CDK-dependent nuclear localization of B-cyclin Clb1 promotes FEAR activation during meiosis I in budding yeast.
Bottom Line: While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity.Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway.We discuss the significance of our results in relation to regulation of exit from meiosis I.
Affiliation: University of Warwick, Coventry, United Kingdom.
Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.
Mentions: Clb1 has been observed to be post-translationally modified and localized to the nucleus during meiosis I , . We first tested whether progression through anaphase I was required for post-translational modification and nuclear localisation of Clb1 during meiosis. We constructed Clb1-tagged strains containing the CDC20 (responsible for activating the anaphase promoting complex) either under the endogenous promoter or the mitosis-specific promoter PCLB2. We induced cells to enter meiosis by transferring them to sporulation medium (SPM). While wild type cells underwent two rounds of nuclear division and formed 60% tetrads after 12 h in SPM, the PCLB2CDC20 cells remained mononucleate during the entire experiment (Figure S1). After 6 h into SPM, Clb1 was modified and localized to the nucleus in both wild type and PCLB2CDC20 cells (Figure 1A and 1B). This indicates that progression through anaphase I is not required for Clb1 modification and nuclear localization. However, in contrast to wild type cells, Clb1 modification and nuclear localization was sustained in PCLB2CDC20 cells (Figure 1A and 1B). This suggests that passage through anaphase I reverses the modification and nuclear localization of Clb1. One possibility is that activation of FEAR during anaphase I might abolish nuclear localization and modification of Clb1. Consistent with this possibility, nuclear localization of Clb1 during meiosis is retained in FEAR mutants . If FEAR was required for delocalization of Clb1, then its premature activation should prevent Clb1 nuclear localization. Since PCLB2CDC55 cells release Cdc14 prematurely from the nucleolus but undergo other cell cycle events like securin degradation, cohesin cleavage and Clb3 expression (specific to meiosis II) normally, we monitored Clb1 nuclear localization and modification in PCLB2CDC55 cells . Clb1 nuclear localisation and modification was maintained in both PCLB2CDC55 and PCLB2CDC55 PCLB2CDC20 strains (Figure 1A and 1B) suggesting that Clb1 nuclear localization and modification is not sensitive to premature FEAR activation .