GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae.
The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.
Foiani
M
M
Section on Molecular Genetics of Lower Eukaryotes, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Cigan
A M
AM
Paddon
C J
CJ
Harashima
S
S
Hinnebusch
A G
AG
D000596:Amino Acids / Q000096:biosynthesis; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D000596:Amino Acids; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D005656:Fungal Proteins; C068999:GCD2 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D007930:Leucine