In vivo site-sp ecific cross-linking analysis of substrate recognition mechanisms of membrane-associated E3 ligase enzymes
| Project/Area Number |
22657034
|
|---|
| Research Category |
Grant-in-Aid for Challenging Exploratory Research
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | Nagoya University |
|---|
Principal Investigator |
|
|---|
| Project Period (FY) |
2010 – 2011
|
| Project Status |
Completed (Fiscal Year 2011)
|
| Budget Amount *help |
¥3,420,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥420,000)
Fiscal Year 2011: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2010: ¥1,600,000 (Direct Cost: ¥1,600,000)
|
|---|
| Keywords | 膜タンパク質 / 小胞体 / 分解 / 光架橋 / 構造 |
|---|
| Research Abstract |
It is important to obtain detailed structural insight into substrate recognition mechanisms by E3 ubiquitin ligases. However, X-ray crystallography and NMR analysis are not suitable for proteins that are prone to aggregate in vitro and recombinant proteins that are not expressed well in bacteria or other organisms. One such E3-ligase enzyme is Doa10 in the yeast ER. Doa10 has 14 transmembrane regions and are thought to recognize both misfolded proteins and N-degron containing proteins. To begin to analyze the mechanism by which Doa10 recognizes these proteins, we tried to introduce the in vivo site-specific crosslinking method. Previous studies have suggested that Doa10 specifically recognizes Deg1, an N-terminal sequence of Matalpha2. In fact, model proteins that are fused with Deg1 at their N-terminus are degraded by the proteasome in Doa10 dependent manner. We first introduced amber codons in Deg1 and tried to introduce photoreactive amino acids. However, in the course of this study, other group reported an essential role of N-acetylation of Deg1 in recognition by Doa10. Currently we are trying to see the effects of depletion or overexpression of N-acetylation enzymes on the recognition of Deg1 by Doa10 using in vivo site-specific crosslinking method. We also started to analyze other ER membrane E3 ligase Hrd1. We have shown that the second transmembrane region of Hrd1 is critical for binding to Hrd3, a substrate recognition subunit of Hrd1. We are currently analyzing how the interaction between Hrd1 and Hrd3 changes during substrate recognition and ubiquitination.
|
Report
(3 results)
Research Products
(11 results)
