Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul;7(3):223.
doi: 10.1007/s13205-017-0857-8. Epub 2017 Jul 4.

Role of Saccharomyces cerevisiae TAN1 (tRNA acetyltransferase) in eukaryotic initiation factor 2B (eIF2B)-mediated translation control and stress response

Sonum Sharma  1 , Anuradha Sourirajan  1 , Kamal Dev  2
Affiliations

Role of Saccharomyces cerevisiae TAN1 (tRNA acetyltransferase) in eukaryotic initiation factor 2B (eIF2B)-mediated translation control and stress response

Sonum Sharma et al. 3 Biotech. 2017 Jul.

Abstract

Eukaryotic initiation factor 2B (eIF2B) controls the first step of translation by catalyzing guanine nucleotide exchange on eukaryotic initiation factor 2 (eIF2). Mutations in the genes encoding eIF2B subunits inhibit the nucleotide exchange and eventually slow down the process of translation, causing vanishing white matter disease. We constructed a Saccharomyces cerevisiae genomic DNA library in YEp24 vector and screened it for the identification of extragenic suppressors of eIF2B mutations, corresponding to human eIF2B mutations. We found a suppressor-II (Sup-II) genomic clone, as suppressor of eIF2Bβ (gcd7-201) mutation. Identification of Sup-II reveals the presence of truncated SEC15, full-length TAN1 (tRNA acetyltransferase), full-length EMC4, full-length YGL230C (putative protein) and truncated SAP4 genes. Full-length TAN1 (tRNA acetyltransferase) gene, subcloned into pEG(KG) vector and overexpressed in gcd7-201 gcn2∆ strain, suppresses the slow-growth (Slg-) and general control derepression (Gcd-) phenotype of gcd7-201 gcn2∆ mutation, but YGL230C did not show any effect. A GST-Tan1p fusion protein of 60 kDa was detected by western blotting using α-GST antibodies. Interestingly, Tan1p overexpression also suppresses the temperature-sensitive (Ts-), Slg- and Gcd- phenotype of eIF2Bγ (gcd1-502) mutant. Role of Tan1p protein in eIF2B-mediated translation regulation was also studied. Results revealed that Tan1p overexpression confers resistance to GCD7 GCN2, gcd7-201 gcn2∆, GCD7 gcn2∆ growth defect under ethanol, H2O2 and caffeine stress. No resistance to DMSO-, NaCl- and DTT-mediated growth defect upon GCD7 gcn2∆, GCD7 GCN2, gcd7-201 gcn2∆ was observed by overexpression of TAN1. Hence, we proposed that Tan1p is involved directly or indirectly in regulating eIF2B-mediated translation.

Keywords: Caffeine; Ethanol; H2O2; Tan1 p; Translation; VWM; eIF2B.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors hereby declare that they have no conflict of interest.

Funding and support

Shoolini University.

