Tsr3 establishes the hierarchy of Rio kinase function. (A) 10%–50% sucrose gradients of lysates from cells of WT BY4741 (+Tsr3, left) or ΔTsr3 (−Tsr3, middle). Shown below the absorbance profiles at 254 nm are northern blots of 20S, 18S, and 25S rRNA. (Right) Quantifications of the data on the left. Fraction of 20S rRNA in 80S (fraction 6–7) and polysome (fraction 8–12) are quantified. n = 2. (B) Normalized doubling times of ΔTsr3, Gal:Pno1 cells supplemented with WT Tsr3 or empty vector and Pno1 or Pno1_KKKF plasmids and grown in glucose. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 13. (C) Normalized doubling times of ΔTsr3, Gal:Rps15 cells supplemented with WT Tsr3 or Tsr3_W114A and WT Rps15 or Rps15_YRR or Rps15_RK plasmids and grown in glucose. Cells with Tsr3_W114A and Rps15_YRR plasmids do not grow and are therefore shown as a box filled with diagonal lines. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n = 6. (D) Normalized doubling times of ΔTsr3, Gal:Fap7 cells supplemented with WT Tsr3 or Tsr3_W114A and WT Fap7 or Fap7_G19S or Fap7_K20R plasmids and grown in glucose. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 15.
