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. 2016 Jul 6;113(29):E4151–E4160. doi: 10.1073/pnas.1605951113

Fig. 2.

Fig. 2.

Ltn1-mediated ubiquitylation of NSPs occurs on ribosomes. (A) Evidence for NSP ubiquitylation on ribosomes. Five hundred nanograms of fGFP-K12-encoding mRNA was translated for 30 min in 50 μL of WT or Δltn1 extracts. Reaction products were separated by centrifugation in 15–40% (wt/vol) sucrose gradient; 20% of each fraction was analyzed by immunoblot against ubiquitin (Ub), flag, or the 60S ribosomal protein, Rpl3 (“Input”). The remaining material from each fraction was used for denaturing flag IPs (as in Fig. 1B) and analyzed by immunoblot against Ub or flag. (B) Five hundred nanograms of fGFP-K12–encoding mRNA was translated for 30 min in 50 μL of WT extract or in Δltn1 extract supplemented or not with 40 nM recombinant yeast Ltn1 (WT or Ltn1-ΔR mutant, as indicated). Reaction products were separated by centrifugation in 15–40% (wt/vol) sucrose gradient. Fractions were analyzed by immunoblot against flag or Rpl3. (C) Evidence for tRNA-conjugated fGFP-K12 in ribosomal pellets. Two hundred nanograms of mRNA encoding fGFP-s or fGFP-K12 proteins were incubated with WT or Δltn1 extracts (20 μL) for 30 min. Reaction products were separated by centrifugation on sucrose cushion; 40% of recovered supernatant (“S”) and 100% of resuspended pellet (“P”) were separated by electrophoresis in Bis-Tris gel containing Mes buffer, pH 6.8 (conditions under which peptidyl-tRNA is more stable) and analyzed by immunoblot against flag or Rpl3. Dashed lines indicate that lanes were removed from the original image. (D) Ribosome-stalled but not free NSPs are modified by Ltn1. Three hundred nanograms of fGFP-K12–encoding mRNA were translated in 30 μL of Δltn1 extract for 30 min, followed by cycloheximide addition. To one set of reactions (lanes 1 and 2), fresh WT or Δltn1 extract was added (1:1) and incubated for another 30 min before denaturing flag IP. In another set of reactions, products of the fGFP-K12 translation reaction in Δltn1 extract were separated by centrifugation on sucrose cushion. Supernatant and the resuspended ribosome-containing pellet were then incubated with fresh WT or Δltn1 extracts in the presence of cycloheximide, for 30 min before denaturing flag IP. The same amount of second extract was added to supernatant and pellet fractions. All reactions (lanes 1–6) were equalized for ∼20% (wt/vol) sucrose concentration. The IPed material was analyzed by immunoblot against Ub or flag.