Iron-Induced Turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 Metal Transporter Requires Lysine Residues    

Abstract

Iron is an essential micronutrient but is toxic if accumulated at high levels. Thus, iron uptake and distribution in plants are controlled by precise regulatory mechanisms. IRON-REGULATED TRANSPORTER1 (IRT1) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis (Arabidopsis thaliana). Previously, we showed that IRT1 is subject to posttranscriptional regulation; when expressed from the constitutive cauliflower mosaic virus 35S promoter, IRT1 protein accumulates only in iron-deficient roots. IRT1 contains an intracellular loop that may be critical for posttranslational regulation by metals. Of particular interest are a histidine (His) motif (HGHGHGH) that might bind metals and two lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutant IRT1 alleles: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple His mutant), IRT1K146R, IRT1K171R, and a double mutant (IRT1K146R,K171R). Mutation of the His or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of each of the IRT1 variants and an IRT1intact construct in plants from the 35S promoter revealed that either K146 or K171 is required for iron-induced protein turnover, and 35S-IRT1K146R,K171R plants contain higher levels of iron as compared to 35S-IRT1 and wild type. Furthermore, accumulation of metals in 35S-IRT1K146R,K171R plants was not associated with an increase in ferric chelate reductase activity; this result indicates that, at least under conditions when iron is abundant, reduction of ferric iron may not be the rate-limiting step in iron uptake by strategy I plants such as Arabidopsis.

IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis

“…Lys-146 or Lys-171 were found to be required for the degradation of IRT1. Plants expressing IRT1 with substitutions at these two residues accumulated more Fe than the wild type (Kerkeb et al, 2008). This report was the first to support the involvement of ubiquitination in the degradation of IRT1.…”

“…IRT1 is a broad substrate range metal ion transporter that can transport Fe 2+ , Zn 2+ , Mn 2+ , Co 2+ , and Cd 2+ either expressed in yeast or in plants (Korshunova et al, 1999). Previous studies have suggested that in Arabidopsis, Fe-induced turnover of IRT1 is regulated by ubiquitination (Kerkeb et al, 2008;Barberon et al, 2011). The second intracellular loop of IRT1 is thought to be an E3 interacting and ubiquitination domain (Kerkeb et al, 2008).…”

“…The second intracellular loop of IRT1 is thought to be an E3 interacting and ubiquitination domain (Kerkeb et al, 2008). Several residues in this domain have been examined for their function in the degradation process (Kerkeb et al, 2008). Lys-146 or Lys-171 were found to be required for the degradation of IRT1.…”

“…Previous studies have suggested that in Arabidopsis, Fe-induced turnover of IRT1 is regulated by ubiquitination (Kerkeb et al, 2008;Barberon et al, 2011). The second intracellular loop of IRT1 is thought to be an E3 interacting and ubiquitination domain (Kerkeb et al, 2008). Several residues in this domain have been examined for their function in the degradation process (Kerkeb et al, 2008).…”

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis

“…A posttranslational control of IRT1 protein by ubiquitination was identified (15,16). IRT1 protein was shown to localize to early endosomes/trans-Golgi network compartments (EE/TGN) as a result of monoubiquitin-and clathrin-dependent endocytosis and is targeted to the vacuole for degradation via late endosomes (LE) (15).…”