Transiently Expressed ATG16L1 Inhibits Autophagosome Biogenesis and Aberrantly Targets RAB11-positive Recycling Endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12-ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.

自噬体生物发生的膜来源是自噬研究领域尚未解决的核心谜题。自噬相关蛋白16L1（ATG16L1）可与ATG12-ATG5结合形成ATG16L1复合物，该复合物被招募至自噬膜以将微管相关蛋白1轻链3B-I（MAP1LC3B-I）转化为微管相关蛋白1轻链3B-II（MAP1LC3B-II）。在自噬体膜闭合前，ATG16L1复合物会从自噬隔离膜（phagophore）解离，因此ATG16L1可作为研究自噬体生物发生的早期事件标志物。我们发现，在ATG16L1复合物的3种蛋白组分中，仅ATG16L1在瞬时过表达时会形成点状聚集体结构，通过瞬时表达形成的ATG16L1⁺点状结构可代表自噬膜结构。我们在多种哺乳动物细胞系中对瞬时表达的ATG16L1进行了全面表征，发现其不仅会抑制自噬体生物发生，还会异常靶向RAB11阳性循环内吞体，进而引发循环内吞体聚集。综上，瞬时表达ATG16L1并非研究自噬的生理模型，因此在审视基于ATG16L1瞬时表达模型得到的研究结果时，需谨慎对待。