All the chemicals and reagents were of molecular biology grade procured from Thermo scientific, Himedia Labs, India; MP Biomedicals, USA; Fermentas Inc. USA; and Bio-Rad. USA.
S. cerevisiae strains and plasmids
S. cerevisiae strains employed in this study (Table S1) were cultured on YPD agar or liquid medium. S. cerevisiae transformants were selected on synthetic complete (SC) medium lacking uracil and supplemented with glucose/galactose/raffinose. S. cerevisiae strains were incubated at 30 °C. E. coli strain DH5α was used for S. cerevisiae genomic DNA library construction and plasmid isolation.
YEp24 (high copy shuttle vector) and pEG(KG) (yeast expression vector) were used for cloning and expression of S. cerevisiae genes respectively. Nutrient broth (NB, Himedia Labs, Mumbai) with 100 μg/ml ampicillin was used to culture the E. coli strain DH5α harboring YEp24 or pEG(KG) at 37 °C. Plasmid DNA of YEp24 and pEG(KG) were isolated and used in transformations of yeast strains [24, 25].
Construction of S. cerevisiae genomic DNA library and transformation into eIF2B mutant S. cerevisiae strains
Genomic DNA from S. cerevisiae strain H4 (Table S1) was isolated and partially digested with Sau3AI enzyme . Fifty nanograms of partially digested and gel purified (gel purification kit Thermo-scientific) genomic DNA was ligated with 20 μg of YEp24 vector at BamHI site using T4 DNA ligase . After ligation at 16 °C for 16 h, E. coli strain DH5α was transformed with the ligation mix by heat shock method . The transformation mix was plated on NA medium containing ampicillin (100 μg/ml). Transformations were selected against ampicillin resistance on NA medium containing ampicillin and were pooled into three groups named as pool-I, pool-II, and pool-III.
Plasmid DNA isolation from three pools indicating ~ 13,575 cfu (colony-forming units) of transformants of DH5α was done . Plasmids isolated from all three pools or vector (YEp24) alone were transformed into S. cerevisiae eIF2B mutant strains (Figure S1). The wild-type strains were transformed with YEp24 vector alone using LiAc method . The nomenclature used for various S. cerevisiae strains used in this study is given in (Table S2). Transformation mix was plated on synthetic complete (SC) medium containing 2% glucose lacking uracil. SC mixture lacking uracil was used as a dropout supplement to select transformants containing uracil-based plasmid. eIF2B mutant S. cerevisiae transformants with normal colony size were compared to that of vector-transformed eIF2B mutant strains and wild-type strains by streaking and spot assay on synthetic complete (SC) medium containing 2% glucose lacking uracil .
Screening of suppressor protein
eIF2Bβ (gcd7-201 gcn2∆) transformants showing Slg+ phenotype as that of isogenic wild type were selected and analyzed for Gcd+ phenotype by spot assay on SC-medium supplemented with 30 mM 3-AT (3-amino triazole). Transformants showing Slg+ and Gcd+ phenotype were further screened by spot assay of 10-fold serially diluted culture and by streaking.
Plasmid DNA from the potential gcd7-201 gcn2∆ transformants (Slg+, Gcd+) were isolated , and gcd7-201 gcn2∆ mutant S. cerevisiae strain were transformed with the rescued plasmid. Simultaneously, the rescued plasmid was sequenced on both the strands at Eurofins Bangalore, (http://www.eurofins.in/) by using YEp24 vector specific primers (S7).
Functional characterization of suppressor protein
EMC4 gene from rescued plasmid was amplified using gene-specific primers (Table S3) followed by sub-cloning into pEG(KG) yeast expression vector (containing a GAL1 promoter and a protease cleavable N-terminal GST tag) at XbaI/SalI restriction sites. Gal promoter is repressed by raffinose and induced by galactose.
DH5α was transformed with recombinant plasmids (100 ng) by heat shock method . Rescued plasmid DNA from transformants was sequenced at Eurofins Bangalore, (http://www.eurofins.in/). An error free nucleotide sequence of EMC4 DNA was obtained. pEG(KG)/EMC4 plasmids were transformed into gcd7-201 gcn2∆ strain by LiAc method in order to confirm the Slg+ and Gcd+ phenotype. The transformation mix was plated on SC medium supplemented with uracil and 2% galactose. gcd7-201 gcn2∆ and GCD7 gcn2∆ transformed with pEG(KG) vector alone were used as control.
Plasmid DNA isolation from the recombinant clones was done as described  and was transformed again in gcd7-201 gcn2∆. Spot assay of pEG(KG)/EMC4 transformants was performed in order to confirm the Slg+ and Gcd+ phenotype. GST-EMC4-based suppression was also confirmed by eviction of pEG(KG) a uracil-based plasmid containing GST-EMC4 on 5-fluoroorotic acid (FOA) containing medium. 5-Flouroorotic acid (5-FOA) is converted to a toxic product (5-floorouracil) by URA3 gene product. Thus, S. cerevisiae cells containing URA3 marker cannot grow on medium containing 5-FOA but are able to grow on medium lacking uracil. Thus, FOA is used to select for the loss of vectors carrying the wild-type URA marker . Colonies from FOA plate were picked and streaked on SC medium without uracil and supplemented with 2% galactose. Plates were incubated at 30 °C for 2 days and were observed for growth phenotype.
