Fig. 7.

Effect of mutations in Ltn1’s SRL-binding surface on ribosome binding and nascent chain ubiquitylation. Dashed lines indicate that lanes were removed from the original image. (A) Mutations in Ltn1’s SRL-binding surface affect ribosome binding as measured by sucrose gradient centrifugation. Flag-tagged Ltn1 WT or NTD mutants (R57A or T61A) were incubated with Δltn1 Neurospora extract and subjected to centrifugation on sucrose cushion, as described in SI Experimental Procedures. Input, supernatant, and pellet were analyzed by immunoblot against Flag (Ltn1), the 60S ribosomal protein Rpl3, or the cytosolic protein Pgk1. Ltn1-independent, anti-Flag antibody cross-reacting bands are marked with asterisks. (B) Mutations in Ltn1’s SRL-binding surface affect stalled nascent chain ubiquitylation. Stalling reporter-encoding mRNA was translated in Δltn1 extract supplemented or not with recombinant Ltn1 (WT, R57A mutant, or T61A mutant) and analyzed for ubiquitylation by denaturing IP (see also Fig. S5). Immunoblots were against Flag (Ltn1), Ubiquitin (Ub), and GFP (stalling reporter). (C) As in A, except that Ltn1 R57A and D68A mutants were analyzed. (D) As in B, except that Ltn1 R57A and D68A mutants were analyzed, the “no template mRNA” control is shown, and the WT extract was also used as positive control.