The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro
The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro
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DOI：
10.1074/jbc.m312745200
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发表时间：
2004-03-05
影响因子：
4.8
通讯作者：
Hershey, JWB
中科院分区：
文献类型：
--
作者：
Fraser, CS;Lee, JY;Hershey, JWB
真核起始因子 3 (eIF3) 是一种 12 亚基蛋白质复合物，在起始甲硫氨酰-tRNA 和 mRNA 与 40 S 核糖体亚基结合形成 40 S 起始复合物中发挥核心作用。 eIF3 发挥这些功能的分子机制尚不清楚。为了更多地了解 eIF3 的结构和功能，我们使用杆状病毒感染的 Sf9 细胞表达并纯化了单个人 eIF3 亚基或 eIF3 亚基复合物。结果表明，与酿酒酵母中具有同源物的人 eIF3 亚基形成的子复合物反映了酵母 eIF3 核心复合物中所见的亚基相互作用。此外，我们还使用体外 40 S 核糖体亚基结合测定来研究 eIF3 子复合物与 40 S 核糖体亚基有效结合的亚基要求。 eIF3j 单独与 40 S 核糖体亚基结合，并且它的存在是 eIF3bgi 亚复合物稳定的 40 S 结合所必需的。此外，缺乏 eIF3j 的纯化 eIF3 与 40 S 核糖体亚基的结合较弱，但在添加 eIF3j 时结合紧密。 caspase-3 从 eIF3j 上切割 16 个残基的 C 端肽，显着降低了 eIF3j 对 40 S 核糖体亚基的亲和力，并且切割形式大大降低了纯化 eIF3-40S 复合物的稳定性。这些结果表明 eIF3j，尤其是其 C 末端，在 eIF3 募集到 40 S 核糖体亚基中发挥着重要作用。
Eukaryotic initiation factor 3 (eIF3) is a 12-subunit protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 40 S initiation complex. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. To learn more about the structure and function of eIF3 we have expressed and purified individual human eIF3 subunits or complexes of eIF3 subunits using baculovirus-infected Sf9 cells. The results indicate that the subunits of human eIF3 that have homologs in Saccharomyces cerevisiae form subcomplexes that reflect the subunit interactions seen in the yeast eIF3 core complex. In addition, we have used an in vitro 40 S ribosomal subunit binding assay to investigate subunit requirements for efficient association of the eIF3 subcomplexes to the 40 S ribosomal subunit. eIF3j alone binds to the 40 S ribosomal subunit, and its presence is required for stable 40 S binding of an eIF3bgi subcomplex. Furthermore, purified eIF3 lacking eIF3j binds 40 S ribosomal subunits weakly, but binds tightly when eIF3j is added. Cleavage of a 16-residue C-terminal peptide from eIF3j by caspase-3 significantly reduces the affinity of eIF3j for the 40 S ribosomal subunit, and the cleaved form provides substantially less stabilization of purified eIF3-40S complexes. These results indicate that eIF3j, and especially its C terminus, play an important role in the recruitment of eIF3 to the 40 S ribosomal subunit.