ABSTRACT

Macroautophagy/autophagy is a multi-step process that leads to cargo degradation via the fusion of hydrolases-containing lysosomes with cargo-loaded autophagosomes. For this process to occur, autophagosomes are directionally transported by molecular motors toward the nucleus, where they fuse with lysosomes for cargo degradation. The molecular basis for this regulation, including the cell machinery required for this directional transport, has not been fully identified. Using a combination of proteomic and live-imaging approaches in mammalian cells, including primary neurons, we describe that the phosphorylation of the autophagosome protein Atg8/LC3B by the Hippo kinase STK4/MST1, an event we previously reported to be required for autophagy completion, reduces the binding of the transport-related protein FYCO1 to MAP1LC3B/LC3B. This event in turn allows the proficient microtubule-based transport of autophagosomes toward the perinuclear area, thus facilitating the contact of autophagosomes with lysosomes. In the absence of LC3B phosphorylation, autophagosomes undergo aberrant transport including increased movement toward the cell periphery resulting in reduced autophagosome-lysosome colocalization. Thus, LC3B phosphorylation modulates the directional transport of autophagosomes to meet with lysosomes in the perinuclear area, a crucial event in ensuring autophagic degradation of cargo.

摘要

巨自噬/自噬是一个多步骤过程，通过含有水解酶的溶酶体与载有货物的自噬体融合，导致货物降解。为了使这一过程发生，自噬体被分子马达定向运输到细胞核，在那里它们与溶酶体融合以进行货物降解。这种调节的分子基础，包括这种定向运输所需的细胞机制，尚未完全确定。在哺乳动物细胞（包括原代神经元）中结合使用蛋白质组学和实时成像方法，我们描述了 Hippo 激酶 STK4/MST1 对自噬体蛋白 Atg8/LC3B 的磷酸化，这是我们之前报道的自噬完成所必需的事件，减少运输相关蛋白 FYCO1 与 MAP1LC3B/LC3B 的结合。这一事件反过来允许自噬体基于微管熟练地转运到核周区域，从而促进自噬体与溶酶体的接触。在没有 LC3B 磷酸化的情况下，自噬体经历异常转运，包括向细胞外围的运动增加，导致自噬体-溶酶体共定位减少。因此，LC3B 磷酸化调节自噬体的定向转运以与核周区域的溶酶体相遇，这是确保货物自噬降解的关键事件。自噬体经历异常转运，包括向细胞外围的运动增加，导致自噬体-溶酶体共定位减少。因此，LC3B 磷酸化调节自噬体的定向转运以与核周区域的溶酶体相遇，这是确保货物自噬降解的关键事件。自噬体经历异常转运，包括向细胞外围的运动增加，导致自噬体-溶酶体共定位减少。因此，LC3B 磷酸化调节自噬体的定向转运以与核周区域的溶酶体相遇，这是确保货物自噬降解的关键事件。