Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress
Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress
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DOI：
10.1023/a:1006449715236
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发表时间：
2000-04-01
影响因子：
5.1
通讯作者：
Dietz, KJ
中科院分区：
文献类型：
--
作者：
Bolte, S;Schiene, K;Dietz, KJ
从兼性CAM植物Mesembryonium stac um中克隆了编码小GTP结合蛋白Ypt/Rab家族成员的cDNA。McAb 5 b包括201个氨基酸的开放阅读框。推导的氨基酸序列与从百脉根中分离的LjRAB 5 b的氨基酸序列相似性为91%。McRAB 5 b的氨基酸序列提供了有趣的特征，表明McRAB 5 b及其来自百脉根的同源物代表了Ypt/Rab蛋白的新亚类。像McRAB 5 b和LjRAB 5 b这样的蛋白质只在植物中发现，而在酵母或脊椎动物中没有发现，这表明它们具有植物特异性功能。通过北方印迹杂交和RT-PCR检测，盐胁迫刺激了Mcrab 5 b的表达。在大肠杆菌中异源表达后，产生了针对重组McRAB 5 b蛋白的抗体。Western blot分析显示McRAB 5 b与细胞膜结合。它在体外和体内以单体和二聚体形式存在。在体外，只有单体蛋白表现出放射性标记的GTP的结合能力，而二聚体是无法做到这一点，表明该活动可能是由单体/二聚体转换调节。
A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b were only found in plants and not in yeast or vertebrates suggests that they have plant-specific functions. The expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress. After heterologous expression in Escherichia coli an antibody was raised against recombinant McRAB5b protein. Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP, while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition.