The interactions between Kre22 and Dsk2. (A) Kre22 binds Dsk2. Co-immunoprecipitation analysis of the interaction between GST-His6-tagged Kre22 and Dsk2-TAP was done as previously described . Briefly, proteins were extracted from cells expressing endogenous Dsk2-TAP and galactose-inducible GST-His6-tagged Kre22 and immunoprecipitated with either IgG or GST-beads as indicated on the left. Immunoprecipitates were resolved by SDS-PAGE and probed with indicated antibodies. The identity of the bands is indicated on the right. The antibodies for IP and immuno-blot (IB) are shown to the left of the panels. (B) Kre22 ubiquitylation involves Lys48-linked Ub chains. GST-His6-tagged Kre22 was co-transformed with myc-tagged wild-type or mutant Ub into wild-type cells. Kre22 was precipitated with GST beads and analyzed by immunoblotting first with anti-myc antibody and later anti-His6 antibody. Ubiquitylated and non-ubiquitylated Kre22 proteins are indicated on the left of the panels. (C) The deletion of KRE22 does not alter DSK2-induced toxicity. The spotting assay was done as described in Figure 1A. The vector plasmid or plasmid bearing DSK2 regulated by the galactose-inducible GAL1 promoter was separately introduced into wild-type or kre22Δ cells as indicated. Exponentially growing yeast cells were spotted onto glucose-containing SD-ura (expression off) and galactose-containing SG-ura (expression on) media in serial five-fold dilutions. The strains used and the plasmid for DSK2 overexpression are labeled on the left of the panels. (D) overexpression of KRE22, but not two other proteasomal substrates RTA and UbV76-V-βgal, suppressed DSK2-trigged slow growth in wild-type cells. The spotting assay was done as described above. The expression of these substrates is also regulated by the GAL1 promoter. The identities of these substrates and the plasmid for DSK2 overexpression are listed to the left of the panels.