Regulatory mechanisms of mitochondria-specific autophagy via the TORC1 signaling pathway

今年，我主要研究了酵母吞丝启动的分子机制。根据对缺乏核心ATG蛋白的细胞的Atg32-Atg11相互作用分析，Atg1是唯一的核心ATG蛋白，在呼吸条件下对有效地启动有丝分裂起关键作用。我还发现，Atg1参与了Atg32的磷酸化。在体外的磷酸化实验中，纯化的Atg1可以使Atg32磷酸化，这增加了Atg1在诱导有丝分裂过程中直接磷酸化Atg32的可能性。在Atg1-Atg8和Atg1-Atg11相互作用中有缺陷的Atg1变体的表达减少了Atg32的磷酸化和有丝分裂，这表明Atg8和Atg11可以将Atg1招募到有丝分裂起始复合体中。此外，Atg1可以与Atg32免疫共沉淀，在缺乏Atg8和Atg11的细胞中，Atg32被显著抑制，表明Atg1通过与Atg8和Atg11的相互作用而靶向线粒体。值得注意的是，这些缺陷通过删除Ppg1修复，Ppg1是一种负面调节Atg32介导的有丝分裂的蛋白磷酸酶。综上所述，Atg1可以作为Atg32磷酸化的直接激酶，在呼吸条件下诱导酵母有丝分裂中起关键作用。

I mainly worked on the molecular mechanisms underlying mitophagy initiation in yeast this year. Based on an Atg32-Atg11 interaction assay for cells lacking core Atg proteins, Atg1 is the sole core Atg protein critical for efficient mitophagy initiation under respiratory conditions. I also found that Atg1 acts in Atg32 phosphorylation. Atg32 can be phosphorylated by purified Atg1 in an in vitro phosphorylation assay, raising the high possibility that Atg1 directly phosphorylates Atg32 during mitophagy induction. Expression of an Atg1 variant defective in Atg1-Atg8 and Atg1-Atg11 interactions reduced Atg32 phosphorylation and mitophagy, suggesting that Atg8 and Atg11 serve to recruit Atg1 to the mitophagy initiation complex. Also, Atg1 can be co-immunoprecipitated with Atg32, which is significantly suppressed in cells lacking Atg8 and Atg11, indicating Atg1 targeted mitochondria via its interactions with Atg8 and Atg11. Notably, these defects were restored by deletion of Ppg1, a protein phosphatase that negatively regulates Atg32-mediated mitophagy. Taken together, Atg1 could serve as the direct kinase for Atg32 phosphorylation and is critical for mitophagy induction in yeast under respiratory conditions.