Imaging and Quantifying the Endocytosis of IRON-REGULATED TRANSPORTER1 from Arabidopsis

Iron plays an essential role in plant metabolism and the regulation of its transport is essential for the plant. In Arabidopsis thaliana, iron uptake in root epidermal cells is mediated by the IRT1 (IRON-REGULATED TRANSPORTER 1) broad-spectrum transporter. The regulation of the IRT1 protein is controlled by sophisticated mechanisms that allow it to fine-tune the amount of transporter found at the plasma membrane and to modulate the uptake of iron and divalent metals transported by IRT1. IRT1 shows low selectivity and transports different metals such as manganese, zinc, cobalt, and cadmium. An excess of these non-iron metal substrates of IRT1 is toxic for the plant. The ability of plants to adapt to non-iron metal stress is based on the sensing of their excess, leading to the internalization and degradation of IRT1. IRT1 acts as a bifunctional transporter/receptor directly sensing metal non-iron excess and then undergoes a series of post-translational modifications of the protein culminating in its endocytosis and vacuolar degradation. To monitor the intracellular dynamics of IRT1, we describe in this chapter a live cell imaging approach to follow and quantify IRT1-mCitrine trafficking from the plasma membrane to the vacuole.