Figure 4. Ssb–ribosome interaction is modulated by RAC and involves rRNA expansion segments.

(a) Ribosome-binding of Ssb is reduced when RAC is absent, non-functional, or if ATP hydrolysis is prevented by the Ssb1-K73A mutation. Total cell extract (tot) was separated into a cytosolic fraction (cyt) and a ribosomal pellet (ribo) under low-salt (LS) or high-salt (HS) conditions. Immunoblots were decorated with αSsb, αZuo1, αSse1 (cytosolic marker) or αRpl35 (ribosomal marker). (b) The ribosome-bound fraction of Ssb in extracts derived from RAC-H128Q, Δzuo1Δssz1 or Ssb1-K73A strains is reduced. Quantification of the ribosome-bound fraction of Ssb under low-salt conditions is based at least on 3 independent experiments (Supplementary Fig. 8), error bars represent the s.d. (c,d) The contact between Ssb1 and Rpl35 or Rpl39 is strongly reduced if RAC is absent, non-functional, or if ATP hydrolysis is prevented by the Ssb1-K73A mutation. Crosslinking was performed in cell extracts of strains expressing Ssb1* (Supplementary Table 1) and (c) wild type RAC, RAC-H128Q, or carrying the Δzuo1Δssz1 mutation or (d) carrying the Ssb1*-K73A mutation. Immunoblots were decorated with antibodies directed against Rpl35 or Rpl39. Crosslink products between Ssb1* and ribosomal proteins (Ssb1*-XL) are indicated with red asterisks. (e) Comparison of crosslinking efficiencies between Ssb1* and Rpl35 or Rpl39 in cell extracts derived from Ssb1* strains expressing wild type RAC, RAC-H128Q, carrying the Δzuo1Δssz1 mutation, or expressing Ssb1*-K73A. The band intensities of crosslink products between Ssb1* and Rpl35 (Ssb1*-Rpl35) or Rpl39 (Ssb1*-Rpl39) were determined in at least 3 independent experiments. The intensity of Ssb1*-Rpl35 and Ssb1*-Rpl39 crosslinks in the Ssb1* strain was set to 100%. Error bars represent the s.d. (f) CRAC analysis on ProtA-TEV-His₆-Ssb1 (red) and negative control (black). The number of hits representing the number of times a nucleotide was mapped onto the 37S rDNA reference sequence of S. cerevisiae is shown. (g) Schematic representation of the expansion segments ES41, ES24 and ES39 of the 25S rRNA crosslinked to Ssb1 (ref. ⁸⁵). Nucleotides found in all hits are highlighted and nucleotides representing the nucleotides frequently mutated or deleted in the CRAC experiments are highlighted in dark colour.