The ubiquitin-activating enzyme (El) gene family in Arabidopsis thaliana
The ubiquitin-activating enzyme (El) gene family in Arabidopsis thaliana
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DOI：
10.1046/j.1365-313x.1997.11020213.x
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发表时间：
1997-02-01
期刊：
影响因子：
7.2
通讯作者：
Vierstra, RD
中科院分区：
文献类型：
--
作者：
Hatfield, PM;Gosink, MM;Vierstra, RD
多个泛素的结合是26 S蛋白酶体降解多种细胞内真核蛋白的关键步骤。结合物通过三酶级联反应形成;初始步骤需要泛素活化酶（E1），其将泛素活化与ATP水解偶联。以前，我们发现许多高等植物含有多种E1蛋白，并描述了小麦中的几个E1基因。为了便于理解不同植物E1的作用，我们对拟南芥E1基因和蛋白家族进行了表征。拟南芥E1 s由两个基因（AtUBA 1和AtUBA 2）编码，这两个基因合成约123 kDa的蛋白质，相互之间具有81%的氨基酸序列同一性，与其他生物体的E1 s具有44-75%的序列同一性。与其他E1蛋白一样，AtUBA 1和2在推定的活性位点含有半胱氨酸残基，用于形成泛素巯基酯中间体。在大肠杆菌中表达的相应蛋白质的酶促分析表明，这两种蛋白质激活泛素在ATP依赖性反应和转移激活的泛素到各种拟南芥E2具有几乎相同的特异性。通过定量RT-PCR和组织化学的表达研究与转基因植物含有AtUBA启动子-β-葡萄糖醛酸酶编码区融合表明，AtUBA 1和2基因共表达在大多数，如果不是全部，拟南芥组织和细胞。总的来说，数据表明，El蛋白，并推测其余的泛素途径，存在于整个拟南芥。他们还表明，AtUBA 1和2基因没有差异表达，也没有编码具有显著不同酶特性的E1。
Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44-75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-beta-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that El proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis. They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.