van Nocker S, Walker J M, Vierstra R D
Department of Horticulture, University of Wisconsin-Madison, 53706, USA.
J Biol Chem. 1996 May 24;271(21):12150-8. doi: 10.1074/jbc.271.21.12150.
Covalent modification of proteins by attachment of multiubiquitin chains serves as an essential signal for selective protein degradation in eukaryotes. The specificity of ubiquitin-protein conjugation is controlled in part by a diverse group of ubiquitin-conjugating enzymes (E2s or UBCs). We have previously reported that the product of the wheat TaUBC7 gene recognizes ubiquitin as a substrate for ubiquitination in vitro, catalyzing the condensation of free ubiquitin into multiubiquitin chains linked via lysine 48 (van Nocker, S., and vierstra, R. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10297-10301). Based on this activity, this E2 may play a central role in the ubiquitin proteolytic pathway by assembling chains in vivo. Here, we describe the cloning and characterization of a three-member gene family from Arabidopsis thaliana (designated AtUBC7/13/14) encoding structural homologs of TaUBC7. Like TaUBC7, recombinant AtUBC7/13/14 proteins formed multiubiquitin chains in vitro. AtUBC7/13/14 mRNAs were found in all tissues examined, and unlike related UBCs from yeast, the levels of mRNA were not elevated by heat stress or cadmium exposure. Transgenic Arabidopsis were engineered to express increased levels of active AtUBC7, for the first time altering the level of an E2 in a higher eukaryote. Plants expressing high levels of AtUBC7 exhibited no phenotypic abnormalities and were not noticeably enriched in multiubiquitinated conjugates. These findings indicate that the in vivo synthesis of multiubiquitin chains is not rate-limited by the abundance of AtUC7 and/or involves other, yet undefined components.
多聚泛素链附着对蛋白质进行共价修饰是真核生物中选择性蛋白质降解的重要信号。泛素 - 蛋白质缀合的特异性部分由多种泛素缀合酶（E2s或UBCs）控制。我们之前报道过，小麦TaUBC7基因的产物在体外将泛素识别为泛素化的底物，催化游离泛素缩合形成通过赖氨酸48连接的多聚泛素链（van Nocker，S.，和vierstra，R.D.（1991年）美国国家科学院院刊88，10297 - 10301）。基于这种活性，这种E2可能通过在体内组装链在泛素蛋白水解途径中发挥核心作用。在这里，我们描述了来自拟南芥的一个由三个成员组成的基因家族（命名为AtUBC7/13/14）的克隆和特征，该家族编码TaUBC7的结构同源物。与TaUBC7一样，重组AtUBC7/13/14蛋白在体外形成多聚泛素链。在所有检测的组织中都发现了AtUBC7/13/14的mRNA，并且与酵母中相关的UBCs不同，mRNA水平不会因热胁迫或镉暴露而升高。首次通过基因工程改造拟南芥以使其表达更高水平的活性AtUBC7，从而改变高等真核生物中E2的水平。表达高水平AtUBC7的植物没有表现出表型异常，并且在多聚泛素化缀合物中也没有明显富集。这些发现表明，多聚泛素链的体内合成不受AtUC7丰度的限速，和/或涉及其他尚未明确的成分。
