The yef3-fs1009 mutation results in a temperature-dependent growth defect, sensitivity to translation inhibitors and reduced amounts of eEF3. (A) The yef3-fs1009 mutation results in a growth defect that is exacerbated at higher temperatures. Serial dilutions of YEF3 wild-type and yef3-fs1009 strains were grown on rich media (YPAD) at the indicated temperatures. (B) The yef3-fs1009 mutation confers sensitivity to translation inhibitors anisomycin and paromomycin. Serial dilutions of strains in (A) were grown on rich (YPAD) media at 37°C with indicated concentrations of anisomycin or paromomycin. (C) Strains with the yef3-fs1009 mutation have reduced levels of eEF3 protein compared to otherwise isogenic strains with wild-type YEF3. Crude extracts, separated by SDS-PAGE, were subjected to western analysis with anti-eEF3 and anti-glucose-6-phosphate dehydrogenase (G6PDH) antibodies and quantified using Image J (https://imagej.nih.gov/ij/). (D) Increased copies of the yef3-fs1009 allele result in increased frameshifting in the yef3-fs1009 mbf1-R89K mutant, but have no effect in YEF3 mbf1-R89K strains. GFP/RFP expression from the (CGA)4+1 reporter was examined in YEF3 and yef3-fs1009 strains bearing CEN plasmids with no insert (EV), YEF3 (wt), or yef3-fs1009 (m). (E) The yef3-fs1009 mutation does not suppress programmed frameshifting in TRM140 mRNA. (**) P < 0.01 ≥ 0.001. (***) P < 0.001.
