Figure 3. A minimal level of ubiquitylation is required for Cdc48/Rad23-independent degradation of a UFD substrate with peptide extension.

(A) Cycloheximide-chase analysis of Ub-GFP, Ub-GFP-15aa and Ub-GFP-20aa turnover in wild-type or in a uba1-ts 26 mutant yeast strain. Quantification of GFP levels of three independent experiments with error bars representing standard error of the mean is shown. (B) Western blot analysis of Ni-NTA pulldown from wild-type or uba1-ts 26 yeast cells co-expressing 6His-Ub and Ub-GFP, Ub-GFP-15aa or Ub-GFP-20aa using GFP antibody. (C) Western blot analysis showing steady-state levels of Ub^K29,48R-GFP, Ub^K29,48R-GFP-15aa and Ub^K29,48R-GFP-20aa in a wild-type yeast strain in the absence or presence of proteasome inhibitor MG132 using GFP antibody. β-actin was detected as loading control. (D) FACS analysis of GFP fluorescence intensities of wild-type yeast cells expressing Ub^K29,48R-GFP, Ub^K29,48R-GFP-15aa and Ub^K29,48R-GFP-20aa. (E) Cycloheximide-chase analysis of Ub^K0-GFP, Ub^K0-GFP-15aa and Ub^K0-GFP-20aa turnover in wild-type yeast. Turnover of Ub^K0-GFP-20aa was analyzed in the absence and presence of MG132. Quantification of GFP levels of three independent experiments with error bars representing standard error of the mean is shown. Solid lines, untreated; dashed lines, MG132-treated.