Abstract
BackgroundEukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg−) and general control derepression (Gcd−) phenotypes in Saccharomyces cerevisiae. ResultsTo suppress eIF2B mutations, S. cerevisiae genomic DNA library was constructed in high-copy vector (YEp24) and transformed into eIF2B mutant S. cerevisiae strains. The library was screened for wild-type genes rescuing S. cerevisiae (Slg−) and (Gcd−) phenotypes. A genomic clone, Suppressor-I (Sup-I), rescued S. cerevisiae Slg− and Gcd− phenotypes (gcd7-201 gcn2∆). The YEp24/Sup-I construct contained truncated TAN1, full length EMC4, full length YGL230C, and truncated SAP4 genes. Full length EMC4 (chaperone protein) gene was sub-cloned into pEG (KG) yeast expression vector and overexpressed in gcd7-201 gcn2∆ strain which suppressed the Slg− and Gcd− phenotype. A GST-Emc4 fusion protein of 47 kDa was detected by western blotting using α-GST antibodies. Suppression was specific to gcd7-201 gcn2∆ mutation in eIF2Bβ and Gcd1-502 gcn2∆ in eIF2Bγ subunit. Emc4p overexpression also protected the wild type and mutant (gcd7-201 gcn2∆, GCD7 gcn2∆, and GCD7 GCN2∆) strains from H2O2, ethanol, and caffeine stress. ConclusionsOur results suggest that Emc4p is involved in eIF2B-mediated translational regulation under stress and could provide an amenable tool to understand the eIF2B-mediated defects.
Highlights
Eukaryotic initiation factor 2B initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg−) and general control derepression (Gcd−) phenotypes in Saccharomyces cerevisiae
Screening of genomic DNA library clones for rescuing slow growth phenotype of S. cerevisiae eIF2B mutant strains Approximately, 30 transformants were observed, showing colony size equivalent to that of isogenic wild-type GCD7 gcn2Δ transformed with vector alone were screened further for Gcd+ phenotype
This data clearly suggests that a genomic clone (Sup-I) suppressed the Slg− and Gcd− phenotype of gcd7-201 gcn2Δ mutant strain
Summary
Eukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. eIF2B gene mutations cause leukoencephalopathy called vanishing white matter disease (VWM) in humans and slow growth (Slg−) and general control derepression (Gcd−) phenotypes in Saccharomyces cerevisiae. Eukaryotic initiation factor 2B (eIF2B) initiates and regulates translation initiation in eukaryotes. Eukaryotic initiation factor 2B (eIF2B) a heterodecameric complex of five non-identical protein subunits (α–ε) initiates/regulates translation [1]. Regulatory subunits of eIF2B are important for eI2BeIF2 interaction under normal and stress conditions. Archeal eIF2B interacts with eukaryotic eIF2α and eIF2Bα indicating the importance of regulatory subunits [20]. EIF2Bβ subunit binds eIF2 which is important for eIF2-eIF2B interaction and translation regulation [21]. Identifying extragenic suppressors, modulators (proteins/chemicals) of mutated eIF2B regulatory subunits, may be useful in curing VWM disease. The chemical modulators, activating either GCN4 or suppressing eIF2B mutations, have been previously identified [23]. The goal of our study was to identify the S. cerevisiae protein that interacts with mutated eIF2B subunit and suppresses the mutation
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