Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.
Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.
复制标题
DOI：
10.1186/s12870-015-0660-9
复制
发表时间：
2015-11-10
影响因子：
5.3
通讯作者：
Sawasaki T
中科院分区：
文献类型：
--
作者：
Ramadan A;Nemoto K;Seki M;Shinozaki K;Takeda H;Takahashi H;Sawasaki T
蛋白质泛素化是真核生物中普遍存在的机制。在拟南芥中，泛素修饰主要由两种泛素激活酶（E1）、37种泛素结合酶（E2）和超过1300种预测的泛素连接酶（E3）介导，其中约470种是RING型E3。 RING E3 的大部分基因产物尚未在体外进行表征，这可能是因为大规模 cDNA 克隆以及蛋白质表达、纯化和表征涉及繁重的工作。此外，一些 E2 可能是某些 E3 连接酶活性所必需的，但大肠杆菌或培养的昆虫细胞不表达这些 E2，因此仍未被表征。利用RIKEN拟南芥全长cDNA文库（RAFL），通过“分割引物”PCR方法和小麦胚芽无细胞系统，我们建立了拟南芥E2和RING E3酶的蛋白质文库。我们表达了 35 个拟南芥 E2，包括 6 种以前未表达过的酶，以及 204 个 RING 蛋白，其中大部分尚未进行功能表征。使用二硫苏糖醇 (DTT) 进行的硫酯测定显示，所有表达的 E2 均形成 DTT 敏感的泛素硫酯。在RING蛋白的表达测定中，31种蛋白显示出高分子涂片，这可能是其功能活性的结果。使用 AtUBC10 和/或一组不同的 E2 评估了另外 27 个 RING 蛋白的活性。所有测试的 27 个 RING E3 都显示出泛素连接酶活性，其中包括 17 个 RING E3。他们的活动首次被报道。我们研究中使用的小麦胚芽无细胞系统是一种真核表达系统，更类似于植物蛋白的内源表达，非常适合以功能形式表达拟南芥E2s和RING E3s。此外，此处描述的蛋白质库可用于进一步了解 E2-E3 特异性并作为蛋白质-蛋白质相互作用筛选的平台。本文的在线版本 (doi:10.1186/s12870-015-0660-9) 包含补充材料，可供授权用户使用。
Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3’s gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, connot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the ‘split-primer’ PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening. The online version of this article (doi:10.1186/s12870-015-0660-9) contains supplementary material, which is available to authorized users.