Regulatory mechanism of cellular functions of yeast by the ubiquitin-proeasome system.

泛素-蛋白酶体系统对酵母细胞功能的调节机制。

• 批准号: 10440225
• 负责人: TOH-E Akio
• 金额: $ 8.58万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10440225/
• 关键词: 26S proteasome; cell cycle; mitosis; metaphase-anaphase transition; RPN9; Saccharomyces cerevisiae; two-hybrid screening; プロテアソーム; 蛋白質分解; Rpn9; Rpn10; 蛋白質の集合; M期中期アレスト; ユビキチン化

We have isolated the RPN9 gene by two hybrid screening using RPN10 (formerly SUN1) as bait, which encodes a multiubiquitin chain receptor residing in the 26S proteasome. A yeast strain carrying a disrupted allele of RPN9 was found to be temperature sensitive for its growth. At the restrictive temperature, the Δrpn9 strain accumulated multiubiquitinated proteins. When the proteasome fractions separated by the glycerol gradient centrifugation were analysed by native PAGE, we found that the 26S proteasome of the Δrpn9 cells was shifted to lighter fractions than expected and that there was a larger amount of the incomplete proteasome complexes in the fractions. Furthermore, Rpn10 was not detected in the 26S proteasome of Δrpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in assembly and/or stability of the 26S proteasome.Δrpn9 cells were found to be arrested at metaphase ; dumbbell shaped, single nucleus at the isthmuth, unseparated sister chromatids. When a Δrpn9 Δpds1 strain was shifted to a restrictive temperature, the cells stopped growing. No anaphase cells were observed, but sister chrmomatids were separated. Judging from these phenotypes, we anticipate the presence of some proteins which should be degraded by the 26S proteasome during anaphase progression.

我们已经分离出RPN 9基因的两个杂交筛选使用RPN 10（以前的SUN 1）作为诱饵，它编码的多泛素链受体驻留在26 S蛋白酶体。发现携带RPN 9的破坏的等位基因的酵母菌株对其生长是温度敏感的。在限制性温度下，Δ rpn 9菌株积累了多泛素化蛋白。用非变性聚丙烯酰胺凝胶电泳（native PAGE）分析甘油梯度离心分离的蛋白酶体组分，发现Δ rpn 9细胞的26 S蛋白酶体向较轻的组分移动，组分中含有较多的不完全蛋白酶体复合物。Δ rpn 9细胞的26 S蛋白酶体中未检测到Rpn 10。这些结果表明Rpn 9参与了26 S蛋白酶体的组装和/或稳定性。Δ rpn 9细胞停滞在中期，呈哑铃形，位于峡部的单核，姐妹染色单体未分离。当Δ rpn 9 Δ粘1菌株被转移到限制性温度时，细胞停止生长。没有观察到后期细胞，但分离出姐妹染色体。从这些表型判断，我们预期的一些蛋白质的存在下，应降解的26 S蛋白酶体在后期进展。

项目成果

Takeuchi, J., & Toh-e, A.: "Genetic dissection of the yeast 26S proteasome : Cell cycle defect caused by the Δrpn9 mutation."Biochemistry. (in press). (2001)

Takeuchi, J., & Toh-e, A.：“酵母 26S 蛋白酶体的基因解剖：Δrpn9 突变引起的细胞周期缺陷。”生物化学（出版中）。

Asakawa,K.,Yoshida,S.,Otake S.,Toh.e.A.: "A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae."Genetics. (印刷中). (2001)

Asakawa, K.、Yoshida, S.、Otake S.、Toh.e.A.：“Cdc15 激酶与酿酒酵母中的 Tem1 GTPase 相互作用需要一个新的功能域。”遗传学（2001 年出版）。

Takeuchi,J,Tohe,A: "Genetic dissection of the yeast 26S proteasume"Biochimie. (印刷中). (2001)

Takeuchi, J, Tohe, A：“酵母 26S 蛋白酶的基因解剖”Biochimie（出版中）。

Toh-e,A. and Oguchi T.: "Las21 participate in extra cellular phenomena in saccharomyces cerevisiae"Gene Genet. Sys.. 74. 241-256 (1999)

东江，A.

Toh-e,A.,Asakama,K.,Yoshida,S.: "Spindle checkpoint in budding yeart"J.Microbiol.. (印刷中). (2001)

Toh-e, A.、Asakama, K.、Yoshida, S.：“萌芽年的纺锤体检查点”J. Microbiol..（出版中）。