Minisymposium 1: Mineral nutrition

Abs # 11003: Identification of amino acid residues critical for post-translational regulation of the IRTI metal transporter by iron in Arabidopsis.

Presenter:	Kerkeb, Loubna , loubna@biol.sc.edu
Authors	Kerkeb, Loubna  (A)   Chatterjee, Indrani  (A)   Ash, Joshua  (A)   Connolly, Erin L. (A)
Affiliations:	(A): Department of Biological Sciences, University of South Carolina

Although iron is an essential nutrient, it is toxic if accumulated at high levels. Hence, iron uptake and sequestration are controlled by precise regulatory mechanisms. Strategy I plants, including the dicots and non-grass monocots, induce expression of a set of genes in response to iron deprivation. The Arabidopsis FRO2 (Ferric Reductase Oxidase) gene encodes the inducible ferric chelate reductase responsible for reduction of iron at the root surface. IRT1 (Iron Regulated Transporter) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis; IRT1 belongs to the ZIP family of metal transporters. Previously, we showed that metals coordinately regulate the transcript abundances of FRO2 and IRT1 and that expression of FRO2 and IRT1 is subject to post-transcriptional regulation by iron. IRT1 protein accumulates only in iron-deficient roots of 35S-IRT1 plants, while root ferric chelate reductase activity is elevated only in iron-deficient roots of 35S-FRO2 plants. IRT1 contains an intracellular loop that may be critical for post-translational regulation by metals; of particular interest is a histidine motif (HGHGHGH) that is found in many ZIP family members. In addition, we targeted two intracellular loop lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutants: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple his mutant), IRT1K146R, IRT1K171R and a double mutant (IRT1K146R,K171R). All mutants were able to rescue the iron- and zinc-limited growth defects of the fet3fet4 and zrt1zrt2 yeast strains, respectively. This result demonstrates that mutation of the his or lys residues does not eliminate the ability of IRT1 to transport iron or zinc. Each of the IRT1 variants and an IRT1intact construct were expressed in plants from the 35S promoter. Mutation of the histidine motif did not alter IRT1 protein stability. However, western analysis revealed that 35S-IRT1K146R,K171R plants accumulate IRT1 protein when grown on iron-sufficient and iron-deficient medium while 35S-IRT1, 35S-IRT1K146R and 35S-IRT1K171R plants accumulate IRT1 protein only when grown on iron-deficient medium. 35S-IRT1K146R,K171R plants also accumulate higher levels of metals than 35S-IRTI, 35S-IRT1K146R, 35S-IRT1K171R and WT. These findings suggest a role for ubiquitination in metal-induced removal of IRT1 from the plasma membrane.