GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3

Abstract

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(i)/(Met) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2α phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.