Yoder-Hill J, Pause A, Sonenberg N, Merrick W C
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 1993 Mar 15;268(8):5566-73.
The p46 subunit of eukaryotic initiation factor (eIF)-4F purified from rabbit reticulocyte lysate has previously been found to be composed of eIF-4AI and eIF-4AII in a 4:1 ratio, respectively, whereas the free form of rabbit eIF-4A is composed solely of eIF-4AI. Using sucrose gradient centrifugation and an m7GTP-Sepharose 4B assay, it was shown that eIF-4A exchanges with the p46 subunit of eIF-4F. Incubation of [14C]eIF-4A and eIF-4F resulted in the incorporation of [14C] eIF-4A into the eIF-4F complex. Conversely, the [14C] p46 subunit of [14C]eIF-4F was shown to dissociate from the [14C]eIF-4F complex in the presence of eIF-4A, presumably due to the incorporation of unlabeled eIF-4A. Similar experiments were conducted in which 14C-labeled initiation factors were incubated with rabbit reticulocyte lysate. When [14C]eIF-4A was incubated with lysate, [14C]eIF-4A became incorporated into the eIF-4F complex present in the lysate. Additionally, when [14C]eIF-4F was incubated with lysate, the [14C]p46 subunit of [14C]eIF-4F dissociated from the [14C]eIF-4F complex, most likely due to the exchange of unlabeled eIF-4A (present in the lysate) with the [14C]p46 subunit. The exchange of mouse eIF-4AI and eIF-4AII expressed in Escherichia coli was also investigated in the presence of eIF-4F and rabbit reticulocyte lysate. Both the sucrose gradient experiments and m7GTP-Sepharose 4B assays demonstrated that the [14C]p46 subunit of [14C]eIF-4F was displaced in the presence of eIF-4AI or eIF-4AII and that mouse [14C]eIF-4AI or [14C]eIF-4AII became incorporated into the eIF-4F complex in the same manner as rabbit reticulocyte eIF-4A.
先前已发现，从兔网织红细胞裂解物中纯化得到的真核起始因子（eIF）-4F的p46亚基分别由eIF-4AI和eIF-4AII以4:1的比例组成，而兔eIF-4A的游离形式仅由eIF-4AI组成。通过蔗糖梯度离心和m7GTP-琼脂糖4B分析表明，eIF-4A与eIF-4F的p46亚基发生了交换。[14C]eIF-4A与eIF-4F一起温育，导致[14C]eIF-4A掺入eIF-4F复合物中。相反，在eIF-4A存在的情况下，[14C]eIF-4F的[14C]p46亚基从[14C]eIF-4F复合物中解离，推测是由于未标记的eIF-4A掺入所致。进行了类似的实验，将14C标记的起始因子与兔网织红细胞裂解物一起温育。当[14C]eIF-4A与裂解物一起温育时，[14C]eIF-4A掺入到裂解物中存在的eIF-4F复合物中。此外，当[14C]eIF-4F与裂解物一起温育时，[14C]eIF-4F的[14C]p46亚基从[14C]eIF-4F复合物中解离，最可能的原因是未标记的eIF-4A（存在于裂解物中）与[14C]p46亚基发生了交换。在eIF-4F和兔网织红细胞裂解物存在的情况下，还研究了在大肠杆菌中表达的小鼠eIF-4AI和eIF-4AII的交换情况。蔗糖梯度实验和m7GTP-琼脂糖4B分析均表明，在eIF-4AI或eIF-4AII存在的情况下，[14C]eIF-4F的[14C]p46亚基被取代，并且小鼠[14C]eIF-4AI或[14C]eIF-4AII以与兔网织红细胞eIF-4A相同的方式掺入eIF-4F复合物中。
