Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the alpha 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-alpha 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-2-GDP + GEF in equilibrium eIF-2-GEF + GDP) was measured by using mixtures of eIF-2(alpha P) X [eH]GDP and eIF-2 X [3H]GDP in different proportions at constant concentration of eIF-2 X GEF. If, for example, the ratio of eIF-2 X GEF to total (phosphorylated and unphosphorylated) eIF-2 X [3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(alpha P) X [3H]GDP in hte mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(alpha P). 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 X GDP. The trapping of GEF by eIF-s(alpha P) is strongly supported by the following observation. Incubation of eIF-2 X GEF with excess [3H]GDP leads to the formation of eIF-2 X [3H] GDP and free GEF and, if eIF-2(alpha 32P) X GDP is also present, all of the GEF is converted to eIF-2(alpha 32P) X GEF. This suggests that, whereas the equilibrium of the reaction eIF-2 X GEF + GDP in equilibrium eIF-2 X GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(alpha P) X GDP + GEF in equilibrium eIF-2(alpha P) X GEF + GDP is in favor of the association of GEF to eIF-2(alpha P).