Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.

INTRODUCTION Macroautophagy (hereafter autophagy) is an evolutionarily conserved lysosomal degradation pathway. It ensures cellular homeostasis and health by removing harmful material from the cytoplasm. Among the many substances that are degraded by autophagy are protein aggregates, damaged organelles, and pathogens. Defects in this pathway can result in diseases such as cancer and neurodegeneration. During autophagy, the harmful material, referred to as cargo, is sequestered by double-membrane vesicles called autophagosomes, which form de novo around the cargo. Autophagosome formation occurs at sites close to the endoplasmic reticulum (ER). The process is catalyzed by a complex machinery that includes protein and lipid kinases, membrane binding and transfer proteins, and ubiquitin-like conjugation systems. How these components and biochemical activities act in concert to mediate autophagosome formation is incompletely understood. Particularly enigmatic are autophagy related protein 9 (Atg9)–containing vesicles that are required for the assembly of the autophagy machinery but do not supply the bulk of the autophagosomal membrane. RATIONALE To understand the mechanism of how the various biochemical activities of the autophagy machinery are orchestrated during the nucleation and expansion of the precursors to autophagosomes at the cargo, we fully reconstituted these events using the yeast machinery. Specifically, we used recombinantly expressed and purified proteins in combination with reconstituted Atg9 proteoliposomes and endogenous Atg9 vesicles isolated from cells. Our reconstituted system included 21 polypeptides, as well as membrane platforms, making up almost the entire yeast core machinery required for selective autophagy. This approach allowed us to exert full control over the biochemical reactions and to define the organization principles of the early autophagy machinery. RESULTS We found that Atg9 vesicles and proteoliposomes were recruited to the autophagy cargo via the Atg19 receptor and Atg11 scaffold axis. The vesicles in turn recruited the Atg2-Atg18 lipid transfer complex and the class III phosphatidylinositol 3-phosphate kinase complex 1(PI3KC3-C1), which produced the signaling lipid phosphatidylinositol 3-phosphate (PI3P). PI3P production triggered the subsequent recruitment of the PI3P-binding protein Atg21, which together with the Atg2-Atg18 complex efficiently attracted the E3-like Atg12–Atg5-Atg16 complex. Together with the E1-like Atg7 and the E2-like Atg3 proteins, the recruitment of the E3-like complex ultimately resulted in the conjugation of the ubiquitin-like Atg8 protein to the headgroup of phosphatidylethanolamine (PE) on the Atg9 vesicles and proteoliposomes. Atg8 conjugation is a hallmark of autophagy and necessary for membrane expansion. Furthermore, we discovered that sustained Atg8 conjugation required the Atg2-mediated transfer of PE from a donor membrane into Atg9 proteoliposomes. CONCLUSION We conclude that Atg9 vesicles form seeds that establish membrane contact sites to initiate the transfer of lipids from donor compartments such as the ER. It has become increasingly clear that lipid transport between different compartments occurs at membrane contact sites and that it is mediated by lipid transfer proteins. Notably, lipid transfer at membrane contact sites requires two preexisting compartments. We propose that during the de novo formation of autophagosomes, the Atg9 vesicles recruit the autophagy machinery and serve as nucleators to establish membrane contact sites with a donor compartment such as the ER. Atg2-mediated lipid transfer in conjunction with energy-consuming reactions such as PI3K-dependent PI3P production and Atg8 lipidation on the Atg9 vesicles drive net flow of lipids into the vesicles, resulting in their expansion for autophagosome formation.