The ubiquitin-binding domain (CUE) of Def1 is required for ribosome binding. (A) Schematic of the Def1 protein with the CUE and polyQ domains illustrated. (B) Def1(1–380)-GFP migration in polysome profiles. Western blotting was performed using anti-GFP antibodies. (C) Polysome fractions of wild-type, CUEmut1, and CUEmut1,2 versions of Def1. The amino acid substitutions are described in Supplemental Table 1 and Wilson et al. (2013b). Western blotting was performed using antiserum to Def1 and Rpl3-s. (D) Hygromycin B (HygB) sensitivity growth assay. As in Figure 2A, except that two concentrations of the drug were used. (E) HIS3 codon optimality assay. The assay was conducted as described in Figure 4C. 0% (black bars) and 100% (gray bars) refer to the codon optimality of the HIS3 construct. Biological triplicates were analyzed (n = 3). (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001.
