Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome
Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome
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DOI：
10.1074/jbc.m808840200
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发表时间：
2009-04-17
影响因子：
4.8
通讯作者：
Inada, Toshifumi
中科院分区：
文献类型：
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作者：
Dimitrova, Lyudmila N.;Kuroha, Kazushige;Inada, Toshifumi
缺乏终止密码子的异常mRNA（非终止mRNA）的潜在有害作用可通过翻译停滞、蛋白酶体介导的蛋白质不稳定和快速mRNA降解来改善。由于通过翻译poly（A）mRNA尾合成多聚赖氨酸导致翻译停滞和蛋白质降解的蛋白酶体，我们研究了其他氨基酸序列的影响。在GFP和HIS 3报告基因之间插入12个连续的碱性氨基酸，而不是茎环结构，导致蛋白酶体降解截短的绿色荧光蛋白（GFP）产物。在not4 Delta突变体中检测到来自GFP-R12-FLAG-HIS3或GFP-K12-FLAG-HIS3 mRNA的翻译停滞产物，并且MG132处理不影响截短的停滞产物的水平。删除Ccr4-Not复合物的其他组分不会增加翻译停滞产物或报告基因mRNA的水平。Not4p环指结构域中的L35A取代破坏了其与Ubc4/Ubc5E2酶的相互作用及其作为泛素蛋白连接酶的活性，也消除了逮捕产物的降解。这些结果表明，Not4p，Ccr4-Not复合物的一个组成部分，可以作为翻译停滞产物的E3泛素蛋白连接酶。结果让我们提出，碱性氨基酸残基与核糖体带负电荷的出口隧道之间的相互作用导致翻译停滞，随后是Not4 p介导的遍在蛋白化和蛋白酶体的蛋白质降解。
The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stem-loop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4 Delta mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome.