REGULATORY MECHANISMS OF THE 26S PROTEASOME ASSEMBLY

26S 蛋白酶体组装的调控机制

• 批准号: 11480175
• 负责人: YOKOSAWA Hideyoshi
• 金额: $ 7.87万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480175/
• 关键词: proteasome; budding yeast; growth; regulatory subunit; ubiquitin receptor

In this study, we investigated the regulatory mechanisms of the 26S proteasome assembly and obtained the following results. 1. We established methods for rapid isolation of the 19S regulatory complex, the lid complex, and the base complex of the 26S proteasome from budding yeasts. Rpn10, a multiubiquitin chain-binding subunit, was found to be as a component of the lid complex. 2. We next investigated the role of Rpn10 in the 26S proteasome assembly. Using, various deletion mutants of RPN10, we analyzed their growth, the amounts of the 26S proteasome, and their suppressive effects on the temperature-sensitivity of rpn12-1 strain, and found that the N-terminal region of Rpn10 plays an important role in the 26S proteasome assembly. 3. In addition to Rpn10, Rad23 and Dsk2, ubiquitin-like proteins, were found to act as multiubiquitin chain-binding proteins to recruit polyubiquitinated proteins to the 26S proteasome. 4. By two-hybrid screening using Rpnl2 as bait, a novel protein, Nob1, was isolated. This protein was detected in the growing stage but not in the stationary stage. In the former stage, it was bound to the 26S proteasome. We proposed that Nob1 is a candidate protein functioning in the 26S proteasome assembly in a growth-dependent manner.

在本研究中，我们对26S蛋白酶体组装的调控机制进行了研究，获得了以下结果。1.建立了从发芽酵母中快速分离26S蛋白酶体的19S调控复合体、LID复合体和碱基复合体的方法。Rpn10是一个多泛素链结合亚单位，被发现是LID复合体的一个组成部分。2.接下来我们研究了Rpn10在26S蛋白酶体组装中的作用。利用RPN10的各种缺失突变体，我们分析了它们的生长、26S蛋白酶体的数量以及它们对rpn12-1菌株温度敏感性的抑制作用，发现Rpn10的N末端区域在26S蛋白酶体组装中起着重要的作用。3.除Rpn10、RAD23和Dsk2外，还发现泛素样蛋白RAD23和Dsk2作为多泛素链结合蛋白将多泛素化蛋白募集到26S蛋白酶体中。4.以Rpn12为诱饵，通过双杂交筛选，分离到一种新的蛋白质Nob1。该蛋白在生长阶段检测到，而在稳定期检测不到。在前一阶段，它与26S蛋白酶体结合。我们提出Nob1是26S蛋白酶体组装中的一个候选蛋白，其功能依赖于生长。

项目成果

Tone, Y., Tanahashi, N., Tanaka, K., Fujimuro, M., Yokosawa, H., Toh-e, A.: "Nob1p, a new essential associates with the 26S proteasome of growing Saccharomyces cerevisiae cells"Gene. 243(1). 37-45 (2000)

Tone, Y.、Tanahashi, N.、Tanaka, K.、Fujimuro, M.、Yokosawa, H.、Toh-e, A.：“Nob1p，一种与生长的酿酒酵母细胞的 26S 蛋白酶体相关的新必需基因”基因

Yuki Yanagawa: "Purification and characterization of the 26S proteasome from cultured rice (Oryza sativa) cells"Plant Science. 149・1. 33-41 (1999)

Yuki Yanakawa：“来自培养稻（Oryza sativa）细胞的 26S 蛋白酶体的纯化和表征”《植物科学》149・1（1999）。

Etsuko Tanaka: "Isolation and characterization of a novel 530kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes"Archives of Biochemistry and Biophysics. 374. 181-188 (2000)

Etsuko Tanaka：“能够与海星卵母细胞 20S 蛋白酶体结合的新型 530kDa 蛋白质复合物 (PC530) 的分离和表征”生物化学和生物物理学档案。

Kazuki Saitoh: "Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit"Biochemistry. 40. 314-319 (2001)

Kazuki Saitoh：“26S 蛋白酶体的组装受 p45/Rpt6 ATP 酶亚基磷酸化的调节”生物化学。

Kazuki Satoh: "Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit"Biochemistry. 40・2. 314-319 (2001)

Kazuki Satoh：“26S蛋白酶体的组装由p45/Rpt6 ATP酶亚基的磷酸化调节”生物化学40・2（2001）。