N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1
N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1
复制标题
N 端半胱氨酸影响变构调节铵转运蛋白 LeAMT1 寡聚物的稳定性；1
DOI：
10.1093/jxb/erq379
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发表时间：
2011-02-01
影响因子：
6.9
通讯作者：
von Wiren, Nicolaus
中科院分区：
文献类型：
--
作者：
Graff, Lucile;Obrdlik, Petr;von Wiren, Nicolaus
铵转运蛋白（AMT）在生命的所有领域都是保守的，并介导铵或氨跨细胞膜的转运。AMTs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，稳定AMT蛋白复合物的结合力还没有得到很好的表征。高温或还原剂释放的单体和二聚体形式从三聚体复合物形成的AMT 1;1从拟南芥和番茄。然而，在paraqueous LeAMT 1;3中，未检测到三聚体复合物。LeAMT 1;3与其他AMTs的不同之处在于异常短的N-末端，这表明N-末端在寡聚体稳定性中的作用。截短LeAMT 1;1中的N-末端使三聚体不稳定，并导致在酵母中表达时功能丧失。LeAMT 1;1和LeAMT 1;3之间的N-末端交换表明，LeAMT 1;1的N-末端中的序列对于稳定亚基之间的相互作用是必要的和足够的。两个N-末端半胱氨酸残基是高度保守的AMT 1转运蛋白在植物中，但缺乏LeAMT 1;3。LeAMT 1;1的C3 S或C27 S变体表现出降低的复合物稳定性，这与底物类似物甲基铵的较低转运能力相一致。两个半胱氨酸取代LeAMT 1;1变体与野生型的相互作用较弱，如通过使用基于交配的分裂泛素测定的复合物稳定性的定量分析所确定的。这些数据表明，AMT 1亚基的结合亲和力是稳定的N-末端的半胱氨酸，并建议通过质外体N-末端半胱氨酸残基的二硫桥形成的作用。
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.