Li Hui, Song Jing-Zhen, He Cheng-Wen, Xie Meng-Xi, Zhang Zheng-Tan, Zhou You, Li Xin-Jing, Cui Li, Zhu Jing, Gong Qingqiu, Xie Zhiping
Shanghai Sixth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, PR China.
State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China.
Cell Death Differ. 2025 May;32(5):866-879. doi: 10.1038/s41418-024-01438-8. Epub 2024 Dec 23.
Autophagosomes are formed by the enlargement and sealing of phagophores. This is accompanied by the recruitment and release of autophagy-related (Atg) proteins that function therein. Presently, the relationship among factors that act after the initial emergence of the phagophore is unclear. The endosomal sorting complexes required for transport (ESCRT) machinery and Atg4 are known to function in phagophore sealing and Atg8 release, respectively. Here we show that biochemically, both Atg4 and ESCRT promoted phagophore sealing. Intriguingly, Atg4-mediated release of Atg8 from the phagophore promoted phagophore sealing even in the absence of ESCRT. This sealing activity could be reconstituted in vitro using cell lysate and purified Atg4. To elucidate the temporal relationship between Atg4 and ESCRT, we charted a timeline of the autophagosome formation cycle based on the trafficking of Atg proteins and mapped the actions of Atg4 and ESCRT to specific stages. The temporal impact of Atg4-mediated release of Atg8 from phagophore was mapped to the stage after the assembly of phagophore assembly site (PAS) scaffold and phosphatidylinositol-3-kinase (PtdIns-3-K) complex; its retardation only extended the duration of Atg8 release stage, leading to delayed phagophore sealing and accumulation of multiple phagophores. The impacts of ESCRT were mapped to two stages. In addition to promoting phagophore sealing, it also dictates whether PtdIns-3-K recruitment can occur by controlling Atg9 trafficking, thereby determining the incidence of autophagosome formation. Accordingly, ESCRT deficiency led to a combination of reduced autophagosome frequency and extended autophagosome formation duration, manifesting as reduced autophagic flux but normal apparent Atg8 puncta number. Our study thus identifies Atg4-mediated Atg8 shedding as a novel membrane scission mechanism and reveals a new early-stage role for ESCRT in autophagy.
自噬体由吞噬泡的扩大和封闭形成。这伴随着自噬相关（Atg）蛋白的募集和释放，这些蛋白在其中发挥作用。目前，吞噬泡最初出现后起作用的各因素之间的关系尚不清楚。已知转运所需的内体分选复合物（ESCRT）机制和Atg4分别在吞噬泡封闭和Atg8释放中起作用。在此我们证明，在生化层面上，Atg4和ESCRT均促进吞噬泡封闭。有趣的是，即使在没有ESCRT的情况下，Atg4介导的Atg8从吞噬泡的释放也促进了吞噬泡封闭。这种封闭活性可以使用细胞裂解物和纯化的Atg4在体外重建。为了阐明Atg4和ESCRT之间的时间关系，我们根据Atg蛋白的运输绘制了自噬体形成周期的时间线，并将Atg4和ESCRT的作用映射到特定阶段。Atg4介导的Atg8从吞噬泡释放的时间影响被映射到吞噬泡组装位点（PAS）支架和磷脂酰肌醇-3-激酶（PtdIns-3-K）复合物组装后的阶段；其延迟仅延长了Atg8释放阶段的持续时间，导致吞噬泡封闭延迟和多个吞噬泡积累。ESCRT的影响被映射到两个阶段。除了促进吞噬泡封闭外，它还通过控制Atg9运输来决定PtdIns-3-K的募集是否能够发生，从而决定自噬体形成的发生率。因此，ESCRT缺陷导致自噬体频率降低和自噬体形成持续时间延长，表现为自噬通量降低但Atg8斑点数量正常。我们的研究因此将Atg4介导的Atg8脱落鉴定为一种新的膜切割机制，并揭示了ESCRT在自噬中的新的早期作用。
