rpn1-D517A mutants exhibit a selective defect in protein degradation. (A) 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 removed at the indicated time points for analysis by immunoblotting. Equal loading of extracts was confirmed by blotting with an anti-tubulin antibody (lower panel). The quantification of these blots is shown. (B) 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 for analysis by immunoblotting. Quantification is shown. (C) rpn1-D517A and ddi1Δ do not tolerate over expression of Ufo1. The indicated strains containing a plasmid that expressed GST-Ufo1 under the control of a galactose inducible promoter were grown on medium containing either glucose (SD, expression OFF) or galactose (SGalactose, expression ON). After two to three days, the plates were scored for growth. (D) Sensitivity of rpn1-D517A to GST-Ufo1 over-expression was specific and was not shown by other rpn1 alleles.