Figure 1. Purification of trapped pre‐40S ribosomes and Hrr25 kinase activity.

• A
  N‐terminally tagged Protein A (pA)‐Nob1 is functional, but requires endonuclease activity for viability. The PGAL1‐NOB1 strain was transformed with indicated plasmids and spotted in 10‐fold dilutions on selective and repressive glucose‐containing plates and grown at 25°C for 4 days.
• B
  RNA composition of 80S ribosomes and purified Protein A (pA)‐tagged Nob1‐D15N particles. RNA was separated by agarose gel electrophoresis and stained with GelRed.
• C
  Protein composition of 80S ribosomes and purified Protein A (pA)‐tagged Nob1‐D15N particles. Proteins were separated by SDS–PAGE and visualized by silver staining. Indicated protein bands were identified by mass spectrometry.
• D, E
  The kinase activity of Hrr25 is essential for cell viability and is required for cytoplasmic release and recycling of Enp1 and Tsr1. (D) The PGAL1‐HRR25 strain was transformed with indicated plasmids and spotted in 10‐fold dilutions on selective and repressive glucose‐containing plates and grown at indicated temperatures for 3–7 days. (E) Yeast strains expressing endogenous Enp1‐GFP or Tsr1‐GFP were transformed with plasmids carrying either a galactose‐inducible wild‐type HRR25 gene or a dominant‐negative hrr25‐K38A kinase dead mutant gene. Strains were then grown on galactose‐containing medium at 25°C, and GFP constructs were visualized by fluorescence microscopy.

Data information: For a detailed description of the strains and plasmids, see Appendix Tables S1 and S2.