College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, Guangdong Engineering Research Center for Marine Algal Biotechnology, College of Life Science and Oceanography, Shenzhen University, Shenzhen, China.
Plant Cell Rep. 2018 Dec;37(12):1653-1666. doi: 10.1007/s00299-018-2336-z. Epub 2018 Aug 24.
TuMTP1 maintains Zn and Co homeostasis by sequestering excess Zn and Co 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 Zn and Co but not Fe, Cu, Ni or Cd in solid and liquid culture and localized on the vacuolar membrane. Furthermore, TuMTP1-expressing yeast accumulated more Zn and Co when treated. TuMTP1 expression in T. urartu roots was significantly increased under Zn and Co 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 Zn, whereas the I119F mutation confined specificity to Co. The mutations H74, D78, H268 and D272 (in the Zn-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 Zn and Co into yeast vacuoles to maintain Zn and Co homeostasis. The NSEDD/VTVTT and I119F mutations are crucially important for restricting the substrate specificity of TuMTP1, and the Zn-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.
TuMTP1 通过将多余的锌和钴螯合到液泡中来维持锌和钴的体内平衡。TuMTP1 的组氨酸环富区中的 NSEDD/VTVTT 突变和跨膜结构域 3 中的 I119F 突变限制了金属选择性。矿物质营养元素，如锌（Zn）和钴（Co），对植物是必需的或有益的，但在高浓度下可能有毒。金属耐受蛋白（MTP）是参与细胞内金属体内平衡的阳离子扩散促进剂（CDF）转运体家族的植物成员。然而，由于不同植物中 MTP1 底物的多样性，底物选择性的决定因素尚不清楚。本研究对小麦族滨麦 MTP1 进行了表征。当在酵母中表达时，TuMTP1 赋予了对 Zn 和 Co 的耐受性，但在固体和液体培养中对 Fe、Cu、Ni 或 Cd 没有耐受性，并且定位于液泡膜上。此外，在用 TuMTP1 处理的酵母中积累了更多的 Zn 和 Co。在 Zn 和 Co 胁迫下，TuMTP1 在滨麦根中的表达显著增加。然后通过定点突变检查了底物选择性的决定因素。TuMTP1 组氨酸环富区中的 NSEDD 与 VTVTT 的交换限制了其对 Zn 的金属选择性，而 I119F 突变则将特异性限制在 Co 上。H74、D78、H268 和 D272（Zn 结合位点）和 Leu322（C 端亮氨酸拉链）突变部分或完全破坏了 TuMTP1 的转运功能。这些结果表明，TuMTP1 可能将多余的细胞质 Zn 和 Co 螯合到酵母液泡中以维持 Zn 和 Co 的体内平衡。NSEDD/VTVTT 和 I119F 突变对于限制 TuMTP1 的底物特异性至关重要，而 Zn 结合位点和 Leu322 对于其离子选择性和转运功能是必需的。这些结果可用于改变金属选择性，以用于生物强化或植物修复应用。
