1 (IRT1) is critical for iron uptake in roots, and its exocytosis to the plasma membrane (PM) is regulated by the iron status sensed by the histidine-rich domain (HRM). However, studies on the fate of IRT1 after fusion with PM in response to iron conditions are still limited. In this study, we found that K165 and K196 regulate the monoubiquitination of MxIRT1 (mUb-MxIRT1), which acts as a receptor delivering signals from HRM to downstream effectors such as clathrin to determine the fate of MxIRT1. Iron supply led MxIRT1 in the PM to monoubiquitin-dependent endocytosis which could be inhibited by endocytosis inhibitor TyrA23 or in the double site-directed mutant K165/K196R. Subsequently, the endocytosis pathway to the vacuole was inhibited by vacuolar protease inhibitor Leupeptin in excessive iron conditions and the inability of being able to respond to iron change, indicated by the protein accumulating in the PM, contributed to iron toxicity in K165/K196R transgenic Arabidopsis. With iron availability decreasing again, MxIRT1 could dock close to the PM waiting for to be recycled. Another monoubiquitination site, K26, was necessary for MxIRT1 Endoplasmic Reticulum (ER) export as site-directed mutant K26R lost the ability of PM targeting, and co-localized with the COPII subunit of the coat protein OsSec24. Therefore, after K26-directed ER export and iron-induced PM fusion, mUb-MxIRT1 determines subsequent vacuolar degradation or recycling to the PM via endocytosis for maintaining iron homeostasis.