Figure 6.

Butanol reduces ternary complex levels and modestly reduces the transit of eIF2 through the 2B body. (A) Myc-immunoprecipitation experiments using extracts prepared from the yMK1125 [bearing GCD1-P180-MYC (BUT^R)] and yMK1126 [bearing GCD1-S180-MYC (BUT^S)] strains grown in rich media and then either incubated in media (−) or media with 1% (vol/vol) butanol (+) for 15 min. Samples were separated into input and pellet fractions, as indicated, and were used for Western blots using antibodies against eIF2Bα to ε. (B) Flag-immunoprecipitation experiments using extracts prepared from strains yMK1613 [GCD1-P180 (BUT^R)] and yMK1615 [GCD1-S180 (BUT^S)] (both bearing a eIF2β-Flag–expressing plasmid) grown in rich media and then either incubated in media (−) or media with 1% (vol/vol) butanol (+) for 15 min. Samples were separated into input and pellet fractions, as indicated, and 20% was used for Western blots using antibodies against Flag, eIF2α to eIF2γ. The remaining 80% was used to prepare RNA that was used to perform Northern blots using an oligonucleotide probe to tRNA_i^Met. (C) A panel of live-cell images from the BUT^R (GCD1-P180) strains: yMK883 (eIF2α-GFP), yMK1211 (eIF2γ-GFP), yMK880 (eIF2Bγ-GFP), yMK882 (eIF2Bε-GFP), yMK1347 (eIF2Bβ-GFP), yMK1356 (eIF2Bα-GFP) and the BUT^S (GCD1-S180) strains: yMK914 (eIF2α-GFP), yMK1212 (eIF2γ-GFP), yMK876 (eIF2Bγ-GFP), yMK878 (eIF2Bε-GFP), yMK1363 (eIF2Bβ-GFP), and yMK1355 (eIF2Bα-GFP) either treated with 1% (vol/vol) butanol for 15 min (+) or untreated (−). (D) Bar chart depicting the mean percentage eIF2 in the 2B body for the strains yMK883 (SUI2-GFP GCD1-P180 BUT^R) and yMK914 (SUI2-GFP GCD1-S180 BUT^S) after 15-min treatments in complete media (−), media with l% (vol/vol) butanol (+but), or media lacking amino acids (−AAs). eIF2 levels in the 2B body were measured using confocal microscopy and densitometry. Fifteen image-merged z-stacks from at least 20 single yeast cells were quantified using ImageJ to obtain the plotted values. Error bars, ±1 SEM; *p < 0.01 from an analysis of variance (ANOVA). (E) Bar chart showing half times of FRAP derived from experiments performed on eIF2α-GFP–bearing strains yMK883 (SUI2-GFP GCD1-P180 BUT^R) and yMK914 (SUI2-GFP GCD1-S180 BUT^S). Strains were grown in SCD media then incubated for 15 min in either complete media (−), media with l% (vol/vol) butanol (+but), or media lacking amino acids (−AAs) before single-cell FRAP studies. Error bars, ±1 SEM (where n is at least 18). *p < 0.05 from analysis of variance (ANOVA).