Ubiquitylation of eS7a by Not4 recruits Def1 to ribosomes. (A) Western blot showing Not1 depletion using the auxin-induced degron system. Antibodies to Not1 and Dhh1 (loading control) were used. Two isolates of the Not1-AID strain are shown. Cells were treated with auxin for 2 h, where indicated. (B) Lysates of untreated and auxin-treated Not1-AID cells were subjected to polysome analysis. Blots were probed with anti-Def1 and Rpl3-s antibodies. (C) Analysis of Def1 binding to polysomes in a not4Δring mutant. Blots were probed with anti-Def1 and Rpl3-s antibodies. (D) Lysates from strains expressing wild-type eS7a or a version with lysine to arginine substitution in the Not4-dependent ubiquitylation sites (eS7KR mut) (Buschauer et al. 2020) were fractionated on sucrose gradients.
