The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo
The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo
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DOI：
10.1017/s1355838200991787
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发表时间：
2000-03-01
期刊：
影响因子：
4.5
通讯作者：
Johnson, AW
中科院分区：
文献类型：
--
作者：
Brown, JT;Bai, XX;Johnson, AW
酵母超级杀伤（SKI）基因最初是从突变中鉴定出来的，这些突变允许增加由M“杀伤”病毒（dsRNA病毒L-A的卫星）编码的杀伤毒素的产生。XRN 1（SKI 1）编码负责大部分细胞质RNA周转的细胞质5 '-核糖核酸外切酶，而SKI 2、SKI 3和SKI 8是mRNA的正常3'-降解和抑制poly（A）-RNA翻译所必需的。Ski 2 p是一种假定的RNA解旋酶，Ski 3 p是一种四肽重复（TPR）蛋白，Ski 8 p含有五个WD-40（β-转导素）重复。xrn 1突变与ski 2、ski 3或ski 8突变的组合是致命的，表明功能冗余。使用功能性表位标记的Ski 2，Ski 3和Ski 8蛋白，我们表明，Ski 2 p，Ski 3 p和Ski 8 p可以作为一个明显的异源三聚体复合物共免疫沉淀。对于表位标记的Ski 2 p，复合物中蛋白质的化学计量比为1：1：1。在Ski 8 p缺失的情况下，Ski 2 p不与Ski 3 p相关，在Ski 3 p缺失的情况下，Ski 2 p也不与Ski 8 p相关。然而，Ski 3 p/Ski 8 p相互作用不需要Ski 2 p。此外，SKI 6 -2或SKI 4 -1突变或SKI 7缺失不影响复合物的形成。由Ski 2 p、Ski 3 p和Ski 8 p组成的复合物的鉴定解释了先前显示SKI 2、SKI 3和SKI 8中突变之间的表型相似性的结果。Ski 3 p的间接免疫荧光和Ski 2 p和Ski 3 p的亚细胞分级表明Ski 2 p和Ski 3 p是细胞质的。这些数据支持Ski 2 p、Ski 3 p和Ski 8 p在细胞质中以3 '-mRNA降解途径起作用的观点。
The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.