|Budget Amount *help
¥13,100,000 (Direct Cost : ¥13,100,000)
Fiscal Year 2005 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 2004 : ¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2003 : ¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2002 : ¥4,400,000 (Direct Cost : ¥4,400,000)
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 ligase
Ull1/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.