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

我们对芽殖酵母细胞周期的m期进行了两个项目的研究。磷酸酶2A b型亚基Cdc55在纺锤体检查点控制中的作用。在nocodazole（一种微管解聚药物）存在下，cdc55突变体与bub2突变体一样降解了m期抑制剂Pds1和m期细胞周期蛋白Clb2。由于Bub2是Tem1-GTPase的GAP，负调控MEN （Mitotic Exit Network，有丝分裂退出网络）通路，我们研究了MEN通路的下游组分Amn1是否表达，但它没有表达。然后，我们检测了调节cdc14磷酸酶的Net1是否被修改，cdc14磷酸酶是MEN通路的激活剂。Net1蛋白在cdc55突变体中出现去磷酸化，表明cdc55间接调节Net1，并参与核仁释放Cdc14以激活MEN途径2。SUMO连接酶ull1 /Siz1的结构域分析，是出芽酵母的SUMO连接酶之一，它以细胞周期依赖的方式改变其在细胞质中的定位，从细胞核到颈部区域，并识别各种底物。我们通过构建不同的缺失突变体对这种由904个氨基酸组成的SUMO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，还需要一个称为PINIT结构域的新区域来保证连接酶的活性。出乎意料的是，通过双杂交实验，一个强大的sumo相互作用结构域，称为SXS-motif，并不是连接酶活性所需要的。此外，缺乏c端一半的Ull1蛋白是稳定的，并且总是定位于细胞核。n端sap结构域，一个假定的dna结合结构域，参与了核定位。因此，RING-like和PINIT结构域是该SUMO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控。

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.