Autophagy is a well-studied catabolic process through which cytoplasmic components are targeted for lysosomal degradation by autophagosomes. A key step in this process is the recruitment, processing and lipidation of LC3 to autophagosomes. Recently it has become increasingly apparent that, through the unconventional use of some autophagy related proteins, LC3 can also become lipidated to distinct non-autophagosomal membranes of the endolysosomal system. This process is termed non-canonical autophagy and occurs independently of conventional autophagy initiation signals. Non-canonical autophagy usually occurs after macro endocytic engulfment events such as macropinocytosis, entosis and LC3 associated phagocytosis (LAP). Certain ionophores and lysosomotropic drugs, such as monensin, can also activate this process and promote LC3 lipidation to lysosomes. This project focuses on Atg16L1, an essential autophagy protein that directs the membrane site where LC3 is lipidated. Atg16L1 is relatively well characterised in autophagy but little is known about the mechanisms underlying its role in non-canonical autophagy. This project used a structure/function approach to assess the importance of different domains of Atg16L1 in the context of autophagy versus non-canonical autophagy. I have demonstrated for the first time, that the Atg16L1 C-terminal WD40 domain (CTD) is dispensable for its role in autophagy but essential for LC3 lipidation during non-canonical autophagy. Furthermore, single point mutants were uncovered in the CTD of Atg16L1 that likewise are dispensable for autophagy but fundamental to LC3 lipidation in non-canonical autophagy. These data provide a novel strategy for dissecting canonical and non-canonical autophagy pathways at a molecular level. This project used an existing mouse model with an Atg16L1 truncation (lacking the CTD and nearby residues) and implicated the lack of non canonical autophagy to a defect in MHCII antigen presentation. Furthermore this project has generated new refined Atg16L1 mutant models ablating non-canonical autophagy without affecting canonical autopahgy. In parallel, proteomic analysis was done to provide mechanistic insights into Atg16L1 binding partners in the context of non-canonical autophagy.