FIGURE 5.

Rio2 is needed for Bud23 release from pre-40S particles. (A) The growth phenotypes of the wild-type (AJY4724) or RIO2-AID (AJY4733) strains as shown by 10-fold serial dilutions of cells on YPD media with or without 0.5 mM auxin and 1 µM β-estradiol. (B) Western blot of time-course of the depletion of Rio2-AID-HA using equivalent amounts of total protein from AJY4733 cells cultured to exponential phase, then collected prior to or after the addition of 0.5 mM auxin and 1 µM β-estradiol for the indicated time points. G6PDH was used as the loading control. The ratio Rio2-AID-HA signal to G6PDH signal for each time point relative to that of the untreated time point (−1) is shown below. (C,D) Bud23 accumulated in Rio2-depleted particles as shown by western blotting for specific factors on pre-40S particles that copurify with Enp1–FTP (C) and Tsr1–TAP (D). Asterisks (*) denote residual Rio2-AID-HA signal that is detected by the anti-Rio2 antibody. For C, strains AJY4724 (Rio2+) and AJY4733 (Rio2−) were cultured in YPD to early exponential phase, then treated with 0.5 mM auxin and 1 µM β-estradiol to deplete Rio2-AID-HA. For D, strains AJY4754 (Rio2+) and AJY4757 (Rio2−) were similarly cultured in YPD. (E) Proteomic composition of Tsr1-associated preribosomal particles as measured by semiquantitative mass spectrometry is shown as the average log₂ fold change (Rio2−/Rio2+ samples) for two independent biological replicates. Error bars denote the standard deviation. Individual factors are ordered by the timing of their approximate known association with 40S precursors. The log₂ fold change values were calculated as described in the Materials and Methods section. The supporting data are provided in Supplemental File 2.