Figure 3

Kre22 and YLL030C are degraded by a Rad23/Dsk2-dependent pathway. (A-B) Degradation of Kre22 and YLL030C is impaired in cells lacking RAD23 and DSK2. Wild-type (Y8835) and mutant cells containing a GAL1 promoter regulated GST and His6 tagged Kre22 or YLL030C were first grown in raffinose-containing media. Protein expression was then induced by the addition of galactose. Samples were taken after expression shutoff at intervals, processed for immunoprecipitation with GST beads, and analyzed by Western blotting using anti-His6 antibody. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in all of the expression shutoff experiments (lower panels). (C-D) Degradation of Kre22 and YLL030C is Ufd2-independent. The stability of Kre22 or YLL030C in wild-type or ufd2Δ mutant cells was determined by an expression shut-off assay as described in A. (E) Kre22 and YLL030C are not glycosylated. GST-tagged Kre22 or YLL030C was expressed in wild-type cells and recovered by immunoprecipitation. The immunoprecipitates were mock treated (-) or digested (+) with Endoglycosidase H (Endo H), resolved by SDS-PAGE, and visualized by immunoblotting. If the proteins are N-glycosylated, the bands are expected to migrate faster on the gel after Endo H treatment. A positive control for EndoH treatment of RTA is also included on the right [26]. (F-G) Degradation of Kre22 and YLL030C appended with other epitope tags is compromised in cells lacking RAD23 and DSK2. The stability of Kre22 or YLL030C tagged with C-terminal Ha epitope and the IgG-binding domain from protein A in wild-type and rad23Δ dsk2Δ mutant cells was determined as described in A, except the immunoprecipitation was done with IgG sepharose and the blots were probed with anti-Ha antibody.