Autophagy constitutes a primary pathway through which bulk proteins are degraded in response to reduced nutrient levels and developmental signals, and defects in this pathway affect many disease states. Autophagy in budding yeast involves more than 30 ATG genes that together, regulate the primary steps in the autophagy process: generation of a double bi-layer membrane vesicle (the autophagosome) that captures cellular contents and organelles and delivery of this autophagosome to the lysosome for degradation. Four major signaling systems have emerged: 1) the Atg1p kinase complex involved in signaling, 2) the Class III PI3 Kinase complex involved in generating PtdIns3P (P3) on the surface of autophagosomal membranes, 3) the Atg8p/Atg12p ubiquitin-like (UBL) conjugation system, which is responsible for conjugating Atg8p to phosphatidylethanolamine thereby facilitating its incorporation into autophagosome, and 4) the pre- autophagosome cycling complex, which is responsible for assembling Atg9p, Atg2p, and Atg18p onto the pre- autophagosome in a pathway that requires Atg1p. Despite the clear role that autophagy plays in mammalian cells, much less is known about the organization of the pathway, the proteins that are involved, and how modules within the pathway interact with each other. We have performed a proteomic analysis of 65 known and candidate human autophagy proteins, revealing an Autophagy Interaction Network (AIN) composed of 763 interactions between 439 candidate proteins, with an experimental validation rate of >70%. This analysis revealed numerous examples of involvement of protein kinases, ubiquitin pathway components, and proteins linked to vesicle trafficking. The C- terminally lipidated UBL protein ATG8 (MAP1LC3 and GABARAP family members in humans) uses a conserved surface to recruit autophagy receptors containing the LC3-interacting region (LIR) to autophagosomes. The six MAP1LC3/GABARAP proteins formed a sub-network containing 182 interactions among 67 proteins in vivo, with extensive overlap between family members and frequent involvement of the LIR-docking site (LDS) measured in vitro. RNAi of 80 AIN components revealed dozens of genes whose depletion reduces or increases autophagosome number, providing functional evidence for involvement in the pathway. In this proposal, we will use systematic approaches to further refine, develop, and disseminate this first generation AIN map and will begin to examine how newly identified components regulate the activities of protein kinase, ubiquitination, and ATG8 sub-networks within the pathway. These studies will focus on defining dynamic changes in the AIN network and modifications of proteins in the network that accompany activation of autophagy, thereby providing a glimpse into the interaction landscape of this important cellular system. PUBLIC HEALTH RELEVANCE: The question of how and why eukaryotic cells "eat" their cytoplasmic contents has fascinated cell biologists for decades and has been the central driving force in the discovery of a signal transduction system referred to generically as "autophagy". This system is linked to many diseases states, as well as to aging and organismal development, yet there are many aspects of this process that remain poorly understood at the molecular level. This work seeks to employ proteomic and genetic approaches to develop a roadmap for understanding the physical and genetic interactions that are important for establishment of this system.