![]() Plants now showed a drastic reduction in growth, likely attributable to an uncontrolled overaccumulation of metals, and the vast majority died prematurely ( Fig. The consequences of this mislocalization of IRT1 were dramatic for the plants. When we looked at the localization of the corresponding mutated IRT1 protein in root hair cells, we found it readily at the plasma membrane. To evaluate if the localization of IRT1 in early endosomes was a consequence of monoubiquitin-dependent trafficking, we created a mutant version of IRT1 in which the amino acid residues that are posttranslationally modified with ubiquitin are modified. Monoubiquitination of plasma membrane proteins was indeed shown to trigger their internalization and their sorting into later endosomal compartments on their way to the vacuole/lysosome. The role of monoubiquitination in the context of plasma membrane proteins was reported years ago in several seminal articles from other model organisms ( 2, 3), guiding us in our quest for the role of IRT1 ubiquitination in plants. We confirmed that IRT1 was indeed ubiquitinated in plants and, using a set of specific antibodies that recognize different forms of ubiquitination, we observed that IRT1 was, in fact, decorated with several monoubiquitin moieties in vivo. This notably requires the covalent linkage of ubiquitin chains under the K48 form to the target protein. Ubiquitination is a posttranslational modification that has multiple roles, but it is mostly famous for tagging soluble proteins for degradation by a multiprotein complex called the proteasome. When searching for factors controlling such a dynamic behavior for IRT1, we came across a usual suspect in cell biology: ubiquitination. More generally, our work demonstrates the existence of monoubiquitin-dependent trafficking to lytic vacuoles in plants and points to proteasome-independent turnover of plasma membrane proteins. Together, these data suggest a model in which monoubiquitin-dependent internalization/sorting and turnover keep the plasma membrane pool of IRT1 low to ensure proper iron uptake and to prevent metal toxicity. We also prove that IRT1 is monoubiquitinated on several cytosol-exposed residues in vivo and that mutation of two putative monoubiquitination target residues in IRT1 triggers stabilization at the plasma membrane and leads to extreme lethality. ![]() Using pharmacological approaches, we show that IRT1 cycles to the plasma membrane to perform iron and metal uptake at the cell surface and is sent to the vacuole for proper turnover. ![]() IRT1 is unexpectedly found in trans-Golgi network/early endosomes of root hair cells, and its levels and localization are unaffected by iron nutrition. Here, we demonstrate that overexpression of Arabidopsis thaliana IRT1 leads to constitutive IRT1 protein accumulation, metal overload, and oxidative stress. Sophisticated regulatory mechanisms allow plants to tightly control the levels of IRT1, ensuring optimal absorption of essential but toxic iron. Plants take up iron from the soil using the IRON-REGULATED TRANSPORTER 1 (IRT1) high-affinity iron transporter at the root surface. ![]()
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