FIG. 2.

eIF2α residues 1 to 180 are necessary and sufficient for efficient phosphorylation of Ser51 both in vivo and in vitro. (A) The C-terminal half of eIF2α is not required for Ser51 phosphorylation in vivo. GST-eIF2α fusion proteins with the indicated C-terminal residue were expressed under the control of a galactose-inducible promoter in the yeast strain H2653, in which the chromosomal SUI2 gene encoding eIF2α contains the nonphosphorylatable mutation S51A. WCEs were prepared from cells treated with 3-AT to activate GCN2, and proteins were separated by SDS-PAGE and analyzed by Western blotting using antibodies specific for the Ser51-phosphorylated form of eIF2α (left) (Quality Controlled Biochemicals, Inc.), as described previously (43). The membrane was stripped and probed with anti-GST antisera to analyze the expression of the GST-eIF2α fusion proteins (right). (B) Fine mapping of eIF2α residues required for Ser51 phosphorylation in vivo. The indicated GST-eIF2α fusion proteins were expressed under the control of a galactose-inducible promoter in SUI2-S51A yeast strains either containing or lacking GCN2 as indicated. WCEs, in 1× and 10× amounts, were separated by SDS-PAGE and analyzed by Western blotting for Ser51 phosphorylation and total GST-eIF2α expression as described for panel A. (C) eIF2α residues 1 to 180 are required for efficient phosphorylation of Ser51 in vitro. The indicated GST-eIF2α fusion proteins were purified from bacteria and incubated with recombinant human PKR purified from yeast and [γ-³³P]ATP for the indicated times. Reaction mixtures were resolved by SDS-PAGE, proteins were detected by Coomassie staining (lower panels), and eIF2α phosphorylation was analyzed by autoradiography (upper panels). (D) PKR phosphorylation of eIF2α in vitro is dependent on Ser51. GST-eIF2α(1-180) and GST-eIF2α-S51A(1-180) fusion proteins were prepared and utilized for in vitro kinase assays with human PKR as described for panel C.