CASEIN KINASE-II MEDIATES MULTIPLE PHOSPHORYLATION OF SACCHAROMYCES-CEREVISIAE EIF-2-ALPHA (ENCODED BY SUI2), WHICH IS REQUIRED FOR OPTIMALEIF-2 FUNCTION IN SACCHAROMYCES-CEREVISIAE

Previous studies have demonstrated that the alpha subunit of eukaryoti c initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the y east Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe th at yeast eIF-2 alpha is a constitutively phosphorylated protein specie s that is multiply phosphorylated by a GCN2-independent mechanism. P-3 2(i) labeling and isoelectric focusing analysis of a SU12(+) Delta gcn 2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectr ic protein species. Treatment of SU12(+) Delta gcn2 strain extracts wi th phosphatase results in the identification of three additional isoel ectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SU12 couple d with biochemical analysis of eIF-2 alpha maps the sites to the carbo xyl region of SU12 that correspond to Ser residues at amino acid posit ions 292, 294, and 301 that compose consensus casein kinase II sequenc es. P-32(i) labeling or isoelectric focusing analysis of eIF-2 alpha f rom conditional casein kinase II mutants indicated that phosphorylatio n of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth, conditions. Furthermore, yeast casein kinase II phosphorylates recombi nant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all thr ee consensus casein kinase II sequences. These data strongly support t he conclusion that casein kinase II directly phosphorylates eIF-2 alph a! at one or all of these Ser amino acids in vivo. Although substituti on of SU12 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 phys iological consequence on eIF-2 function in S. cerevisiae. Haploid stra ins constructed to contain Ser-to-Ala mutations at the consensus casei n kinase II sequences in SU12 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. The se genetic data appear to indicate that the modifications that we desc ribe at the carboxyl end of the eIF-2 alpha protein are required for o ptimal eIF-2 function in S. cerevisiae.