The binding of Rio2 displaces Bud23 from pre-40S particles. (A) The addition of recombinant Rio2 (Rio2-HA) to affinity-purified pre-40S particles depleted of Rio2 induces the release of Bud23 as shown by western blotting for specific factors on pre-40S particles that copurified with Tsr1–TAP. The Coomassie-stained gel is shown for reference. The absence or presence of Rio2-HA is indicated by a hollow or filled circle, respectively. The reactions were incubated for 10 min at 20°C and performed in the absence or presence of 0.5 mM ATP and AMP–PNP. The reactions were subsequently overlayed on sucrose cushions and subjected to ultracentrifugation. The pellet (P) and supernatant (S) fractions, respectively, containing preribosome-bound and extraribosomal proteins, were collected, TCA precipitated, and separated on SDS-PAGE gels. See the Materials and Methods section for a full description of experimental details. In the Coomassie stained gel, asterisks (*) denote residual uncleaved recombinant MBP-Rio2-HA that remain in the supernatant fraction. (B) Bud23 does not accumulate on preribosomes containing Rio2–D253A as shown by western blotting for specific factors on pre-40S particles that copurify with Enp1–FTP. Strain AJY4733 was transformed with an empty vector (pAJ5103) or vectors encoding RIO2 (pAJ4657) or rio2–D253A (pAJ4698). Cells were cultured in SD Leu- media to early exponential phase, then cultured for 2 h in the presence of 0.5 mM auxin and 1 µM β-estradiol.
