eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange
eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange
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DOI：
10.1101/gad.12.4.514
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发表时间：
1998-02-15
影响因子：
10.5
通讯作者：
Hinnebusch, AG
中科院分区：
文献类型：
--
作者：
Pavitt, GD;Ramaiah, KVA;Hinnebusch, AG
eIF 2B是真核生物中蛋白质合成起始所必需的异五聚鸟嘌呤-核苷酸交换因子。其活性在饥饿或应激反应中受到其底物（翻译起始因子eIF 2）α亚基磷酸化的抑制，导致翻译速率和细胞生长速率降低。我们已经使用了体外核苷酸交换试验，以显示野生型酵母eIF 2B被磷酸化eIF 2 [eIF 2（α P）]抑制，并表征eIF 2B调控突变，使翻译起始对体内eIF 2磷酸化不敏感。与野生型eIF 2B不同，eIF 2B与突变的GCN 3或GCD 7亚基的复合物使用eIF 2（α P）作为底物有效地催化GDP交换。使用亲和力结合试验，我们表明，eIF 2B亚复合物的GCN 3，GCD 7，和GCD 2亚基结合eIF 2，并具有较高的亲和力eIF 2（α P），但它缺乏核苷酸交换活性。相比之下，GCD 1和GCD 6亚基形成eIF 2B亚复合物，其等同地结合eIF 2和eIF 2（α P）。值得注意的是，该第二亚复合物具有比野生型eIF 2B更高的核苷酸交换活性，其不被eIF 2（α P）抑制。监管和催化eIF 2B亚复合物的鉴定导致我们提出，eIF 2（α P）的监管亚复合物的结合，防止生产性的相互作用与催化亚复合物，从而抑制核苷酸交换。
eIF2B is a heteropentameric guanine-nucleotide exchange factor essential for protein synthesis initiation in eukaryotes. Its activity is inhibited in response to starvation or stress by phosphorylation of the alpha subunit of its substrate, translation initiation factor eIF2, resulting in reduced rates of translation and cell growth. We have used an in vitro nucleotide-exchange assay to show that wild-type yeast eIF2B is inhibited by phosphorylated eIF2 [eIF2(alpha P)] and to characterize eIF2B regulatory mutations that render translation initiation insensitive to eIF2 phosphorylation in vivo. Unlike wild-type eIF2B, eIF2B complexes with mutated GCN3 or GCD7 subunits efficiently catalyzed GDP exchange using eIF2(alpha P) as a substrate. Using an affinity-binding assay, we show that an eIF2B subcomplex of the GCN3, GCD7, and GCD2 subunits binds to eIF2 and has a higher affinity for eIF2(alpha P), but it lacks nucleotide-exchange activity. In contrast, the GCD1 and GCD6 subunits form an eIF2B subcomplex that binds equally to eIF2 and eIF2(alpha P). Remarkably, this second subcomplex has higher nucleotide-exchange activity than wild-type eIF2B that is not inhibited by eIF2(alpha P). The identification of regulatory and catalytic eIF2B subcomplexes leads us to propose that binding of eIF2(alpha P) to the regulatory subcomplex prevents a productive interaction with the catalytic subcomplex, thereby inhibiting nucleotide exchange.