Pore Mutations in Ammonium Transporter AMT1 with Increased Electrogenic Ammonium Transport Activity
Pore Mutations in Ammonium Transporter AMT1 with Increased Electrogenic Ammonium Transport Activity
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DOI：
10.1074/jbc.m109.020842
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发表时间：
2009-09-11
影响因子：
4.8
通讯作者：
Frommer, Wolf B.
中科院分区：
文献类型：
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作者：
Loque, Dominique;Mora, Silvia I.;Frommer, Wolf B.
AMT/Mep铵转运蛋白介导细菌、真菌和植物的高亲和力铵/氨摄取。拟南芥AMT 1蛋白介导离子形式的铵的摄取。AMT转运活性通过与三聚体中相邻亚基相互作用的高度保守的胞质C末端变构控制。因此，C末端能够调节孔的电导率。为了深入了解潜在的机制，孔突变体抑制C-末端反式激活结构域的突变的抑制作用进行了表征。在跨膜螺旋I（TMH I）中携带突变Q57 H的AMT 1; 1显示增加的铵摄取，但降低的摄取甲基铵的能力。为了探索转运机制是否被改变，AMT 1; 1-Q57 H突变体在非洲爪蟾卵母细胞中表达并进行电生理学分析。AMT 1; 1-Q57 H的特征在于增加铵诱导的电流和减少甲基铵诱导的电流。与野生型形式相比，AMT 1; 1-Q57 H具有低100倍的铵亲和力（K-m）和高10倍的V-max。为了测试反式调节机制是否在古细菌同源物中是保守的，在酵母中表达了来自闪烁古生菌的Aftergobus-2。Afghanistan-2的转运功能也依赖于C末端的反式激活，并且对应于AMT 1; 1的Q57 H的孔残基中的突变抑制缺乏激活C末端的非功能性Afghanistan-2突变体。总之，我们的数据表明，细菌和植物的AMT使用一个保守的变构机制来控制铵通量，可能使用一个门控机制，限制通量，以防止铵毒性。
AMT/Mep ammonium transporters mediate high affinity ammonium/ammonia uptake in bacteria, fungi, and plants. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer. The C terminus is thus capable of modulating the conductivity of the pore. To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. AMT1; 1 carrying the mutation Q57H in transmembrane helix I (TMH I) showed increased ammonium uptake but reduced capacity to take up methylammonium. To explore whether the transport mechanism was altered, the AMT1; 1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1; 1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1; 1-Q57H possesses a 100x lower affinity for ammonium (K-m) and a 10-fold higher V-max as compared with the wild type form. To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1; 1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.