Additional file 5.

Figure S4. rpn1-D517A mutants exhibit a selective defect in protein degradation. (A) Mutant rpn1-D517A cells degrade the Dsk2 substrate Kre22 with normal kinetics. Strains carrying a plasmid that expressed GST-Kre22 from the GAL1 promoter were grown in raffinose medium and then induced with 2% galactose for three hours. Dextrose was added at time zero to extinguish expression and samples were taken at the indicated time points for immunoblot analysis. Below, cells were plated in five-fold serial dilutions onto either glucosoe or galactose containing medium and monitored for growth after two to three days at 30°C. (B) Mutant rpn1-D517A cells degrade the Ufd1/Rad23/Dsk2 substrate CPY* with normal kinetics in a cycloheximide chase. Cycloheximide was added at time zero and samples were taken at the indicated time points for immunoblot analysis. Equal loading of extracts was confirmed by blotting with an anti-tubulin antibody (lower panel). The quantification of these blots is shown in the right panel. This is a replicate of the experiment shown in Figure 5A. (C) Ufo1 is stabilized in rpn1-D517A and ddi1Δ mutants. Wild type and mutant cells carrying a plasmid that expressed GST-Ufo1 from the GAL1 promoter were grown in raffinose medium and then induced with 2% galactose for 14 h. Dextrose was added at T0 to extinguish expression and samples were taken at the indicated time points and analyzed by immuoblot. Quantification is shown in the right-hand panel. This is a replicate of the experiment shown in Figure 5B

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Gomez et al. BMC Biology 2011 9:33   doi:10.1186/1741-7007-9-33