Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

Abstract

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecularweight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30%o identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2i phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.In response to starvation for an amino acid, the budding yeast Saccharomyces cerevisiae increases expression of the GCN4 protein, which in turn activates transcription of more than 30 genes encoding enzymes involved in amino acid biosynthesis (general amino acid control). The increase in GCN4 expression occurs primarily at the level of translation initiation (for a review, see references 22, 24, and 25) and involves a pathway of positive and negative regulatory factors, some of which have general functions in translation initiation (Fig. 1).A reduction in the activity of eukaryotic translation initiation factor 2 (eIF-2) appears to be responsible for increasing GCN4 translation in amino acid-starved cells (25 Exchange factor eIF-2B isolated from rabbit reticulocytes is composed of five subunits and copurifies with a fraction of eIF-2 (29, 36). A complex associated with eIF-2 that contains GCD1, GCD2, and GCN3 was postulated to be the GTP-GDP exchange factor for eIF-2 in S. cerevisiae (6,15). The GCD1 and GCD2 subunits of this "GCD complex" have essential functions in translation initiation in S. cerevisiae (6,15,56). Nonlethal mutations in these factors lead to increased GCN4 expression under nonstarvation conditions, as would be expected of mutations in subunits of eIF-2B that reduce guanine nucleotide exchange on eIF-2 and thereby lower eIF-2 activity.Four upstream open reading frames (uORFs) in the leader of GCN4 mRNA couple GCN4 translation to the level of eIF-2 activity (22,23). Under nonstarvation conditions, it appears that ribosomes scanning from the 5' end of GCN4 mRNA translate the first ORF (uORF1) and reinitiate at one of the remaining uORFs, -2, -3, or -4, instead of at GCN4.Because of the low level of eIF-2. GTP. tRN...

Structure of the Catalytic Fragment of Translation Initiation Factor 2B and Identification of a Critically Important Catalytic Residue

“…Plasmid shuffling used 5-fluoro-orotic acid (21). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) to introduce mutations into the coding region of eIF2B⑀ (GCD6) in the GCD6 LEU2 CEN plasmid pJB102 (22). Details of all site-directed mutagenesis oligonucleotides used are provided in Supplementary Materials Table I.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Yeast eIF2B was discovered through studies examining the translational control of the GCN4 mRNA. Mutations in the eIF2B subunits cause Gcd 2 phenotypes (Harashima and Hinnebusch 1986) and also confer slow-growth and reduced rates of translation initiation (Hannig et al 1990;Foiani et al 1991;Bushman et al 1993a).…”

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

“…Regulation of protein transport in the 14q24.3 endoplasmic reticulum (19) Subunit of a GDP-GTP exchange factor (20) 14q24 Ordering of the human FOS, S31iiil25, and S20i15 cosmids on chromosome 14q24.3 was performed by FISH to extended human DNA fibers. On the extended DNA fibers, the hybridized S20i15 cosmid (p126H2) shows a red signal (A), the S31iiil25 cosmid (1:66-4) shows a green signal (B), and the FOS cosmid (A6-7) shows an orange signal (a combination of red and green fluorescence) (C).…”

“…The comparative sequence data suggests that the fos-like protein gene may represent a duplication event in the Fugu genome of the cFOS gene. Functions for human S31iiil25 and S20ilS are postulated, based upon amino acid sequence homologies to canine glycoprotein gp25L (19) and the yeast GCD7 subunit of a GDP-GTP exchange factor (20), respectively. The chromosomal location of DLST and FOS have previously been reported to 14q24.2-q24.3 (16) and 14q21-q31 (22), respectively, and were also confirmed to be in the AD3 locus on 14q24.3, together with S31iiil25 and S20ilS by Sherrington et al (12).…”