Casein Kinase II Mediates Multiple Phosphorylation of Saccharomyces cerevisiae eIF-2α (Encoded by SUI2), Which Is Required for Optimal eIF-2 Function in S. cerevisiae

Abstract

Previous studies have demonstrated that the α subunit of eukaryotic initiation factor 2 (eIF-2α), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2α is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. ³²P_i labeling and isoelectric focusing analysis of a SUI2⁺ Δgcn2 strain identifies eIF-2α as radiolabeled and a single isoelectric protein species. Treatment of SUI2⁺ Δgcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2α that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2α maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292,294, and 301 that compose consensus casein kinase II sequences. ³²P_i labeling or isoelectric focusing analysis of eIF-2α from conditional casein kinase II mutants indicated that phosphorylation of eIF-2α is abolished or dephosphorylated forms of eIF-2α are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2α protein in vitro but does not phosphorylate recombinant eIF-2α that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2α at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2α protein are required for optimal eIF-2 function in S. cerevisiae.