Biochemical Isolation and Characterization of the Tubulovesicular LC3-positive Autophagosomal Compartment

Autophagosomes and their precursors are best defined by electron microscopy but may also be traced in living cells based on the distribution of specific autophagy molecules. LC3, the most commonly examined autophagy marker in mammalian cells, labels structures that are frequently manifested as dots or rings using light microscopy; however, the nature of these structures is not entirely clear. We reported here a novel approach to examine the LC3-positive compartment in cell-free lysates, which revealed that they were actually tubulovesicular structures with considerable heterogeneity. Using affinity purification, we isolated these membranes for electron microscopy, which indicated that they possessed ultrastructural features consistent with autophagosomal membranes at various maturation stages. Further biochemical and proteomics analyses demonstrated the presence of multiple autophagy-related and other functional molecules. The different distribution patterns of Atg5, Atg16, Atg9, and p62/SQSTM1 on the LC3-positive compartment provided new clues on how these molecules might be involved in the dynamics of the autophagosomal membranes. Finally, several morphologically unique groups of LC3-positive membranes were categorized. Their topological configurations suggested that double-membrane vesicles could be derived from single membrane compartments via different means, including tubule-to-vesicle conversion, whose presence was supported by live cell imaging. These findings thus provide new information on the dynamics of the autophagosomal compartment.