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首页> 外文期刊>Molecular and Cellular Biology >Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5
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Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

机译：鉴定酵母和哺乳动物中保守的与eIF5相互作用的翻译起始因子3（eIF3）核心复合物

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Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in theSaccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAi Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.
机译：人类真核翻译起始因子3（eIF3）的9个亚基中只有5个具有可识别的同源基因，这些基因在啤酒酵母（Saccharomyces cerevisiae）基因组中编码，并且以前只有两个（Prt1p和Tif34p）被鉴定为酵母的亚基。 eIF3。我们通过Ni 2 + 亲和力和凝胶过滤色谱法纯化了带有Phist1p（His-Prt1p）的多组氨酸标签形式，并获得了约600 kDa的复合物，该复合物由六个多肽组成，它们的共纯化完全依赖于聚组氨酸标签在His-Prt1p上。通过质谱鉴定了与His-Prt1p相关的所有五种多肽，发现其中四种是 S编码的人eIF3亚基的其他推定同源物。酿酒酵母：YBR079c / Tif32p，Nip1p，Tif34p和YDR429c / Tif35p。第五个与Prt1p相关的蛋白是eIF5，这是以前未知的与eIF3相互作用的起始因子。纯化的复合物可以挽救 prt1 突变体缺陷提取物中或Nip1p耗尽的提取物中Met-tRNA i Met 与40S核糖体的结合，表明它具有已知的eIF3生化活性。这些发现表明，Tif32p，Nip1p，Prt1p，Tif34p和Tif35p包含一个eIF3核心复合物，该复合物在酵母和哺乳动物之间保守，并与eIF5稳定相互作用。在酵母双杂交和体外蛋白质结合测定中，Nip1p与eIF5结合。有趣的是，Sui1p也与Nip1p相互作用，并且eIF5和Sui1p都与AUG起始密码子的准确识别有关。因此，eIF5和Sui1p可通过与eIF3的Nip1p亚基的物理相互作用而募集到40S核糖体中。

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