Figures

Fig. 1
Fig. 1
Screening for extragenic suppressors of gcd7-201 gcn2∆ mutant strain transformed with a genomic DNA library. b (YEp24) vector and c GCD7 gcn2∆ transformed with (YEp24) vector. S. cerevisiae strains were plated on SC medium lacking uracil and incubated at 30 °C for 2 days. d gcd7-201 gcn2 ∆ transformant showing Slg+ phenotype (Sup-II) was streaked along with gcd7-201 gcn2∆ and GCD7 gcn2∆ transformed with YEp24. e Serial dilutions of the transformant (Sup-II) showing Slg+ phenotype were spotted on SC-Ura and SC medium containing 30 mM-3AT. GCD7 gcn2∆ and GCD7 GCN2∆ transformed with vector were spotted as control. f The transformants (Sup-II) showing Slg+ and Gcd + phenotype were used for rescuing plasmid as indicated in lane 2. g The rescued plasmid was retransformed into gcd7-201 gcn2∆ mutant strain and h further verified by streaking on SC-Ura medium as indicated
Fig. 2
Fig. 2
PCR amplification of TAN1 gene. a Schematic representation of Sup-II clone showing complete ORF of EMC4, TAN1, YGL230C and truncated SEC15 and SAP4 genes on chromosome VII, b PCR amplification of the TAN1 gene from genomic clone (Sup-II) and separated on 1% agarose gel, c pEG(KG) plasmid was isolated (lane 3) and digested with XbaI and SalI (lane 1). b, c Lane 1 represents molecular size marker (kb)
Fig. 3
Fig. 3
gcd7-201 gcn2∆ rescued Slg+ and Gcd+ phenotype when transformed with pEG(KG)/TAN1 plasmid. GCD7 gcn2∆ and gcd7-201 Gcn2∆ harboring pEG(KG)/TAN1 or empty vector pEG(KG) were streaked in parallel on SC medium laking uracil, but either containing a raffinose or b galactose. Uracil-based plasmid pEG(KG)/TAN1 was evicted on SC medium containing, c FOA and further streaked on d SC-Ura medium. e Spotting on SC medium containing 2% galactose and lacking uracil or SC medium containing 2% galactose, 30 mM 3-AT and lacking uracil. Plates were incubated at 30 °C for 2 days. f Western analysis of GST-Tan1p expression in gcd7-201 gcn2∆ strain with anti-GST antibody. The whole-cell protein extracts (20 µg) were prepared from uninduced and 2% galactose-induced cultures of the strain harboring pEG(KG)/TAN1. Samples were separated on 10% SDS gel followed by western blotting using anti-GST for GST-Tan1 and anti-Gcd6 antibodies (loading control). UI uninduced, I induced
Fig. 4
Fig. 4
Tan1p overexpression rescues Slg−, Gcd− and Ts− phenotype of gcd1-502 gcn2-101 strain. a gcd1-502 gcn2-101 transformants showing Slg+ phenotype were streaked along with gcd1-502 gcn2-101 transformed with pEG(KG) vector alone. b Serial dilutions of the gcd1-502 gcn2-101 transformants showing Slg+ phenotype were spotted on SC-Ura or SC medium containing 3-AT (30 mM). gcd1-502 gcn2-101 transformed with pEG(KG) was spotted as control. Plates were incubated for 2 days at 30 °C
Fig. 5
Fig. 5
S. cerevisiae strains overexpressing TAN1 showed enhanced stress tolerance. a gcd7-201 gcn2∆, GCD7 gcn2∆ and GCN2 gcn2∆ S. cerevisiae strains were streaked on YPD agar plates containing 4 mM H2O2, 10% ethanol and 30 mM caffeine. Number of plus signs (+) indicates comparative visible growth. Negative signs (−) indicates no visible growth. SC medium supplemented with b 4 mM H2O2, c 10% ethanol and d 30 mM caffeine in the presence of raffinose or galactose. Cultures were grown for 16 h at 30 °C. Cultures with raffinose supplementation were served as control. The cell density was obsereved by measuring absorbance at 600 nm (A 600). Data of three independent experiments were plotted with standard deviation
Fig. 6
Fig. 6
Halo assay to test the effect of H2O2, ethanol and caffeine on the growth of eIF2B mutants. Hydrogen peroxide, ethanol and caffeine halo assays were performed with gcd7-201 gcn2∆, GCD7 gcn2∆ and GCD7 GCN2. S. cerevisiae strains harboring either the empty vector or the TAN1 gene-expressing plasmid were spread on SC medium, containing filter disks soaked in a H2O2 (4 mM), b ethanol (10%) and c caffeine (30 mM) in the presence of either raffinose or galactose. The plates were incubated at 30 °C for 2–3 days
Fig. 7
Fig. 7
Spot assay to test the effect of H2O2, ethanol and caffeine on the growth of eIF2B mutants. Hydrogen peroxide, ethanol and caffeine halo assays were performed with gcd7-201 gcn2∆, GCD7 gcn2∆ and GCD7 GCN2. Tenfold serially diluted transformants harboring empty vector (pEG(KG) or pEG(KG)-TAN1 were spotted on SC-Ura or SC-Ura/3-AT agar plates supplemented with a H2O2 (4 mM), b ethanol (10%) and c caffeine (30 mM) in the presence of either raffinose or galactose. Cultures were grown for 2 days at 30 °C
Fig. 8
Fig. 8
Schematic model indicating the repression of GCN4 by overexpression of GST-Tan1p via eIF2B. Panel I indicates that in the presence of amino acids (+a.a), Gcn2p is inhibited in wild-type cells that in turn inhibits Gcn4p expression. In contrast, amino acid starvation (−a.a) in wild-type cells leads to eIF2α phosphorylation and Gcn4p induction via activation of Gcn2p kinase. Similarly, the presence of H2O2, ethanol and caffeine in the growth medium inhibits eIF2B activity that in turn activates GCN4p. On the other hand, in panel II, eIF2B mutants grown in the presence or absence of a.a, H2O2, ethanol and caffeine inhibit eIF2B activity that can lead to Gcn4p activation. In panel III, the eIF2B mutants gcd7-201 gcn2∆, GCD7 gcn2∆ and GCD7 GCN2∆ overexpressing Tan1p repress Gcn4p in the presence or absence of a.a, H2O2, ethanol and caffeine and rescue their Slg−, Gcd− and Ts− phenotype
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ashe MP, Slaven JW, Susan K, Ibrahimo S, Sachs AB. A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols. EMBO J. 2001;20:6464–6474. doi: 10.1093/emboj/20.22.6464. - DOI - PMC - PubMed
    1. Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, Hall MN. TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell. 1996;7:25–42. doi: 10.1091/mbc.7.1.25. - DOI - PMC - PubMed
    1. Becker HF, Motorin Y, Planta RJ, Grosjean H. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of Ψ55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res. 1997;25:4493–4499. doi: 10.1093/nar/25.22.4493. - DOI - PMC - PubMed
    1. Cavaillé JEROME, Chetouani FARID, Bachellerie JP. The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs. RNA. 1999;5:66–81. doi: 10.1017/S1355838299981475. - DOI - PMC - PubMed
    1. Cherkasova V, Hinnebusch AG. Translational control by TOR and TAP42 through dephosphorylation of eIF2α kinase GCN2. Genes Dev. 2003;17:859–872. doi: 10.1101/gad.1069003. - DOI - PMC - PubMed

LinkOut - more resources