Mechanism of the G2/M transition of the budding yeast cell cycle

我们研究了发芽酵母细胞周期中M期的两个项目：1.纺锤体检查点控制中磷酸酶2A的B型亚基CDC55的作用。在微管解聚药物诺可达唑的存在下，M期抑制物Pds1和M期细胞周期蛋白Clb2在cdc55突变体中的降解类似于bub2突变体。由于Bub2是TEM1-GTPase的一个缺口，负调控MAN(有丝分裂退出网络)途径，我们研究了MAN途径的下游成分Amn1是否表达，但没有表达。然后，我们检测了Net1调节MEN途径的激活物--CDC14-磷酸酶是否被修饰。Net1蛋白在cdc55突变体中似乎被去磷酸化，这表明CDc55间接调节Net1，并参与从核仁中释放CDc14以激活MAN途径。2.相扑连接酶的结构域分析作为芽生酵母的相扑连接酶之一，Ull1/Siz1以细胞周期依赖的方式改变其在细胞质中从细胞核到颈部的定位，并识别其各种底物。我们通过构建不同的缺失突变体对这个由904个氨基酸组成的相扑连接酶进行了结构域分析，并检测了每种酶的活性和定位。除了环状结构域外，连接酶活性还需要一个新的区域，称为Pinit结构域。出乎意料的是，连接酶的活性并不需要通过双杂交实验获得的被称为SXS-Motif的强大的相扑相互作用结构域。此外，缺少C-末端一半的Ull1蛋白是稳定的，并且总是定位于细胞核。N端的SAP结构域参与了细胞核的定位，这是一个可能的DNA结合域。因此，环状结构域和Pinit结构域是该相扑连接酶的核心区域，其他结构域通过其定位和蛋白质稳定性参与调节。

We investigated two projects on the M-phase of the budding yeast cell cycle.1.Role of Cdc55, B-type subunit of phosphatase 2A in spindle checkpoint control.In the presence of nocodazole, a depolymerizing drug of microtubules, Pds1, an M-phase inhibitor and Clb2, M-phase cyclin, were degraded in the cdc55 mutant similarly to the bub2 mutant. Since Bub2 is a GAP of Tem1-GTPase and negatively regulates MEN (Mitotic Exit Network) pathway, we investigated whether Amn1, a downstream component of the MEN pathway was expressed, but it was not expressed. Then, we examined whether Net1 regulating Cdc14-phosphatase, an activator of the MEN pathway was modified. The Net1 protein appeared to be dephosphorylated in the cdc55 mutant, suggesting that Cdc55 regulates Net1 indirectly and is involved in release of the Cdc14 from the nucleolus to activate the MEN pathway.2.Domain analysis of the SUMO ligaseUll1/Siz1, one of the SUMO ligases of the budding yeast changes its localization from the nucleus to the neck region in the cytoplasm in a cell-cycle dependent way, and recognizes its various substrates. We performed the domain analysis of this SUMO ligase composing of 904 amino acids by constructing various deletion mutants and examined each enzymatic activity and localization. A new region called as PINIT domain besides the RING-like domain was necessary for the ligase activity. Unexpectedly, a strong SUMO-interacting domain by the two-hybrid assay, called as SXS-motif, was not needed for the ligase activity. Furthermore, the Ull1 protein lacking the C-terminal half was stable and always localized to the nucleus. The N-terminal SAP-domain, a putative DNA-binding domain, was involved in the nuclear localization. Thus, the RING-like and PINIT domains are core regions of this SUMO ligase and the other domains are involved in the regulation through its localization and protein stability.