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.
Related in: MedlinePlus
Mentions: To determine the functional significance of Clb1 nuclear localisation, the protein was tagged with ectopic Nuclear Localisation Sequences (NLS) or Nuclear Export Sequences (NES), and 6 copies of HA epitope. In each case, two sequences were added in tandem to overcome any endogenous localisation signals (Figure 5A). The tandem NLS tag leads to nuclear localisation of Clb1 during mitosis (Figure 5C). In contrast the tandem NES severely reduced the nuclear localisation of Clb1 during meiosis I (Figure 5B, D). Crucially, the fusion of NLS/NES had little or no effect on Clb1-CDK activity (Figure 5E, Figure S4B). The clb1Δ strains sporulate poorly, producing only 15% tetrads in comparison to 70% for wild-type cells (Figure S4C). Expression of NES/NLS-tagged Clb1 does not affect sporulation efficiency in otherwise wild-type cells (Figure S4C), suggesting that the localization mutants are not inactivated, and that nuclear localisation of Clb1 is not essential for meiotic nuclear divisions. Spore viability was also largely unaffected by the mutations: CLB1-ha and CLB1-NES-ha strains have viabilities of 97%, whereas the CLB1-NLS-ha strain has a spore viability of 85%.