TMD1 domain and CRAC motif determine the association and disassociation of MxIRT1 with detergent‐resistant membranes

Iron is essential for most living organisms. The iron‐regulated transporter1 (IRT1) plays a major role in iron uptake in roots, and its trafficking from endoplasmic reticulum (ER) to plasma membrane (PM) is tightly coordinated with changes in iron environment. However, studies on the IRT1 response are limited. Here, we report that Malus xiaojinesis IRT1 (MxIRT1) associates with detergent‐resistant membranes (DRMs, a biochemical counterpart of PM microdomains), whereas the PM microdomains are known platforms for signal transduction in the PM. Depending on the shift of MxIRT1 from microdomains to homogeneous regions in PM, MxIRT1‐mediated iron absorption is activated by the cholesterol recognition/interaction amino acid consensus (CRAC) motif of MxIRT1. MxIRT1 initially associates with DRMs in ER via its transmembrane domain 1 (TMD1), and thus begins DRMs‐dependent intracellular trafficking. Subsequently, MxIRT1 is sequestered in COPII vesicles via the ER export signal sequence in MxIRT1. These studies suggest that iron homeostasis is influenced by the CRAC motif and TMD1 domain due to their determination of MxIRT1‐DRMs association. Malus xiaojinesis IRT1 (MxIRT1) is a plasma membrane (PM)‐localized Fe2+ transporter. We found that MxIRT1 PM targeting and function depend on the association with detergent‐resistant membranes (DRMs, a biochemical counterpart of PM microdomains). This association occurs in endoplasmic reticulum (ER), and it is determined by transmembrane domain 1 (TMD1) of MxIRT1. After intracellular traffic and exocytosis into PM via vesicle, cholesterol recognition/interaction amino acid consensus (CRAC) motif of MxIRT1 regulates the shift of MxIRT1 from PM microdomains to PM homogeneous regions, and thus activates MxIRT1‐mediated Fe2+ uptake.