2020 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 / yeast / TORC1 |
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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.
In previous study, we demonstrated that in cells lacking SEACIT component Npr2, mitophagy was significantly suppressed and the interactions of Atg32-Atg11 were also impaired. It has been reported that the Atg32-Atg11 interaction is a prerequisite step to promote mitophagy. Therefore, I applied NanoLuc Binary Technology (NanoBiT) using a split luciferase consisting of Large BiT and Small BiT to a fast, small scale assay system for Atg32-Atg11 interactions in live cells.
I confirmed that by using NanoBiT system, the Atg32-Atg11 interactions in npr2-null cells were decreased and recovered to wild-type level with rapamycin treatment. Next, I sought to ask whether loss of core Atg proteins alters Atg32-Atg11 interactions. Specifically, I generated several single gene-deletion mutants lacking each of core Atg proteins including Atg1 complex (Atg1, Atg13, Atg17, Atg29, and Atg31), Atg12 and Atg8 ubiquitin-like systems (Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, and Atg16), PI3K complex (Atg6, Atg14, Atg38, Vps15 and Vps34), Atg2-Atg18 complex (Atg2 and Atg18) and Atg9, and subjected to NanoBiT assays. Among all the core Atg protein mutants, only in cells lacking Atg1, the Atg32-Atg11 interactions were significantly decreased to about 40% compared to wild type cells, while other mutants were mostly wild-type like or slightly increased in Atg32-Atg11 interactions.
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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
Previously I found Atg32-Atg11 interactions were impaired in cells lacking Npr2, which caused mitophagy defects. In this year, I decided to investigate the interactions of Atg32-Atg11 via NanoBiT system because it is fast and small scale to examine Atg32-Atg11 interactions in live cells.
I successfully constructed the strains for Atg32-Atg11 NanoBiT assay and confirmed Atg32-Atg11 interactions in npr2-null cells were affected and rescued by treating with rapamycin. In addition, I examined several core autophagy proteins mutants using the NanoBiT system including Atg1 complex (Atg1, Atg13, Atg17, Atg29, and Atg31), Atg12 and Atg8 ubiquitin-like systems (Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, and Atg16), PI3K complex (Atg6, Atg14, Atg38, Vps15 and Vps34), Atg2-Atg18 complex (Atg2 and Atg18) and Atg9, and among them only cells lacking Atg1 displayed defects in Atg32-Atg11 interactions in the NanoBiT assay.
I will continue investigating how Atg1 regulates Atg32-Atg11 interactions and I am looking forward to unraveling the molecular mechanism of mitophagy.
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| Strategy for Future Research Activity |
In this year, I decided to focus on investigation of Atg32-Atg11 via NanoBiT system because Atg32-Atg11 and Atg32-Atg8 interactions are the critical steps of mitophagy induction. By now I established the NanoBiT system of Atg32-Atg11 interactions and found that Atg1 is essential for efficient interactions of Atg32-Atg11. It has been proved that Atg1 is involved in Atg32 phosphorylations upon mitophagy induction. I will next generate mutants in Atg1-Atg11 interactions, Atg1-Atg8 interactions and the double mutants of Atg1-Atg11 and Atg1-Atg8 interactions, and subject them in NanoBiT assay to understand how Atg1 regulate mitophagy specifically. With the NanoBiT system, I also want to investigate if Atg32 localization affects Atg32-Atg11 interactions by constructing a Atg32 variant lacking transmembrane domain, Atg32(1-388). Previous research found this variant failed to induce mitophagy but can be phosphorylated upon mitophagy induction. In addition, I will also examine Atg32-Atg8 interactions using NanoBiT system in the next year because Atg32-Atg8 interactions is also important in mitophagy initiation.
Taken together, I will utilize the NanoBiT system, which is a fast, small scale system to detect protein-protein interactions in live cells, to examine Atg32-Atg11/Atg8 interactions during mitophagy induction.
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