MAMMALIAN EUKARYOTIC INITIATION FACTOR-2-ALPHA KINASES FUNCTIONALLY SUBSTITUTE FOR GCN2 PROTEIN-KINASE IN THE GCN4 TRANSLATIONAL CONTROL MECHANISM OF YEAST

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) in Saccharomyces cerevisiae by the GCN2 protein kinase stimulates the translation of GCN4 mRNA. The protein kinases heme-regulated inhibitor of translation (HRI) and double-stranded RNA-dependent eIF-2alpha protein kinase (dsRNA-PK) inhibit initiation of translation in mammalian cells by phosphorylating Ser-51 of eIF-2alpha. We show that HRI and dsRNA-PK phosphorylate yeast eIF-2alpha in vitro and in vivo and functionally substitute for GCN2 protein to stimulate GCN4 translation in yeast. In addition, high-level expression of either mammalian kinase in yeast decreases the growth rate, a finding analogous to the inhibition of total protein synthesis by these kinases in mammalian cells. Phosphorylation of eIF-2alpha inhibits initiation in mammalian cells by sequestering eIF-2B, the factor required for exchange of GTP for GDP on eIF-2. Mutations in the GCN3 gene, encoding a subunit of the yeast eIF-2B complex, eliminate the effects of HRI and dsRNA-PK on global and GCN4-specific translation in yeast. These results provide further in vivo evidence that phosphorylation of eIF-2alpha inhibits translation by impairing eIF-2B function and identify GCN3 as a regulatory subunit of eIF-2B. These results also suggest that GCN4 translational control will be a good model system to study how mammalian eIF-2alpha kinases are modulated by environmental signals and viral regulatory factors.