In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing
In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing
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DOI：
10.7554/elife.27949
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发表时间：
2017-07-18
期刊：
影响因子：
7.7
通讯作者：
Weinberg, David E.
中科院分区：
文献类型：
--
作者：
Osuna, Beatriz A.;Howard, Conor J.;Weinberg, David E.
由于mRNA模板或翻译机制的缺陷，核糖体在翻译过程中可能会停滞，导致产生不完整的蛋白质。核糖体相关质量控制复合物（RQC）接合停滞的核糖体并靶向新生多肽用于蛋白酶体降解。然而，每个RQC组件如何促进这一过程仍不清楚。在这里，我们证明了关键的RQC活动Ltn1p依赖的泛素化和Rqc2p介导的羧基末端丙氨酸和苏氨酸（CAT）尾延伸可以在体外重演与酵母无细胞系统。使用这种方法，我们确定CAT加尾是机械上不同于典型的翻译，Ltn1p介导的泛素化依赖于表征不佳的RQC组件Rqc1p，CAT加尾的过程使新生多肽具有强大的泛素化。这些发现建立了一个新的系统来研究RQC，并提供了一个框架，了解RQC因子如何协调其活动，以促进不完全合成的蛋白质的清除。
Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.