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

我们研究了芽殖酵母细胞周期M期的两个问题：1.磷酸酶2A的B型亚基Cdc55在纺锤体检查点控制中的作用：在微管解聚剂诺考达唑存在下，Cdc55突变体中的M期抑制剂Pds 1和M期细胞周期蛋白Clb 2与bub2突变体类似地被降解。由于Bub2是Tem1-GTP酶的GAP，负调节MEN（有丝分裂出口网络）通路，我们研究了MEN通路的下游组分Amn 1是否表达，但它不表达。然后，我们检查是否Net1调节Cdc14-磷酸酶，MEN途径的激活剂被修改。在cdc55突变体中，Net1蛋白被脱磷酸化，表明Cdc55间接调控Net1，并参与Cdc14从细胞核释放，激活MEN通路。2. SUMO连接酶Ull 1/Siz 1的结构域分析表明，SUMO连接酶Ull 1/Siz 1在胞质中的定位与细胞周期有关，并识别其各种基质。我们通过构建各种缺失突变体对由904个氨基酸组成的SUMO连接酶进行了结构域分析，并检查了每个酶的活性和定位。除了RING样结构域之外，一个新的区域称为PINIT结构域是连接酶活性所必需的。出乎意料的是，一个强大的SUMO相互作用域的双杂交测定，称为SXS基序，是不需要的连接酶活性。此外，缺乏C-末端一半的Ull 1蛋白是稳定的，并且总是定位于细胞核。N端SAP结构域是一个DNA结合结构域，参与核定位。因此，RING样和PINIT结构域是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.