Dim1 stabilizes h24 at the 80S-like ribosome interface. (A) Local classification followed by refinement and local B-factor sharpening in Relion 3.0 revealed a bilobed density at the 60S-pre-40S interface that matches the Dim1 structure (blue). The position of Dim1 in an earlier cytoplasmic pre-40S intermediate (red, Dim1 from PDB ID 6RBD, Mitterer et al. 2019) is also shown, demonstrating its repositioning at the interface. (B) The Dim1-IKN residues (orange) face h24. (C) In pre-40S subunits lacking 60S (PDB ID 6RBD, Mitterer et al. 2019), all altered amino acids are solvent-exposed. (D) Dim1-IKN impairs subunit joining. Whole cell extracts depleted of endogenous Dim1 and Fap7 (Gal::Dim1;Gal::Fap7) for 16–20 h and supplemented with plasmids encoding wild-type or mutant Dim1 and Fap7_K20R were fractionated on 10%–50% sucrose gradients. The sedimentation of pre-40S ribosomes containing 20S rRNA was probed by northern blot analysis using a probe directed against the unique extension of 20S pre-rRNA. Mature 18S and 25S were probed with oligos directed against the mature rRNA. Note that the 18S probe also picks up 20S rRNA in strains where preribosomes accumulate strongly (like the Fap7 mutants here). 80S-like ribosomes sediment in fractions 6–7 and contain 20S and 25S rRNA, while pre-40S sediment in fractions 3–4 (left). Dim1 from total cell lysates was probed by western blot to demonstrate equal expression wild-type Dim1 and Dim1_IKN. Dim1 from total cell lysates was probed by western blot to demonstrate equal expression of wild-type Dim1 and Dim1_IKN from two biological replicates (right). (E) Fraction of 20S in 80S-like ribosomes (fractions 6–7) compared with total 20S was calculated from data in D and replicates. Data were normalized to wild-type Dim1. Three biological replicates were obtained. Error bars indicate the standard error of the mean (SEM).
