His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity
His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity
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DOI：
10.1073/pnas.95.15.9025
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发表时间：
1998-07-21
影响因子：
11.1
通讯作者：
Bush, DR
中科院分区：
文献类型：
--
作者：
Lu, JMY;Bush, DR
质子-蔗糖同向转运体是许多高等植物同化物分配的重要组成部分，它介导韧皮部的装载。以前的生化研究表明，焦碳酸二乙酯敏感的组氨酸残基是在或附近的同向转运体的底物结合位点。在迄今为止克隆的质子-蔗糖共转运蛋白中，只有拟南芥AtSUC 1的65位组氨酸残基在物种间是保守的。为了测试His-65是否参与转运反应，我们使用了定点诱变和酵母中的功能表达来确定该残基在反应机制中的意义。His-65突变的共转运体表现出一系列的活性;例如，H65 C突变体导致转运能力的完全丧失，而H65 Q几乎与野生型一样活跃。令人惊讶的是，H65 K和H65 R同向转运体转运蔗糖的速率显著高于野生型同向转运体（V-max增加），这表明His-65可能与转运反应中的限速步骤有关。RNA凝胶印迹和蛋白质印迹分析表明，除了H65 C，运输活性的变化不是因为mRNA或共转运蛋白的稳态水平的改变。值得注意的是，那些替换His-65的共转运蛋白，保持运输能力不再敏感的失活焦碳酸二乙酯，这表明这是对乙酰氨基酚敏感的组氨酸残基，两者结合我们以前的结果，这些数据表明，His-65参与蔗糖结合，并增加运输率牵连该地区的蛋白质在运输反应。
The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased V-max) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue, Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.