Background: 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. Results: To 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. Conclusions: Our 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.
Bibliographical noteFunding Information:
Authors are thankful to Dr. Alan G. Hinnebusch, NICHD, USA, for providing yeast strains, ?-GCD6 antibodies, and plasmids used in this study. Authors are also thankful to Shoolini University, Bajhol, Solan, Himachal Pradesh, India, for providing financial and infrastructural support for this study and all the members of Yeast Biology Lab for moral support.
© 2020, The Author(s).
Copyright 2020 Elsevier B.V., All rights reserved.
- General Control Derepressed (GCD)
- General Control Nonderepressible (GCN)
- S. cerevisiae