Small ARF-like 2 GTPase TITAN 5 is linked with the dynamic regulation of IRON-REGULATED TRANSPORTER 1

Abstract

The endomembrane system is the central sorting machinery for membrane proteins where signals can be integrated to control plant nutritional responses. IRON-REGULATED TRANSPORTER1 (IRT1) is tightly controlled through trafficking between the endomembrane system and the plasma membrane to balance the demands for iron (Fe) and toxic effects that may arise from excess of this micronutrient in roots. Small GTPases of the Ras superfamily regulate signaling inputs in endomembrane trafficking, but their functions and activities are poorly known in plants. One plant endomembrane system-associated regulatory component is the small ARF-like GTPase TITAN5/ARL2/ARLC1/HALLIMASCH (hereafter termed TTN5). TTN5 is required during early embryo development but many open questions remain with regard to its physiological function during the later life cycle or in response to environmental cues. Here, we demonstrate that TTN5 interacts with IRT1vr, the large cytoplasmic variable region and protein-regulatory platform of IRT1. TTN5 also interacts with peripheral membrane proteins that are components of the IRT1 regulation machinery and can be involved in protein trafficking, like SNX1, the C2 domain protein EHB1/CAR6 and the SEC14-GOLD domain-containing PATL2. We show that heterozygous ttn5-1+/- plants have a Fe reduction phenotype. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at the plasma membrane and in endosomes/multivesicular bodies, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. Hence, this work links IRT1 and iron nutritional regulation with a small GTPase. This opens up the possibility that iron nutrient levels are controlled via the cellular regulation of the endomembrane system.