Grace M, Bagchi M, Ahmad F, Yeager T, Olson C, Chakravarty I, Nasrin N, Banerjee A, Gupta N K
Proc Natl Acad Sci U S A. 1984 Sep;81(17):5379-83. doi: 10.1073/pnas.81.17.5379.
A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.
真核起始因子2（eIF-2）辅助蛋白因子Co-eIF-2促进GDP从eIF-2·GDP上解离，并在Mg2+存在的情况下促进三元复合物（Met-tRNAf·eIF-2·GTP）的形成。血红素调节蛋白合成抑制剂HRI使eIF-2的α亚基磷酸化，从而抑制三元复合物的形成，因为Co-eIF-2不能从eIF-2α（P）·GDP上置换GDP。RF是一种高分子量细胞上清因子，可逆转血红素缺乏的网织红细胞裂解物中的蛋白质合成抑制，也可逆转HRI对三元复合物形成 的抑制。RF具有Co-eIF-2活性。此外，活性RF制剂含有过量的游离且未磷酸化形式的eIF-2α亚基，该eIF-2α亚基不会被HRI和ATP磷酸化。在本文中，我们报道：（i）活性RF制剂含有过量的eIF-2α亚基，并且该α亚基在GDP存在的情况下可被HRI和ATP磷酸化；（ii）RF通过eIF-2·[3H]GDP促进三元复合物的形成，并伴随GDP的置换；（iii）在HRI和ATP存在的情况下，RF通过eIF-2·[3H]GDP促进三元复合物的形成，但不伴随GDP的置换；（iv）在HRI和ATP存在的情况下，使用RF形成的三元复合物在Met-tRNAf·40S起始复合物形成中具有活性；（v）在HRI和ATP存在的情况下，三元复合物和Met-tRNAf·40S复合物的形成在RF与GDP预先温育后被完全抑制；（vi）对活性RF组分进一步分级分离后，可获得一种含有对HRI敏感的Co-eIF-2活性的制剂。然而，该制剂不能有效地逆转血红素缺乏的网织红细胞裂解物中的蛋白质合成抑制，并且不含有过量的eIF-2α亚基。基于这些观察结果，我们提出：（a）RF为eIF-2α（P）·GDP提供未磷酸化的α亚基并恢复eIF-2活性。随着RF制剂中的α亚基在GDP存在的情况下被HRI和ATP磷酸化，这种RF活性受到抑制；（b）RF具有Co-eIF-2活性，其具有双重功能：（i）刺激eIF-2形成三元复合物；（ii）在三元复合物形成过程中从eIF-2·GDP上置换GDP。在HRI和ATP存在的情况下，Co-eIF-2不能从eIF-2α（P）·GDP上置换GDP。
