Autophagy (literally, self-eating) is a conserved pathway by which eukaryotic cells protect themselves from starvation, intracellular pathogens, and accumulation of protein aggregates or damaged organelles. This process involves the formation of a large, double-membrane vesicle termed an autophagosome, which envelops either bulk cytoplasm (in the case of nutrient-recycling during starvation), or specific targets such as damaged organelles or protein aggregates. The vesicle then fuses with the vacuole/lysosomes, delivering its cargo for degradation. This study focuses on gaining a mechanistic understanding of the process of autophagosome formation. Because defects in autophagy are associated with many human diseases, including neurodegenerative diseases, Crohn's disease, and some cancers, understanding its basic mechanisms should lead to new treatments for those diseases. Most of the core components of the autophagosome formation machinery are conserved between the model organism S. cerevisiae (baker's yeast) and humans, and the genetic tractability of yeast makes it ideal for the study of basic cell biological processes. Therefore, we will use yeast mutants arrested at different steps of autophagosome assembly to analyze the membrane morphology of the forming autophagosome by electron microscopy and determine its protein and lipid composition by affinity-purification followed by mass spectrometry. This will allow us to tie the functions of specific protein and lipid complexes to specific stages of the autophagosome formation process, and likely identify new components of the relevant machinery. We expect our findings to be generalizable to other organisms in which this process is conserved, including humans. This information will allow us to create more accurate, mechanistic models of autophagosome formation, thereby providing a critical foundation for translational studies aimed at modulating autophagy for therapeutic purposes. PUBLIC HEALTH RELEVANCE: Cells use autophagy (literally, self-eating) to survive starvation, defend themselves against pathogens, and get rid of damaged and disease-causing proteins and other internal machinery. Defects in autophagy contribute to Parkinson's disease and other neurodegenerative diseases, gastrointestinal and cardiovascular disorders, and some cancers. The goal of this project is to better understand the mechanisms by which autophagy works, which may lead to new treatments for autophagy-related diseases.