2019 Fiscal Year Annual Research Report
Regulatory mechanisms of mitochondria-specific autophagy via the TORC1 signaling pathway
| Project/Area Number |
19J20223
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| Research Institution | Osaka University |
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Principal Investigator |
LIU YANG 大阪大学, 生命機能研究科, 特別研究員(DC1)
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| Project Period (FY) |
2019-04-25 – 2022-03-31
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| Keywords | mitophagy / TORC1 / yeast |
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| Outline of Annual Research Achievements |
In this year, I mainly focused on my project of TORC1 signaling pathway in regulation of respiration-inducing mitophagy in yeast from following aspects.
We propose that mitophagy in respiring yeast is initiated through SEACIT (Iml1, Npr2 and Npr3)-dependent TORC1 inactivation. The AGC kinase Sch9 is a substrate of yeast TORC1, and directly phosphorylated by TORC1, which is utilized to monitor TORC1 activity. A Sch9 antibody and Sch9 phosphorylation antibody of Thr737 are adjusted to western blotting of npr2-null cells. We detected that Sch9 expression and phosphorylation were not significantly altered in npr2-null cells compared with wild-type cells, and Sch9 was strongly induced and phosphorylated in cells under respiratory media. Our hypothesis was that cells should inhibit TORC1 to induce mitophagy. However, from Sch9 phosphorylation status, TORC1 seemed to be activated under respiratory conditions. More investigations are needed to understand this issue.
To identify the potential factors that interact with the SEACIT, as well as involving in TORC1-mediating mitophagy pathway, we will construct a new system that using proximity-dependent labelling techniques. We plan to utilize the engineered ascorbate peroxidase (APEX2), which is fused with SEACIT component and covalently tags nearby proteins with biotin-phenol (BP). Western blotting and mass spectrometry will be performed for protein analysis. If we can identify a new protein or complex interacted with SEACIT, we will then investigate it for specific functions in the regulation of Atg32 and mitophagy.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
My project is TORC1 signaling pathway in regulation of respiration-inducing mitophagy in yeast. The ultimate goal of this project is to clarify the regulation pathway of mitophagy through TOCR1, including the signal transmission to SEACIT, TORC1 activity changes, the effectors of TORC1, and regulations of mitophagy specific factors. In the first year, I mainly focused on investigating TORC1 activity changes and the effectors of TORC1 in mitophagy regulating pathway. Although some of the experimental results are remained to be repeated and discussed, I accumulated more evidence to understand how TORC1 regulates mitophagy in yeast.
Some preliminary experiments to identify the signal transmission factors of SEACIT in respiration-inducing mitophagy have been set in this year. Hopefully, it can be performed in the next year, and I am looking forward to obtain some candidates and conduct more investigations.
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| Strategy for Future Research Activity |
In this project, I would like to address the regulation pathway of mitophagy through TOCR1, including the signal transmission to SEACIT, TORC1 activity changes, the effectors of TORC1, and regulations of mitophagy specific factors. I have performed some experiments in the first year to understand the TORC1 activity changes to regulate mitophagy, however, some results are slightly difficult to explain. In the future, I will firstly repeat these experiments and summary the molecular mechanisms of TORC1 activity in this pathway. In addition, a new screening system has been settled to identify the potential factors that interact with the SEACIT complex, as well as involving in TORC1-mediating mitophagy pathway, which will be my primary focus of this project. I hope to discover some novel factors participating mitophagy initiation to further clarify the regulation mechanism of mitophagy.
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