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首页> 外文期刊>Journal of Experimental Botany >N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1
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N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1

机译：N端半胱氨酸影响变构调节的铵转运蛋白LeAMT1; 1的低聚物稳定性。

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AMMONIUM TRANSPORTER (AMT) proteins are conserved in all domains of life and mediate the transport of ammonium or ammonia across cell membranes. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized. High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato. However, in the paralogue LeAMT1;3, trimeric complexes were not detected. LeAMT1;3 differs from the other AMTs by an unusually short N-terminus, suggesting a role for the N-terminus in oligomer stability. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. Swapping of the N-terminus between LeAMT1;1 and LeAMT1;3 showed that sequences in the N-terminus of LeAMT1;1 are necessary and sufficient for stabilization of the interaction among the subunits. Two N-terminal cysteine residues are highly conserved among AMT1 transporters in plants but are lacking in LeAMT1;3. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.
机译：铵转运蛋白（AMT）蛋白在生命的所有域中都是保守的，并介导铵或氨跨细胞膜的转运。 AMT形成三聚体并利用亚基之间的分子间相互作用来调节活性。到目前为止，还没有很好地表征稳定AMT蛋白复合物的结合力。高温或还原剂从拟南芥和番茄的AMT1; 1形成的三聚体复合物中释放出单聚体和二聚体形式。但是，在旁观者LeAMT1; 3中，未检测到三聚体复合物。 LeAMT1; 3与其他AMT的不同之处在于N末端异常短，这表明N末端在低聚物稳定性中发挥了作用。当在酵母中表达时，LeAMT1; 1中N末端的截短使三聚体不稳定并导致功能丧失。 LeAMT1; 1和LeAMT1; 3之间的N末端交换显示，LeAMT1; 1的N末端的序列对于稳定亚基之间的相互作用是必要的和充分的。在植物的AMT1转运蛋白中，两个N端半胱氨酸残基高度保守，但LeAMT1; 3中缺少。 LeAMT1; 1的C3S或C27S变体显示出降低的复杂稳定性，这与底物类似物甲基铵的较低运输能力相吻合。两个半胱氨酸取代的LeAMT1; 1变体均显示出与野生型的较弱相互作用，这是通过使用基于交配的分裂泛素测定对复杂稳定性进行定量分析确定的。这些数据表明，AMT1亚基的结合亲和力在N端被半胱氨酸稳定，并暗示了通过质外性N端半胱氨酸残基形成二硫键的作用。

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