MUTATIONS IN THE GCD7 SUBUNIT OF YEAST GUANINE-NUCLEOTIDE EXCHANGE FACTOR EIF-2B OVERCOME THE INHIBITORY EFFECTS OF PHOSPHORYLATED EIF-2 ONTRANSLATION INITIATION

Phosphorylation of the a subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting th e guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Sa ccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in additi on to reducing general protein synthesis. We isolated mutations in sev eral unlinked genes that suppress the growth-inhibitory effect of eIF- 2 alpha phosphorylation catalyzed by mutationally activated forms of G CN2. These suppressor mutations, affecting eIF-2 alpha and the essenti al subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the leve l of eIF-2 alpha phosphorylation in cells expressing the activated GCN 2(c) kinase. Four GCD7 suppressors were shown to reduce the derepressi on of GCN4 translation in cells containing wild-type GCN2 under starva tion conditions or in GCN2(c) strains. A fifth GCD7 allele, constructe d in vitro by combining two of the GCD7 suppressors mutations, complet ely impaired the derepression of GCN4 translation, a phenotype charact eristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammal ian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the trans lational machinery had been rendered completely insensitive to phospho rylated eIF-2. None of the GCD7 mutations had any detrimental effect o n cell growth under nonstarvation conditions, suggesting that recyclin g of eIF-2 occurs efficiently in the suppressor strains. We propose th at GCD7 and GCD2 play important roles in the regulatory interaction be tween eIF-2 and eIF-2B and that the suppressor mutations we isolated i n these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential cataly tic function of eIF-2B in translation initiation.