The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae - PubMed (original) (raw)

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

J de la Cruz et al. Proc Natl Acad Sci U S A. 1997.

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.

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Figures

Figure 1

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

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

Figure 3

The PET56-DED1 region of chromosome XV. The original fragment carrying the suppressor activity and four relevant subclones are shown. B, _Bam_HI; H, _Hin_dIII; P, _Pst_I; S, _Sal_I; X, _Xba_I. The _Sal_I site belongs to the polylinker of YEplac181.

Figure 4

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

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.

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