Figure 3.

Proteasome- and Ubiquitin-Dependent Def1 Processing

(A) Western blot of extracts from cells treated with (or without) MG132 prior to incubation with 4-NQO for the indicated times.

(B) Left: Def1 processing reconstituted using pure Def1 and 26S proteasome (see Figure S3A), and its sensitivity to MG132. Right: western blot comparing pr-Def1 with Def1 processed by pure proteasome.

(C) Western blot of extract from WT and rsp5-1 cells grown at 37°C for 2 hr before addition of 4-NQO for the indicated times. Lanes 4 and 5 are underloaded, giving the false impression that Def1 disappears at those time points. Normal processing was observed in rsp5-1 at the permissive temperature (data not shown).

(D) Western blots of Def1 ubiquitylation, reconstituted using highly purified proteins (Def1, ubiquitin, Uba1, Ubc5, Rsp5, and Ubp2; see Figure S3A).

(E) Western blots of ubiquitylated proteins (upper three panels) and extracts (input; lower three panels) from UV-irradiated WT and Def1 ubiquitylation site mutant (4xUbm) cells, respectively.

See also Figure S3.