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.

• PMID: 30167804
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Journal Article

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Wang FH, Qiao K, Liang S, Tian SQ, Tian YB, Wang H, Chai TY

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