Cellular Degradation Systems Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK; Institute of Biochemistry, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland.
Institute of Molecular Systems Biology, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland; School of Biological Sciences, Queen's University of Belfast, 19 Chlorine Gardens, BT9 5DL Belfast, UK.
Mol Cell. 2021 Dec 16;81(24):5066-5081.e10. doi: 10.1016/j.molcel.2021.10.024. Epub 2021 Nov 18.
Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
自噬是一种保守的细胞内降解途径，通过新形成的双层膜（自噬体）来靶向细胞质中的多种物质进行液泡/溶酶体降解，从而发挥多种细胞保护和动态平衡功能。Atg1 激酶是其关键调控因子之一，协调复杂的信号程序来协调自噬体的形成。通过体外重构和基于细胞的方法，我们证明 Atg1 被脂化的 Atg8（Atg8-PE）激活，刺激沿生长的自噬体膜上的底物磷酸化。Atg13 的 Atg1 依赖性磷酸化触发 Atg1 复合物解离，使 PAS 上的 Atg1 复合物亚基能够快速周转。此外，Atg8-PE 通过磷酸化从而抑制 Atg8 特异性 E2 和 E3，来招募 Atg1，从而自我调节生长中的自噬体膜中的 Atg8-PE 水平。我们的工作揭示了 Atg1 施加的正反馈和负反馈的分子基础，以及相反的磷酸化和去磷酸化事件如何为自噬的时空调节奠定基础。
