Tsr3 ensures that Rio2 release is irreversible. (A) Normalized doubling times of yeast cells encoding WT Rio2/Rio2_K105E/Rio2_Δloop, WT S20/S20_RK/S20_Δloop, WT S15/S15_YRR/S15_RK in the presence and absence of Tsr3. WT or mutant proteins were expressed from plasmids in strains where the endogenous protein is under the GAL-promoter and depleted by growth in glucose. Shown in the figure are doubling times of mutants normalized to corresponding WT protein in the same Tsr3 background. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 6. (B) Scheme showing the order of steps required for the formation of 80S-like ribosomes (Huang et al. 2020) and the effects of the mutants in A on these steps. (C) A composite structure of yeast pre-40S (PDBID 6FAI) and mature 40S (PDBID 3JAQ) obtained by overlay on Rps18. Mutations in Rio2, Rps15, and Rps20 are highlighted in sphere and colored in the same color as the corresponding text. The loop containing the residues deleted in Rio2_Δloop is unresolved in all structures and the flanking residues, R129 and S145, are highlighted in sphere in cyan. l31 from mature 40S, which is not resolved in pre-40S, is highlighted in green. (D) Normalized doubling times of yeast cells encoding WT Rio2/Rio2_K105E/Rio2_Δloop, WT S20/S20_RK/S20_Δloop, WT S15/S15_YRR/S15_RK in the presence of normal levels of Rio2 (expressed under the CYC1 or native promoter) or high levels of Rio2 (expressed under the TEF promoter). WT or mutant proteins were expressed from plasmids in strains where the endogenous protein is under the GAL-promoter and depleted by growth in glucose. Shown in the figure are doubling times of mutants normalized to corresponding WT protein in the same Rio2 background. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 9. (E) Doubling times of yeast cells encoding Rio2 or Rio2_RQQR expressed from plasmids with the lower expressing CYC1 or the higher expressing TEF promoter in the presence and absence of Tsr3. Significance was tested using a two-way ANOVA test. (****) Padj < 0.0001; n ≥ 6. WT or mutant Rio2 was expressed from plasmids in a strain where the endogenous Rio2 is under the GAL-promoter and depleted by growth in glucose. (F) Normalized doubling times of BY4741 (+Tsr3) and ΔTsr3 (−Tsr3) cells grown at 30°C (normal temperature) or 26°C (low temperature), respectively. Significance was tested using a two-way ANOVA test. (***) Padj < 0.001; n ≥ 6. (G) (Left) Western blots for Rio2, Rio1, Tsr1, Pno1, and Dim1 from gradients in Figure 1C. Asterisk notes the upper band corresponding to Pno1. (Middle) Quantification of the fraction of Rio2 in 80S-like ribosomes. Significance was tested using an unpaired t-test. (*) P < 0.05; n = 3. (Right) Quantification of fraction of Rio1 in free 40S or 80S fractions, respectively. n = 2.
