Ddi1 homodimerization is required for rescue of pds1–128 temperature sensitivitity. (A) Ddi1 sequence with the UBL and UBA domains in green and orange, respectively. Residues 184–285 are displayed in plum and C276 and C300 in blue. (B) Yeast two hybrid assay reveals that residues 184–285 are important for homodimerization of Ddi1 but not for its interaction with Rad23. Production of blue color in x-gal containing medium signals for a positive interaction between two proteins . ddi1Δ184–285 interaction with DDI1 is highly reduced but ddi1Δ184–285 interaction with RAD23 is similar to that of wild type DDI1. (C) Western blots showing levels of different Ddi1 variants. Cells were grown on YEPR overnight, diluted 1:10 and expression of DDI1 constructs was induced from the GAL1 promoter by Galactose addition. These strains corresponded to the pds1–128 DDI1 strains assayed in D and expressed the indicated constructs exogenously from GAL1 in addition to expressing endogenous DDI1 (which is approximately 47 kD) from its native locus. The leftmost lane contains a vector control, not expressing DDI1 exogenously. The Western blot was probed with poly-clonal anti-sera against Ddi1 that recognized all of the mutant forms tested. Where wild type DDI1 is expressed from the GAL1 promoter, the upper band contains endogenous and exogenous Ddi1. (D) Rescue of pds1–128 temperature sensitivity. Serial dilutions of the indicated strains (corresponding to those tested in D) were spotted onto YEPD or YEPG plates and incubated for 24 hr at the indicated temperatures.