Phan L, Zhang X, Asano K, Anderson J, Vornlocher H P, Greenberg J R, Qin J, Hinnebusch A G
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1998 Aug;18(8):4935-46. doi: 10.1128/MCB.18.8.4935.
Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces 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 approximately 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-tRNAiMet 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.
人类真核生物翻译起始因子3（eIF3）的9个亚基中，只有5个在酿酒酵母基因组中编码有可识别的同源物，且其中只有2个（Prt1p和Tif34p）先前被鉴定为酵母eIF3的亚基。我们通过Ni2+亲和层析和凝胶过滤层析纯化了一种带有多聚组氨酸标签的Prt1p形式（His-Prt1p），并获得了一个由6种多肽组成的约600 kDa的复合物，其共纯化完全依赖于His-Prt1p上的多聚组氨酸标签。通过质谱鉴定了与His-Prt1p相关的所有5种多肽，发现其中4种是酿酒酵母中编码的人类eIF3亚基的其他假定同源物：YBR079c/Tif32p、Nip1p、Tif34p和YDR429c/Tif35p。与Prt1p相关的第5种蛋白质是eIF5，一种先前未知与eIF3相互作用的起始因子。纯化的复合物可以挽救Met-tRNAiMet与prt1突变体的缺陷提取物或Nip1p已被耗尽的提取物中的40S核糖体的结合，表明它具有eIF3已知的生化活性。这些发现表明，Tif32p、Nip1p、Prt1p、Tif34p和Tif35p组成了一个在酵母和哺乳动物之间保守的eIF3核心复合物，该复合物与eIF5稳定相互作用。在酵母双杂交和体外蛋白质结合试验中，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可能通过与eIF3的Nip1p亚基的物理相互作用被招募到40S核糖体上。
