Kirdök İdil, Çaydaşi Ayşe Kosa
Department of Molecular Biology and Genetics, College of Sciences, Koç University, İstanbul, Turkiye.
Turk J Biol. 2024 Sep 17;48(5):308-318. doi: 10.55730/1300-0152.2707. eCollection 2024.
BACKGROUND/AIM: The conserved phosphatase Cdc14 facilitates mitotic exit in budding yeast by counteracting mitotic cyclin-dependent kinase activity. Cdc14 is kept in the nucleolus until anaphase onset, when it is released transiently into the nucleoplasm. In late anaphase, Cdc14 is fully released into the cytoplasm upon activation of the mitotic exit network (MEN) to trigger mitotic exit. Cdc14 also localizes to yeast spindle pole bodies (SPBs) in anaphase and dephosphorylates key targets residing on SPBs to allow SPB duplication and prime the MEN. Protein phosphatase 1 (Glc7) with regulatory subunit Bud14 is another phosphatase that plays a key role in the spatiotemporal control of mitotic exit. In this study, we investigated the regulation of Cdc14 localization by Bud14-Glc7.
We used fluorescence microscopy to analyze Cdc14 localization in wildtype and knockout cells () as well as in cells expressing a mutant allele of () that cannot bind Glc7. We also utilized a yeast two-hybrid (Y2H) system to examine the interaction of Bud14 with Cdc14.
We found that Cdc14 remains at the SPBs longer in and compared to wildtype cells. This effect is limited to the SPB that has migrated to the daughter cell (dSPB). Cdc14 localizes to both SPBs shortly after anaphase onset. In mid-to-late anaphase, levels of Cdc14 increase at the dSPB in both wildtype and cells. With mitotic exit, Cdc14 disappears from the dSPB in wildtype cells but not in cells. Accordingly, 50% of cells in G1 have Cdc14 at their SPBs. We also found that Cdc14 localization at the dSPB was largely, but not entirely, dependent on Bfa1 in cells. Furthermore, Bud14 interacted with Cdc14 in the Y2H system.
Our results suggest that Glc7-Bud14 is part of a mechanism that promotes Cdc14 disappearance from the dSPB.
背景/目的：保守的磷酸酶Cdc14通过抵消有丝分裂周期蛋白依赖性激酶活性来促进芽殖酵母中的有丝分裂退出。Cdc14一直保留在核仁中直到后期开始，此时它会短暂释放到核质中。在后期晚期，Cdc14在有丝分裂退出网络（MEN）激活后完全释放到细胞质中以触发有丝分裂退出。Cdc14在后期也定位于酵母纺锤体极体（SPB），并使位于SPB上的关键靶点去磷酸化，以允许SPB复制并启动MEN。具有调节亚基Bud14的蛋白磷酸酶1（Glc7）是另一种在有丝分裂退出的时空控制中起关键作用的磷酸酶。在本研究中，我们研究了Bud14-Glc7对Cdc14定位的调控。
我们使用荧光显微镜分析野生型和基因敲除细胞（）以及表达不能结合Glc7的（）突变等位基因的细胞中Cdc14的定位。我们还利用酵母双杂交（Y2H）系统检测Bud14与Cdc14的相互作用。
我们发现，与野生型细胞相比，在和细胞中Cdc14在SPB上停留的时间更长。这种效应仅限于已迁移到子细胞的SPB（dSPB）。后期开始后不久，Cdc14定位于两个SPB。在后期中期到后期晚期，野生型和细胞中dSPB上的Cdc14水平均升高。随着有丝分裂退出，野生型细胞中Cdc14从dSPB消失，但在细胞中没有。因此，50%的G1期细胞在其SPB上有Cdc14。我们还发现，在细胞中，dSPB上的Cdc14定位在很大程度上但并非完全依赖于Bfa1。此外，在Y2H系统中Bud14与Cdc14相互作用。
我们的结果表明，Glc7-Bud14是促进Cdc14从dSPB消失的机制的一部分。
