The dissociation of Tsr3 is required for Rio1 binding to pre-40S ribosomes. (A) Northern blots of 10%–50% sucrose gradients from ΔTsr3 cells expressing plasmid-encoded wild-type (WT) HA-Tsr3 (left) or HA-Tsr3_W114A (middle) proteins. The positions where 40S and 80S ribosomes sediment are indicated. (Right) Quantifications of the data on the left indicate the efficiency of formation of 80S-like ribosome formation. Data are shown as mean with standard deviation. Significance was tested using an unpaired t-test. (*) P < 0.05; n = 3. (B) (Left) 80S-like ribosomes were purified from Tsr1-TAP, ΔTsr3, Gal:Fap7 cells supplemented with HA-Tsr3 or HA-Tsr3_W114A, and probed for bound Rio1, Pno1, Nob1, Rps8 (S8), and Tsr1 from purified pre-40S via Tsr1-TAP from Tsr1-TAP, ΔTsr3, Gal:Fap7 cells supplemented with HA-Tsr3 or HA-Tsr3_W114A. Fap7 were depleted in glucose for more than 16 h. Rio1 signal was normalized to S8 (from the same gel) first before further normalizing Tsr3_W114A to WT Tsr3. Pno1 and Nob1 signal were normalized to Tsr1 first before further normalizing Tsr3_W114A to WT Tsr3. (Right) Quantification of normalized Rio1, Pno1, and Nob1 from the left and additional replicates. Two biological replicates were tested for each cell. (C) Doubling times of ΔTsr3, Gal:Pno1 cells supplemented with WT Tsr3 or Tsr3_W114A and Pno1 or Pno1_KKKF plasmids and grown in glucose. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 12. (D) Doubling times of ΔTsr3, Gal:Pno1 cells supplemented with WT Tsr3 or Tsr3_W114A and Pno1 or Pno1_T212N or Pno1_VD plasmids and grown in glucose. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n = 6.
