FIG. 7.

Ufo1 interacts with Ddi1. (A) Two-hybrid interaction between three independent cotransformants of Ufo1 fused to the Gal4 activating domain (AD) and Ddi1 fused to the Gal4 DNA-binding domain (BD). Activation of the HIS3 and ADE2 reporter genes from the GAL promoter promotes growth on selective plates. Parallel cotransformants of Ufo1-AD with Rad23- or Dsk2-BD fusion proteins did not grow on the selective plates (not shown). (B) Interaction of Ufo1 with Ddi1 requires the Ufo1 UIMs. Extracts from cells producing full-length ^GFPUfo1 or ^GFPUfo1ΔUIM were incubated with extracts from wild-type (DDI1) or Δddi1 cells and immunoprecipitated with anti-Ddi1 antiserum. The Western blots were analyzed with anti-GFP antiserum. Control anti-GFP immunoprecipitations on 3% input indicate the presence of both full-length ^GFPUfo1 (U) and ^GFPUfo1ΔUIM (Δ). The Western blot was then incubated with anti-Ddi1 antiserum to detect the presence of Ddi1, which runs very close to the immunoglobulin G chains. (C) The UbL of Ddi1 is essential for interaction with Ufo1; the UbA domain is also very important. Extract from cells producing full-length ^GFPUfo1 was incubated with extracts from wild-type (DDI1), Δddi1, or Δddi1 cells expressing Ddi1ΔUbL or Ddi1ΔUbA as a fusion protein to the LexA activating domain. The mixtures were immunoprecipitated with anti-Ddi1 antiserum, and the Western blots were analyzed with anti-GFP antiserum. Control anti-GFP immunoprecipitations on 3% input indicate the presence of ^GFPUfo1; the presence of the different Ddi1 proteins was detected by Western blotting with anti-Ddi1 antiserum. (D) Percentage of arrested wild-type and Δddi1 cells expressing UFO1 or UFO1ΔUIM. Long buds were observed in cultures of Δddi1 cells expressing full-length UFO1. Compare these results with those for wild-type cells expressing UFO1 in Fig. 3C. (E) Ddi1 is not a degradation substrate of Ufo1. The half-life of Ddi1 was determined by pulse-chase and immunoprecipitation experiments in wild-type and Δufo1 cells.