The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase binding in 40S ribosome assembly

The acp modification activity of Tsr3 is required for its release. (A) Doubling times of ΔTsr3 cells supplemented with a plasmid encoding WT Tsr3 (Tsr3), an empty vector (ΔTsr3), or a plasmid encoding Tsr3_W114A (Tsr3_W114A). Significance was tested using one-way ANOVA (Sidak's multiple comparisons test). (****) P_adj < 0.0001; n ≥ 6. (B) Doubling times of BY4741 cells supplemented with plasmids encoding WT Tsr3 or Tsr3_W114A. Significance was tested using an unpaired t-test. (***) P < 0.001; n ≥ 5. (C) Doubling times of ΔTsr3 cells supplemented with plasmids encoding WT Tsr3 (WT), Tsr3_R131A (R131A), Tsr3_R60A, K65A (RK), Tsr3_ R60A, K65A, R131A (RK/R131A), Tsr3_W114A (W114A), Tsr3_W114A/R131A (W114A/R131A), Tsr3_W114A/R60A, K65A (W114A/RK), or Tsr3_ W114A/R60A, K65A/R131A (W114A/RK/R131A). Significance was tested using one-way ANOVA (Sidak's multiple comparisons test). (****) P_adj < 0.0001; n ≥ 4. (D) Western blots for HA-tagged Tsr3, Tsr1, and Pno1 from 10% to 50% sucrose gradients from ΔTsr3 cells supplemented with HA-Tsr3 or HA-Tsr3_W114A plasmid. 0.8% formaldehyde was added to the cells when harvesting to stabilize the binding of Tsr3 on ribosomes. Asterisk denotes the upper band corresponding to Pno1.