Project/Area Number |
17K18230
|
Research Category |
Grant-in-Aid for Young Scientists (B)
|
Allocation Type | Multi-year Fund |
Research Field |
Structural biochemistry
Functional biochemistry
|
Research Institution | Kyoto Sangyo University |
Principal Investigator |
KAWANO Shin 京都産業大学, 総合生命科学部, 研究助教 (90431676)
|
Project Period (FY) |
2017-04-01 – 2019-03-31
|
Project Status |
Completed (Fiscal Year 2019)
|
Budget Amount *help |
¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
|
Keywords | リン脂質輸送 / 再構成 / コンタクトサイト / 出芽酵母 / オルガネラ膜間リン脂質輸送 / ナノディスク |
Outline of Final Research Achievements |
ERMES predominantly consists of 4 proteins (Mmm1, Mdm10, Mdm12, and Mdm34) and we have already reported the reconstitution assay using Mmm1 soluble domain and Mdm12. Here, we tried the preparation and reconstitution of Mdm10 and Mdm34 for the complete understandings of the ERMES complex. Mdm10 is found to be refoled from inclusion body fraction in Mdm10 overexpressing E. coli cell. The inclusion body was washed, solubilized with Gdn-HCl and urea, and then regenerated in an ionic detergent. Mdm34, unfortunately, was failed to establish the purification system. However, genetic approach in which C-anchored protein in the mitochondrial outermembrane, i. e., Fis1 was fused to the C-terminus of Mmm1, indicated that the fusion protein can compensate for the deletion of 4 ERMES proteins in vivo. The architecture of Mmm1-Fis1 fusion protein will give a cornerstone for exploring the equivalent protein/complex in higher eukaryote such as a human.
|
Academic Significance and Societal Importance of the Research Achievements |
本研究ではERMES構成因子4つをすべて精製し、再構成実験を行うことを試みた。その結果、残念ながらMdm34について発現、精製系を確立することはできなかったものの、Mdm10については巻き戻しを行うことで大量精製系の確立に成功した。今後Mdm34の発現、精製系が確立すれば、すぐにでも再構成実験を進めることが可能である。また、Mmm1とFis1の融合タンパク質がERMES構成因子のすべての欠損を相補できるという遺伝的解析により、ヒトを始めとする高等真核生物ではまだ未確認の、ERMES相当因子、複合体を探索する上で、重要なヒントとなることが強く期待される。
|