The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae

Abstract

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5' end of the mRNA. In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the CAF20 gene. Here, we show a negative regulatory role for the p20 protein in translation initiation. Deletion of CAF20 partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the DED1 gene, which we have isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The DED1 gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in S. cerevisiae.

Figure 1

Suppression of the Δstm1 phenotype by Δcaf20. CDK36–1A (Δcaf20) and RCB1–1C (Δstm1) were crossed and subsequently sporulated. A representative tetratype tetrad is shown on rich medium (yeast peptone dextrose) at 30°C and 18°C. The plates were incubated for 3 and 6 days, respectively.

Figure 2

Inhibition of Δstm1 growth by p20 overexpression. The RCB1–1C (Δstm1) strain was transformed with either the pGAL-p20 (CEN-URA3) or YEPL1-p20 (2μ-URA3) plasmids that contain the CAF20 gene under a CYC1-GAL1 promoter or as a control with pGAL or YEPL1. Transformants were grown for 4 days on SD-Ura or on SGal-Ura at 30°C.

Figure 3

The PET56-DED1 region of chromosome XV. The original fragment carrying the suppressor activity and four relevant subclones are shown. B, BamHI; H, HindIII; P, PstI; S, SalI; X, XbaI. The SalI site belongs to the polylinker of YEplac181.

Figure 4

Suppression of the cdc33–42 mutation by DED1 and DBP1. CDK35–4A was transformed with either YEplac181, YEplac181-CDC33, YEplac181-DED1, or YEplac181-DBP1. Transformants were grown on SD-Leu at 30°C or 35°C for 3 and 6 days, respectively.

Figure 5

Polysome analysis of ded1 and dbp1 mutants. Cells were grown in yeast peptone dextrose at 30°C and harvested at an OD₆₀₀ of 0.8. The peaks of free 40S and 60S ribosomal subunits, 80S, and polysomes are indicated. (A) DBY747, wild-type strain. (B) CDK112–1A, Δdpb1 strain. (C) DJY105, ded1/spp81–3 mutant. (D) DJY106, ded1/spp81–2 mutant.