An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat
An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat
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DOI：
10.1111/j.1365-313x.2010.04309.x
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发表时间：
2010-10-01
期刊：
影响因子：
7.2
通讯作者：
Yamamoto, Yoko
中科院分区：
文献类型：
--
作者：
Furuichi, Takuya;Sasaki, Takayuki;Yamamoto, Yoko
P>抗Al ~（3+）小麦品种（Triticum aestivum L.）通过铝激活的阴离子转运蛋白小麦铝激活的苹果酸转运蛋白1（TaALMT 1）释放苹果酸。TaALMT 1在非洲爪蟾卵母细胞和烟草悬浮细胞中的表达增强了基础运输活性（在没有外部Al 3+的情况下存在的内向和外向电流），并产生与在小麦细胞中观察到的相同的Al 3+激活电流（反映Al 3+依赖的运输功能）。我们研究了氨基酸残基参与这种铝3+依赖的运输活动，通过产生一系列的突变的TaALMT 1蛋白。我们针对TaALMT 1的亲水性C-末端结构域上的酸性残基，并通过定点突变将其改变为不带电荷的残基。这些突变蛋白在非洲爪蟾卵母细胞中表达，并在添加Al 3+之前和之后测量它们的运输活性。三个突变（E274 Q，D275 N和E284 Q）取消铝激活的运输活动，而不影响基础运输活动。截断的亲水性C-末端结构域取消了基础和Al 3+激活的运输活动。通过将TaALMT 1的N-末端区域与AtALMT 1的C-末端区域（来自拟南芥的同源物）融合来恢复Al 3+依赖的转运活性。这些发现表明，细胞外C-末端结构域是基础和Al 3+依赖性TaALMT 1活性所必需的。此外，我们确定了三个酸性氨基酸在这个域中，是专门需要由外部Al 3+的运输功能的激活。
P>Al3+-resistant cultivars of wheat (Triticum aestivum L.) release malate through the Al3+-activated anion transport protein Triticum aestivum aluminum-activated malate transporter 1 (TaALMT1). Expression of TaALMT1 in Xenopus oocytes and tobacco suspension cells enhances the basal transport activity (inward and outward currents present in the absence of external Al3+), and generates the same Al3+-activated currents (reflecting the Al3+-dependent transport function) as observed in wheat cells. We investigated the amino acid residues involved in this Al3+-dependent transport activity by generating a series of mutations to the TaALMT1 protein. We targeted the acidic residues on the hydrophilic C-terminal domain of TaALMT1 and changed them to uncharged residues by site-directed mutagenesis. These mutant proteins were expressed in Xenopus oocytes and their transport activity was measured before and after Al3+ addition. Three mutations (E274Q, D275N and E284Q) abolished the Al3+-activated transport activity without affecting the basal transport activity. Truncation of the hydrophilic C-terminal domain abolished both basal and Al3+-activated transport activities. Al3+-dependent transport activity was recovered by fusing the N-terminal region of TaALMT1 with the C-terminal region of AtALMT1, a homolog from Arabidopsis. These findings demonstrate that the extracellular C-terminal domain is required for both basal and Al3+-dependent TaALMT1 activity. Furthermore, we identified three acidic amino acids within this domain that are specifically required for the activation of transport function by external Al3+.