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.
Highlights
Iron is an essential micronutrient but is toxic if accumulated at high levels
We focused on regulation of protein turnover, in part because it is known that the yeast ZIP family member ZRT1 is subject to metalinduced ubiquitination and endocytosis (Gitan et al, 1998; Gitan and Eide, 2000; Gitan et al, 2003)
The data presented here demonstrate that the Arabidopsis IRON-REGULATED TRANSPORTER1 (IRT1) metal transporter is subject to posttranslational regulation by iron
Summary
Iron is an essential micronutrient but is toxic if accumulated at high levels. 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). Studies in many different organisms have shown that iron deficiency induces a carefully regulated set of responses that function to increase the amount of iron available for metabolism. These responses can include increased uptake of iron from the environment and release of intracellular iron stores. A plasma membrane ferric chelate reductase enzyme serves to convert ferric iron to ferrous iron at the root surface; the Arabidopsis. The Arabidopsis IRONREGULATED TRANSPORTER1 (IRT1) gene encodes the primary high affinity ferrous iron transporter necessary for iron uptake from the soil (Eide et al, 1996; Henriques et al, 2002; Varotto et al, 2002; Vert et al, 2002)
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