Mechanisms of autophagosome biogenesis

Summary Intracellular environments are constantly producing local stress in the form of aging organelles or accumulations of redundant or misfolded components. During disease, these processes are exacerbated as, for example, pathogen invasion contributes another source of cytoplasmic toxicity. Our cells deal with these various challenges by creating a new organelle called the autophagosome which grows around the intracellular toxin and eventually fully encapsulates it. In this way, the dying organelle or the invading virus is sequestered from the rest of the cytoplasm. A similar process plays out when cells are starving, as autophagosomes can be used to harvest redundant material as a source of nutrients. Each autophagosome requires millions of lipids to complete its construction and in high stress, hundreds of autophagosomes may be made over tens of minutes. In just the last four years, my lab and several others in the field, have discovered the primary machinery needed to harvest most or all of the lipids involved in autophagosome construction. This machinery includes a lipid transporter and associated transmembrane proteins that distribute these lipids to both leaflets of the connecting membranes. This “bulk lipid transport” system is unprecedented and thus its discovery has raised many important next questions. Most critically, we know which machines harvest the lipids, but we do not understand how the lipids are pulled from their source membrane. We also do not have an absolute understanding of which membrane is the source. These two questions are closely connected as the ability to flux lipids out of a membrane is probably related to physico-chemical attributes of the donor membrane. In addition, the decision to flux potentially 100,000,000's of lipids out of a donor may impact the normal biology at that site and so we need to understand how autophagosome biogenesis is coupled to changes in the lipid donor membrane compartment. The other surprising element of autophagosomes is their shape. They need to adopt a bowl-like structure in order to encapsulate random bits of cytoplasm during starvation. How this occurs is not known, but several models have postulated that the harvesting of lipids alone might suffice, while others suggest key proteins that recognize extreme elements of this unique-shape will stabilize those elements driving the production of bowls. In this proposal, we build on our recent discoveries to explore where lipids are harvested from, how the flux is generated and then ask how these two activities are coupled to the formation of the bowl-like intermediates in autophagosome biogenesis.