Triticum urartu MTP1: its ability to maintain Zn2+ and Co2+ homeostasis and metal selectivity determinants.

KEY MESSAGE: TuMTP1 maintains Zn2+ and Co2+ homeostasis by sequestering excess Zn2+ and Co2+ into vacuoles. The mutations NSEDD/VTVTT in the His-rich loop and I119F in TMD3 of TuMTP1 restrict metal selectivity. Mineral nutrients, such as zinc (Zn) and cobalt (Co), are essential or beneficial for plants but can be toxic at elevated levels. Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. However, the determinants of substrate selectivity have not been clarified due to the diversity of MTP1 substrates in various plants. In this study, Triticum urartu MTP1 was characterized. When expressed in yeast, TuMTP1 conferred tolerance to Zn2+ and Co2+ but not Fe2+, Cu2+, Ni2+ or Cd2+ in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn2+ and Co2+ when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn2+ and Co2+ stresses. Determinants of substrate selectivity were then examined through site-directed mutagenesis. The exchange of NSEDD with VTVTT in the His-rich loop of TuMTP1 restricted its metal selectivity to Zn2+, whereas the I119F mutation confined specificity to Co2+. The mutations H74, D78, H268 and D272 (in the Zn2+-binding site) and Leu322 (in the C-terminal Leu-zipper) partially or completely abolished the transport function of TuMTP1. These results show that TuMTP1 might sequester excess cytosolic Zn2+ and Co2+ into yeast vacuoles to maintain Zn2+ and Co2+ homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn2+-binding site and Leu322 are essential for its ion selectivity and transport function. These results can be employed to change metal selectivity for biofortification or phytoremediation applications.