GUANINE-NUCLEOTIDE EXCHANGE FACTOR FOR EUKARYOTIC TRANSLATION INITIATION FACTOR-II IN SACCHAROMYCES-CEREVISIAE - INTERACTIONS BETWEEN THE ESSENTIAL SUBUNITS GCD2, GCD6, AND GCD7 AND THE REGULATORY SUBUNIT GCN3

Phosphorylation of eukarYotic translation initiation factor 2(eIF-2) i n amino acid-starved cells of the yeast saccharomyces cerevisiae reduc es general protein synthesis but specifically stimulates translation o f GCN4 mRNA. This regulatory mechanism is dependent on the nonessentia l GCN3 protein and multiple essential proteins encoded by GCD genes. P revious genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently show n to be the yeast equivalent of the mammalian guanine nucleotide excha nge factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 i s an integral component of the GCD-eIF.2B complex that, while dispensa ble, can be mutationally altered to have a substantial inhibitory effe ct on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and parti cular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B comp lex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eEF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmu noprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confir med that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fac t that all five subunits of yeast eIF-2B were first identified as tran slational regulators of GCN4 strongly suggests that regulation of guan ine nucleotide exchange on eIF-2 is a key control point for translatio n in yeast cells just as in mammalian cells.