Figure 6.

Peak-equivalent undegradable Clb2 yields fully active Clb2–Cdk. (A) Strains from Figure 5A and B, MET-CDC20 CLB2^WT-YFP (BD128-41A) control were pulsed with deoxycorticosterone, released from metaphase block, and sampled for protein concentration and YFP-associated in-vitro HH1 kinase activity. [Clb2^WT–YFP] and [Clb2kd–YFP] levels measured in units of peak [Clb2–YFP]. Kinase activity is standardized to the metaphase-arrested CLB2^WT-YFP level. (B) Mcm2–GFP nuclear localization as an in vivo Clb2–CDK activity marker. Cells bearing Mcm2–GFP and Htb2–mCherry (BD142a-13C) were released from metaphase arrest (MET-CDC20) with or without a Clb2kd–YFP pulse. Coefficient of variation (CV) of Mcm2–GFP signal in individual cells was used as a proxy for Mcm2 nuclear localization (higher values represent higher nuclear concentration, see Materials and methods section). Representative traces from cells with indicated Clb2kd–YFP concentration (numbers in the upper right corner) are shown (n=35). Blue bars=nuclear separation (anaphase); green bars=budding.