Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate
Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate
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DOI：
10.1093/oxfordjournals.jbchem.a022435
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发表时间：
1999-07-01
影响因子：
2.7
通讯作者：
Kato, S
中科院分区：
文献类型：
--
作者：
Iwamuro, S;Saeki, M;Kato, S
在对人全长cDNA文库进行大规模体外翻译分析时，我们发现许多产生体外翻译产物的克隆在荧光图上显示梯状条带，其等间距约为9 kDa，位置大于开放阅读框预期的分子量。我们分析了一个显示典型梯状带模式的克隆。该克隆编码含有推定的跨膜结构域的188个氨基酸的多肽。一个绿色荧光蛋白标记的多肽在COS 7细胞中表达的定位在内质网和高尔基体。在兔网织红细胞裂解物系统中观察到梯形带，而在麦胚提取物系统中未观察到。加入谷胱甘肽S-转移酶融合的泛素到裂解液中引起的梯形带向上移位。微粒体膜的添加阻止了梯带的形成。时程实验表明，在体外翻译产物的微粒体膜的存在下增加，但在他们的情况下逐渐降解。这些结果表明，梯状结构的形成是由于错误折叠的多肽未能移位到其正确位置而引起的泛素化，并且错误折叠的膜蛋白在兔网织红细胞裂解物系统中起排斥作用。
During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.