The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase
The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase
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DOI：
10.1073/pnas.94.8.3656
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发表时间：
1997-04-15
影响因子：
11.1
通讯作者：
Beaudenon, SL
中科院分区：
文献类型：
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作者：
Huibregtse, JM;Yang, JC;Beaudenon, SL
E3泛素-蛋白连接酶在控制泛素蛋白水解系统的底物特异性中起重要作用。采用生物化学方法鉴定了酿酒酵母（Saccharomycescerevisiae）E3的必需hect（与E6-AP羧基端同源）Rsp 5的底物。我们发现Rsp 5在体外结合并泛素化RNA聚合酶II的最大亚基（Rpb 1）。在酵母细胞提取物中也检测到Rsp 5和Rpbl之间稳定的复合物形成，并且RSP 5在体内表达的抑制导致Rpb 1的稳态水平升高。Rsp 5的氨基末端结构域介导与Rpb 1的结合，而Rpb 1的羧基末端结构域含有聚合酶II的七肽重复特征，是与Rsp 5结合所必需的。Rpb 1羧基末端结构域与另一种蛋白质的融合也会导致该蛋白质被Rsp 5泛素化。这些研究结果表明，Rsp 5的目标至少是一个子集的细胞Rpb 1分子的泛素依赖性降解，因此可能在调节聚合酶II的活动中发挥作用。此外，结果支持hect E3功能的模型，其中氨基末端结构域介导底物结合，而羧基末端hect结构域催化结合底物的泛素化。
The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpbl was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The aminoterminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.