A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols - PubMed (original) (raw)

A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols

M P Ashe et al. EMBO J. 2001.

Abstract

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

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Figures

Fig. 1. Butanol inhibits growth and translation in specific genetic backgrounds. (A) Yeast strains yMK36 (_BUT_S) and yMK23 (_BUT_R) serially diluted on YPD (2 days at 30°C) or YPD + 1% butanol agar plates (3 days at 30°C). (B) Growth curves for yMK36 are shown either after the addition of 1% butanol at time zero or after no addition. (C) Polyribosome traces from yMK36. Yeast were grown in YPD and 1% butanol was added for the time periods indicated. Polyribosomes were analysed as described in Materials and methods. The 40S (small ribosomal subunit), 60S (large ribosomal subunit), 80S (monosome) and polysomes are labelled. (D) [35S]methionine incorporation into proteins over time in the presence or absence of 1% butanol. yMK36 was grown in synthetic complete media without methionine, split in two and at _t_0 [35S]methionine was added to each aliquot. After 5 min, 1% butanol was added to one of the aliquots. The level of [35S]methionine (c.p.m. × 105) incorporated into protein samples removed at the time points indicated was determined.

Fig. 2. Butanol inhibits translation in yeast by a previously unidentified mechanism. (A) Polyribosome traces from strains with wild-type SUI2 (yMK129) or mutant SUI2 S51A (yMK127). Yeast were grown in SCD-Leu and washed in SCD-Leu, SCD minus all amino acids (–amino acids) or SCD-Leu + 1% butanol (+butanol) for 10 min. (B) Polyribosome traces from strains with wild-type TOR1 (yMK449) or mutant TOR1 S1972I (yMK450). Strains were grown in SCD-Leu followed by the addition of either drug vehicle for 1 h, 0.2 µg/ml rapamycin (+rap) for 1 h or 1% butanol for 10 min (+butanol). (C) Polyribosome traces from butanol-sensitive (yMK36) and butanol-resistant (yMK23) strains. Yeast were grown in YPD and washed in either YPD (+glucose) or YP (–glucose) for 10 min. (D) Polyribosome traces from yMK441 (caf20Δ _BUT_S). Yeast were grown in YPD and 1% butanol was added to half the culture for 10 min (+butanol).

Fig. 3. Different alleles of the GCD1 gene account for the strain differences in butanol sensitivity/resistance. (A) A serial dilution plate assay and polyribosome traces from the butanol-sensitive background (_BUT_S strain) overexpressing either vector (yMK443), GCD1 isolated from the butanol-sensitive strain (_GCD1_S) (yMK444) or GCD1 isolated from the butanol-resistant strain (_GCD1_R) (yMK445). (B) A serial dilution plate assay and polyribosome traces from the butanol-resistant background (_BUT_R strain) overexpressing either vector (yMK446), GCD1 isolated from the butanol-sensitive strain (_GCD1_S) (yMK447) or GCD1 isolated from the butanol-resistant strain (_GCD1_R) (yMK448). For the serial dilution assay, SCD-Trp plates were incubated for 2 days at 30°C, whereas the butanol plates were incubated for 3–4 days at 30°C. For the polysome analyses, the strains were grown in SCD-Trp and there was either no addition or 1% butanol was added for 10 min (+but). (C) A summary of the growth and translational sensitivity to butanol for strains overexpressing GCD1 S180,E563 (yMK444, yMK447), GCD1 P180,K563 (yMK445, yMK448), GCD1 S180,K563 (yMK503, yMK505) and GCD1 P180,E563 (yMK504, yMK506).

Fig. 4. The translational sensitivity to butanol does not require reversible phosphorylation at Ser180. (A) Gcd1p levels are unaffected by butanol treatment. Protein extracts from the yMK602 (GCD1-P180), yMK526 (Flag-GCD1-S180) and yMK527 (Flag-GCD1-P180) strains were blotted and probed with an antibody to the flag epitope. Strains were treated with 1% butanol where indicated. (B) Total Gcd1p phosphorylation is not changed measurably in response to butanol treatment. A [32P]orthophosphate metabolic labelling experiment using the same strains as in (A). [32P]orthophosphate was added to yeast 5 min prior to treatment with or without 1% butanol for 10 min. The lower panel shows Coomassie Blue-stained immunoprecipitated Flag-Gcd1p and a non-specific band (diamond). The upper panel shows an autoradiograph of the same gel where the predominant phosphate-labelled species are Flag-Gcd1p and a non-specific phosphoprotein (*). (C) Serial dilution plate assays and polyribosome traces from GCD1 disrupted strains harbouring either GCD1-S180 (yMK601), GCD1-P180 (yMK602) or GCD1-A180 (yMK603) plasmids. For serial dilutions, SCD-Trp plates were incubated at 30°C for 2 days, whereas butanol-containing plates were incubated for 3–4 days at 30°C. For the polysome analyses, the strains were grown in SCD-Trp and there was either no addition or 1% butanol was added for 10 min (+but).

Fig. 5. Gcn4p activity is increased upon butanol addition to the GCD1-S180 strain. (A) Diagram summarizing how 3-AT is thought to induce the general control pathway via Gcn4p activation at the translational level. (B) A GCD1-S180 gcn2 strain grows on 3-AT plates in the presence of 1% butanol. Serial dilution plate assays of butanol-resistant (_BUT_R) (yMK430), butanol-sensitive (_BUT_S) (yMK429), butanol-resistant gcn2 deleted (gcn2Δ _BUT_R) (yMK515) and butanol-sensitive gcn2 deleted (gcn2Δ _BUT_S) (yMK516) strains. The plates were grown at 30°C as follows: YPD (2 days); YPD + 1% butanol (3 days) (minor variations in the growth of butanol-sensitive strains occur on these plates due to slight differences in butanol concentration across plates); SCD-His + 10 mM 3-AT (4 days); SCD-His + 1% butanol + 10 mM 3-AT (4 days). (C–F) The GCD1-S180 allele permits increased HIS4-lacZ and GCN4-lacZ expression in response to butanol. β-galactosidase assays measured in Miller units from extracts prepared from the strains (C) yMK526 and yMK527 (HIS4-lacZ), (D) yMK597 and yMK598 (p[_GCN4-lacZ_]), (E) yMK599 and yMK600 (p[_ΔuORFs-GCN4-lacZ_]) and (F) yMK629–632 (<_GCN4-lacZ_>).

Fig. 6. Butanol is likely to inhibit translation via effects on eIF2B. (A) Polysome analyses from butanol-sensitive and -resistant strains carrying either the appropriate empty high copy vectors (pRS424 and pRS425) or the eIF2B high copy plasmids: sensitive pMK31 (p[_GCD1_S _GCD6_]); or resistant pMK32 (p[_GCD1_R _GCD6 LEU2 2_µ])] as well as pAV1492 p[_GCD7 GCD2 GCN3 TRP1 2µ_]. Strains were grown in SCD-Trp-Leu and 1% butanol was added to half the culture for 10 min (+but). (B) Serial dilution plate assays for butanol-resistant (_BUT_R) (yMK23), butanol-sensitive (_BUT_S) (yMK36) and various gcd mutant strains (yMK641–647). The plates were grown at 24°C (due to the temperature sensitivity of the gcd mutants) for 3 days (YPD) or 4 days (YPD + 1% butanol). The lower panels show the effects of a high copy eIF2 plasmid (p1780) or vector in the gcd1-101 strain background (yMK639 and yMK640). Growth was for 3 days (SCD-Ura) or 4 days (SCD-Ura + 1% butanol).

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A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols - PubMed (original) (raw)