Altered selectivity in anArabidopsismetal transporter

Abstract

Plants require metals for essential functions ranging from respiration to photosynthesis. These metals also contribute to the nutritional value of plants for both humans and livestock. Additionally, plants have the ability to accumulate nonessential metals such as cadmium and lead, and this ability could be harnessed to remove pollutant metals from the environment. Designing a transporter that specifically accumulates certain cations while excluding others has exciting applications in all of these areas. The Arabidopsis root membrane protein IRT1 is likely to be responsible for uptake of iron from the soil. Like other Fe(II) transporters identified to date, IRT1 transports a variety of other cations, including the essential metals zinc and manganese as well as the toxic metal cadmium. By heterologous expression in yeast, we show here that the replacement of a glutamic acid residue at position 103 in wild-type IRT1 with alanine increases the substrate specificity of the transporter by selectively eliminating its ability to transport zinc. Two other mutations, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increase IRT1 metal selectivity by eliminating transport of both iron and manganese. A number of other conserved residues in or near transmembrane domains appear to be essential for all transport function. Therefore, this study identifies at least some of the residues important for substrate selection and transport in a protein belonging to the ZIP gene family, a large transporter family found in a wide variety of organisms. T he primary control point for metal ion homeostasis appears to be regulation of metal uptake across the plasma membrane. One important question that arises is whether there are separate transporters for each metal and, if not, which metals share the same transporter system. We have recently identified a group of metal transporters, the ZIP gene family, whose members have been implicated in the transport of a variety of metals (1). IRT1, the first ZIP gene to be identified, was discovered by functional complementation of the fet3 fet4 iron uptake mutant of Saccharomyces cerevisiae (2). IRT1 is also capable of complementing the metal uptake defects of the S. cerevisiae zrt1 zrt2 zinc uptake mutant and the S. cerevisiae smf1 manganese uptake mutant (3). This IRT1 complementation data, along with results from radioactive uptake assays (2, 3), demonstrate that IRT1 is capable of transporting Fe, Zn, and Mn; sensitivity and accumulation studies show that IRT1 can also transport cadmium (E. L. Connolly and M.L.G., unpublished data and this work). In this respect, IRT1 is similar to other iron transporters described to date (4, 5).The broad cation range of IRT1 makes it an excellent model system for the study of residues involved metal recognition and transport. To determine the importance of individual amino acid side chains for metal transport, we substituted alanine for a number of amino acid residues that are highly conserved among ZIP family members and ...

Monoubiquitin-dependent endocytosis of the IRON-REGULATED TRANSPORTER 1 (IRT1) transporter controls iron uptake in plants

“…This includes plasma membrane proteins undergoing ubiquitin-dependent endocytosis and degradation, such as the Can1 arginine transporter (29). Consistent with the ability of IRT1 to transport cadmium (3,30,31), IRT1 expression in yeast conferred hypersensitivity to external cadmium (Fig. S7).…”

Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein

“…9, A and B). Under limiting conditions, we also saw the expected increase in the transcript of the major root iron transporter IRT1, which is highly upregulated under iron limitation to increase the supply of iron to the plant cell (Eide et al, 1996;Korshunova et al, 1999;Rogers et al, 2000;Connolly et al, 2002). This up-regulation leads to an increase in cytoplasmic iron, but due to the poor substrate specificity of IRT1, it also results in increasing cytoplasmic levels of other toxic divalent metal cations, such as nickel.…”

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response

“…2B). This is the aqueous cavity of the protein through which cations are predicted to pass (30). The evidence accumulated to date suggests Zip transporters function to elevate the zinc content of constituents found within the cytosolic compartment of cells (29,31).…”