Figure 6

^Gst 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 ^FlagSCF^Cdc4 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 Ubp15^C354A 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.