Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit
Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit
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DOI：
10.1074/jbc.m605418200
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发表时间：
2006-08-11
影响因子：
4.8
通讯作者：
Rhoads, Robert E.
中科院分区：
文献类型：
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作者：
LeFebvre, Aaron K.;Korneeva, Nadejda L.;Rhoads, Robert E.
eIF3是哺乳动物中最大的翻译起始因子（类似于800 kDa），由13个不同的亚基组成，命名为eIF3a-m。哺乳动物eIF3在48 S复合物组装中的作用通过与eIF4G的高亲和力结合而发生。eIF4G与eIF4E、eIF4A、eIF3、多聚腺苷酸结合蛋白和Mnk 1/2的相互作用已被定位到eIF4G上的离散结构域，相反，除了一个配体之外，所有这些配体上的eIF4G结合位点都已确定。唯一尚未确定的eIF4G配体是eIF3。在这项研究中，我们试图确定哺乳动物eIF3亚基（S）直接相互作用（S）与eIF4G。已建立的检测蛋白质-蛋白质相互作用的程序给出了模糊的结果。然而，部分蛋白水解的HeLa eIF3与人eIF4G-1的eIF3结合结构域的结合，随后用新的肽匹配算法对质谱数据进行高通量分析，鉴定出单一亚基eIF3e（p48/Int-6）。此外，重组FLAG-eIF3e在体外与HeLa eIF3特异性竞争结合eIF4G。将FLAG-eIF3e添加到无细胞翻译系统中（i）抑制蛋白质合成，（ii）导致mRNA从重多聚核糖体向轻多聚核糖体转移，（iii）比依赖于HCV或CSFV内部核糖体进入位点（不需要eIF4G）的翻译更严重地抑制帽依赖性翻译，以及（iv）导致eIF4G和eIF2 α从类似于40 S沉降的复合物中急剧损失。这些数据表明，一个特定的，直接的，和功能的相互作用的eIF3e与eIF4G在帽依赖性翻译起始的过程中，虽然他们不排除其他eIF3亚基的参与。
eIF3 in mammals is the largest translation initiation factor (similar to 800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2 alpha from complexes sedimenting at similar to 40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.