In Vitro Dissection of Autophagy.

Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure sequesters a portion of the cytoplasm by the formation of a double-membrane compartment called the autophagosome. The autophagosome then delivers the cargo to the lysosome to complete degradation. The molecular mechanism accounting for the generation of the autophagic membrane is a longstanding question. Here, a cell-free approach that has been established to understand the mechanism of early autophagic membrane generation is described. This system has provided insight into the membrane source of the autophagosome, the early protein-membrane associations, and the membrane remodeling that generates the autophagosomal precursors. The cell-free assay, in combination with other established approaches (e.g., cell imaging), will facilitate a deeper understanding of the mechanism of autophagy. © 2017 by John Wiley & Sons, Inc.