| Project/Area Number |
13480225
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Molecular biology
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
AKIO Toh-c The University of Tokyo, Graduate School of Science, Department of Biological Sciences, Professor, 大学院・理学系研究科, 教授 (90029249)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YOKOSAWA Hideyoshi Hokkaido University, Graduate School of Pharmaceutical Sciences, Department of Biochemistry, Professor, 大学院・薬学系研究科, 教授 (90012765)
|
|---|
| Project Period (FY) |
2001 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2002: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥8,800,000 (Direct Cost: ¥8,800,000)
|
|---|
| Keywords | 26S proteasome / budding yeast / RPN7 / RPN5 / polyubiguitinated SicI / 19S調節因子 / Nob1 / Pno1 / 核移行 / Rpn7 |
|---|
| Research Abstract |
The yeast 26S proteasome, a ca. 200kDa protein complex, consists of 20S proteasome and the 19S regulatory particle. This protein complex abundantly exists in the yeast cells, but because of its large size, it was difficult to purify holoenzyme intact. During this project, we devised a quick and easy method to purify the 26S proeasome; we constructed yeast strains, one of whose proteasome subunits, Rpnl, Rpnl 1, and Prel, had been tagged with 3xFlag and proteasomes produced in these strains were pulled down with anti-Hag antibody. By exploiting this purification method, we characterized proteasomes produced by rpnZ temperature-sensitive mutants and found that a new subcomplex existed in the extract of the rpn7-3 mutant cells. A major obstacle for biochemical study of the 26S proteasome is the absence of proper substrate, polyubiquitinated protein. In this study, we developed a method to produce polyubiquitinated Sici by in vitro reaction by using Rps5 as an E3 enzyme. A key idea is to introduce a PY motif, that attract Rsp5, into the Sici sequence. We confirmed that polyLibiquitinated Sici is a very good substrate of' the 26S proteasome.
|