Western blot analysis
The whole cell extract of gcd7-201 gcn2∆ harboring either pEG(KG) or pEG(KG)/EMC4 was prepared by glass bead lysis method using Fast Prep (MP Biomedicals). gcd7-201 gcn2∆ harboring either pEG(KG) or pEG(KG)/EMC4 were incubated in SC-medium (5 ml) supplemented with 2% raffinose (w/v) at 30 °C for 18 h. Ten milliliters of SC medium supplemented with 2% raffinose (w/v) was inoculated with 1% of overnight grown primary culture followed by incubation at 30 °C. At an absorbance of A 600 of ~ 0.5, an aliquot was collected as the uninduced control, and the remaining culture was induced by 2% galactose (w/v). Both induced and uninduced cultures were incubated for an additional 3 h at 30 °C.
After incubation, cells were harvested by centrifugation (6000 rpm for 10 min). Protein extraction of both induced and uninduced culture was carried using 20% tri-chloroacetic acid (TCA), and 20 μg of extracted proteins were resolved on SDS–PAGE followed by the transfer to the nitrocellulose membrane (Millipore, Immobilon P 0.45 μm) by electroblotting. The blot was incubated at 4 °C for 1 h in blocking solution containing 5% non-fat dried milk. After incubation, membrane was further incubated with anti-GST antibodies (1:5000, Abcam) overnight at 4 °C. Immunoreactive proteins were detected by using anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000, Abcam) for 1 h. Blots were washed by using PBST (phosphate buffer saline containing TritonX-100) buffer. Finally, the blots were developed using enhanced chemiluminescence kit (ECL, Bio-Rad, Inc. USA).
Expression of Emc4p in eIF2Bγ, eIF2Bδ, eIF2ε, and GCN2 mutant S. cerevisiae strains
Suppression analysis by Emc4p in other eIF2B mutants was done. eIF2Bγ (H70), eIF2Bδ (H750), eIF2Bε (H1792), and GCN2 (H591) mutants (Table S1) were transformed with pEG(KG) or pEG(KG)/Emc4. All the transformants were plated on SC-medium lacking uracil supplemented with 2% galactose, and the plates were incubated for 2 days at 30 °C. The transformants were selected and analyzed for Slg+ and Gcd+ phenotype by streaking and spot assay on SC-medium lacking uracil and containing 2% galactose or SC-medium lacking uracil and containing 2% galactose and 30 mM 3-AT respectively. Plates were incubated for 2 days at 30 °C. eIF2Bγ (H70) a Ts− mutant was also checked for suppression of temperature sensitive (Ts+) phenotype by Emc4p at 37 °C.
Effect of Emc4 protein overexpression on H2O2-, ethanol-, and caffeine-mediated cell death of eIF2B mutant and wild type S. cerevisiae strains
Three different sets of experiments (quantitative assay, spot assay, and halo assay) were performed. Wild-type GCD7 GCN2, GCD7 gcn2∆, and mutant gcd7-201 gcn2∆ strains of S. cerevisiae containing pEG(KG) or pEG(KG)/Emc4 were incubated for 16 h at 30 °C with shaking in the SC-medium supplemented with either 2% galactose or raffinose (lacking uracil). SC-medium also contained 4 mM H2O2 , 10% ethanol , 20 mM caffeine , 1.6% DMSO , 35 mM Dithiothreitol (DTT) , and 1 M NaCl  separately. After 16 h of growth, cell density was measured at A 600 nm using a UV visible spectrophotometer. Spot and halo assays were performed as given by [35, 36].
Ten-fold serially diluted cultures were spotted to check Slg and Gcd phenotypes on SC-medium supplemented with either 2% galactose or raffinose (lacking uracil). The medium was also supplemented 30 mM 3-AT for Gcd- phenotype, 4 mM H2O2, 10% ethanol, 20 mM caffeine, 1.6% DMSO, 35 mM Dithiothreitol (DTT), and 1 M NaCl. The plates were incubated at 30 °C for 2 days and observed for pattern of yeast cell growth.
For halo assay, filter disks containing 4 mM H2O2, 10% ethanol, 20 mM caffeine, 1.6% DMSO, 35 mM Dithiothreitol (DTT), and 1 M NaCl were placed on SC agar medium supplemented with either 2% galactose or raffinose (lacking uracil) with uniformly spread culture of S. cerevisiae mutant and wild-type strains containing pEG(KG) or pEG(KG)/Emc4. Plates were incubated at 30 °C for 2 days and observed for zone of inhibition. The S.cerevisiae strains GCD7 GCN2, GCD7 gcn2∆, and gcd7-201 gcn2∆ (Supplementary Table 1) were streaked on YPD plates with or without H2O2, ethanol, caffeine, DMSO, Dithiothreitol (DTT), and NaCl.