Datta B, Chakrabarti D, Roy A L, Gupta N K
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Proc Natl Acad Sci U S A. 1988 May;85(10):3324-8. doi: 10.1073/pnas.85.10.3324.
During heme deficiency in reticulocyte lysates, the heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2) and thus inhibits protein synthesis. Two factors, eIF-2 and a reticulocyte-lysate supernatant factor that we term RF, reverse this inhibition. We now report the following. (i) An active eIF-2 preparation contained, in addition to the three subunits (alpha, beta, and gamma), a 67-kDa polypeptide. Pretreatment of eIF-2 with polyclonal antibodies against either isolated alpha subunit or 67-kDa polypeptide almost completely inhibited the reversal activity. Upon further fractionation, three-subunit eIF-2 and the 67-kDa polypeptide were resolved. Neither the three-subunit eIF-2 nor the 67-kDa polypeptide alone was active in protein synthesis inhibition reversal. The activity was, however, restored by combining both the three-subunit eIF-2 and the 67-kDa polypeptide. (ii) Active RF preparations contained eIF-2 alpha (unphosphorylated) and beta subunits and the 67-kDa polypeptide. As with eIF-2, prior treatment of the RF preparation with antibodies to either the alpha subunit or the 67-kDa polypeptide almost completely inhibited the reversal activity. The RF preparation devoid of eIF-2 gamma subunit did not form ternary complex (Met-tRNA(fMet).eIF-2.GTP). The eIF-2 gamma subunit in the free form was isolated, and addition of this isolated gamma subunit to RF promoted significant ternary-complex formation. (iii) Purified HRI efficiently phosphorylated the alpha subunit in the three subunit eIF-2. However, the extent of such phosphorylation was significantly reduced when eIF-2 containing the 67-kDa polypeptide was used. The 67-kDa polypeptide apparently protected eIF-2 alpha subunit from HRI-catalyzed phosphorylation but did not inhibit HRI activity. Based on these results, we suggest that the protein synthesis inhibition reversal activity in both eIF-2 and RF is due to the same components--namely, eIF-2 alpha subunit and the 67-kDa polypeptide. The 67-kDa polypeptide protects eIF-2 alpha subunit from HRI-catalyzed phosphorylation and may also be a necessary component of the functioning eIF-2 molecule.
在网织红细胞裂解物中血红素缺乏时，血红素调节的蛋白质合成抑制剂HRI会使真核起始因子2（eIF-2）的α亚基磷酸化，从而抑制蛋白质合成。有两个因子，即eIF-2和一种我们称为RF的网织红细胞裂解物上清因子，可逆转这种抑制作用。我们现在报告以下内容。（i）一种活性eIF-2制剂除了含有三个亚基（α、β和γ）外，还含有一种67 kDa的多肽。用针对分离的α亚基或67 kDa多肽的多克隆抗体对eIF-2进行预处理，几乎完全抑制了其逆转活性。进一步分级分离后，分离出了三个亚基的eIF-2和67 kDa的多肽。单独的三个亚基的eIF-2或67 kDa多肽在逆转蛋白质合成抑制方面均无活性。然而，将三个亚基的eIF-2和67 kDa多肽结合起来可恢复其活性。（ii）活性RF制剂含有eIF-2α（未磷酸化）、β亚基和67 kDa的多肽。与eIF-2一样，用针对α亚基或67 kDa多肽的抗体对RF制剂进行预处理，几乎完全抑制了其逆转活性。不含eIF-2γ亚基的RF制剂不能形成三元复合物（Met-tRNA(fMet).eIF-2.GTP）。分离出游离形式的eIF-2γ亚基，并将这种分离出的γ亚基添加到RF中可促进显著的三元复合物形成。（iii）纯化的HRI能有效地使三个亚基的eIF-2中的α亚基磷酸化。然而，当使用含有67 kDa多肽的eIF-2时，这种磷酸化的程度会显著降低。67 kDa的多肽显然保护eIF-2α亚基免受HRI催化的磷酸化，但不抑制HRI的活性。基于这些结果，我们认为eIF-2和RF中的蛋白质合成抑制逆转活性归因于相同的成分，即eIF-2α亚基和67 kDa的多肽。67 kDa的多肽保护eIF-2α亚基免受HRI催化的磷酸化，并且可能也是有功能的eIF-2分子的必要成分。
