Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This “packaging” is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane coordinates the capture of cytosolic toxins with its own expansion and closure is an area of intense study. One factor implicated in both cargo-capture and autophagosome dynamics is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE decorates the earliest autophagosome membrane progenitor structure and plays an integral role in organizing the accumulation of membranes that presages the eventual growth of the autophagosome itself. These activities depend upon the ability of Atg8-PE to tether individual vesicles into an underlying support. Critically, Atg8 must remain associated with the membranes throughout the growth of the autophagosome. Our results now also describe how this pool of Atg8-PE is protected from recycling proteases that otherwise function to constitutively remove Atg8 at other sites. Our discoveries are made possible by two important technological advances. First, we have developed a variety of in vitro reconstitution approaches to study how Atg8- PE and other autophagy proteins influence membrane deformation and structure. Second, we are now able to image autophagosome intermediate structures at super resolution in three dimensions and identify the same structures in micron deep electron tomograms revealing key features of the earliest Atg8-decorated membranes. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins organize the proteins and membranes that support autophagosome membrane expansion.