Mechanism of Translational Control by Partial Phosphorylation of the Α Subunit of Eukaryotic Initiation Factor 2

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Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the EIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-α phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF + GEF ⇆ eIF-2·GEF + GDP) was measured by using mixtures of eIF-2(αP)·[3H]GDP and eIF-2·[3H]GDP in different proportions at constant concentration of eIF-2·GEF. If, for example, the ratio of eIF-2·GEF to total (phosphorylated and unphosphorylated) eIF-2·[3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(αP)·[3H]GDP in the mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(αP). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2·GDP. The trapping of GEF by eIF-2(αP) is strongly supported by the following observation. Incubation of eIF-2·GEF with excess [3H]GDP leads to the formation of eIF-2·[3H]GDP and free GEF and, if eIF-2(α32P)·GDP is also present, all of the GEF is converted to eIF-2(α32P)·GEF. This suggests that, whereas the equilibrium of the reaction eIF-2·GEF + GDP ⇆ eIF-2·GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(αP)·GDP + GEF ⇆ eIF-2(αP)·GEF + GDP is in favor of the association of GEF to eIF-2(αP).



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