Cka2 inhibits synthesis of free polyubiquitin chains by Cdc34 constructs. (A). Effects of GstCka2 on Sic1 ubiquitination and synthesis of free polyubiquitin chains by Cdc34 proteins. 5 pmol of Cdc34 or tmCdc34 were phosphorylated at 25°C for 1 hour, as in 5B. The assays were then supplemented with 1 pmol E1, 660 pmol of ubiquitin, 2 pmol of FlagSCFCdc4 and 2 pmol of Sic1/Cln5/GstCdc28, where indicated, and incubated at 25°C for the times indicated, followed by α-Sic1 and α-Ub WBs. (B). Ectopic expression of Ubp14 or Ubp15C354A does not rescue growth of tm
CDC34 cka2Δ yeast. The heterozygous diploid RC174 was transformed with the indicated plasmids that overexpress Ubp14 (pUBP14) or catalytically inactive Ubp14 (pUBP14-C354A) from the ADH1 promoter. These transformed diploids as well as RC173 were patched onto sporulation media and incubated at 26°C for five days. Haploids with the indicated genotypes were selected by streaking the heterozygous diploids on haploid selection media with G418 and nourseothricin. Plates were incubated at 30°C for three days.