Identification of the N‐terminal region of TjZNT2, a Zrt/Irt‐like protein family metal transporter, as a novel functional region involved in metal ion selectivity

Abstract

The Zrt/Irt‐like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N‐terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn2+ and Mn2+ transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn2+ uptake of yeast cells expressing them. As a result, the extracellular N‐terminal ends were identified as regions involved in Zn2+ selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N‐terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N‐terminal extension was shown to possess Zn2+ uptake activity. This suggests that the extended N‐terminal region inhibits Zn2+ transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N‐terminus that is important for the inhibition of Zn2+ transport. Furthermore, the N‐terminal truncated TjZNT2 lacked Mn2+ uptake activity. These findings suggest that the N‐terminal region is a novel substrate selector in the ZIP family of transporters.

AtIRT1, the Primary Iron Uptake Transporter in the Root, Mediates Excess Nickel Accumulation in Arabidopsis thaliana

“…The Ni uptake assay was performed as described previously (Nishida et al 2008, Nishida et al 2011, except that 50 mM CdCl 2 was substituted for 500 mM NiCl 2 .…”

The specificity of interaction of Zn2+, Ni2+ and Cu2+ ions with the histidine-rich domain of the TjZNT1 ZIP family transporter

“…for Zn 2+ and Mn 2+ ) in the ZIP family proteins. 10 As far as functional regions involved in metal ion selectivity are concerned, the loop between the transmembrane domains II and III should be considered. It has been shown that the replacement of a Glu residue at position 103 in wild-type IRT1 protein (ZIP family) with Ala changes the substrate specificity of the transporter by selectively eliminating its ability to transport Zn 2+ .…”