ATG9 scrambles lipids in concert with ATG2 lipid delivery to directly grow the autophagosome

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Project Summary/Abstract The autophagosome encapsulates cellular debris into double-membrane vesicles responsible for trafficking this cargo to the lysosome for degradation, thereby maintaining cellular homeostasis. How the autophagosome grows and particularly the method by which lipid is delivered to the growing structure has been a key area of dispute. The model favored by our lab is direct delivery of lipid from a source membrane to the expanding phagophore by the early-acting autophagy-related protein ATG2 that we have defined as a bulk lipid transporter. If ATG2 is growing the phagophore through direct lipid transport, then there must be lipid flipping activity that allows for the population of the inner leaflet of the bilayer. Due to the nature of lipid-flipping proteins, they must span the membrane at least once to facilitate lipid crossover. ATG9 is the only transmembrane protein required for autophagy, and its function remains to be determined. Preliminary data from our collaborator shows that ATG9 is capable of flipping lipids in vitro without the presence of ATP, making it a potential scramblase. This data, the reported direct interaction with ATG2, and the fact that ATG9 accumulates with ATG2 genes knocked out led to the hypothesis that ATG9 is a lipid scramblase on the seed membrane that facilitates membrane expansion of the phagophore through direct lipid delivery by ATG2. This project is designed to provide the training necessary to achieve a future career in independent research. Further, this project will increase the understanding of a crucial cell biological process and in turn lay the foundation for future therapeutic lines of inquiry. This proposal outlines two aims – Aim 1: Determine whether ATG9 functions as a scramblase in vivo, and if so, whether it is embedded in the donor or acceptor membrane during autophagosome expansion; Aim 2: Evaluate the lipid composition of the autophagosomal membrane and ATG9 vesicles to trace the origin of the lipids required for autophagosome formation. Aim 1 will assess changes in the biology correlating to manipulation of the ATG9 protein by a powerful combination of biochemistry and microscopy techniques. Disruptions in autophagy will be evaluated by several classic readouts of autophagy progression such as LC3 puncta formation and autophagy factor turnover (i.e. p62), and will allow for efficient screening of ATG9 mutants before submitting them to in vitro scramblase activity assessment. Aim 2 will leverage high resolution quantitative lipidomics and several knockout cell lines that trap autophagosome morphological intermediates to determine the membrane composition of the mammalian autophagosome for the first time. The composition of the autophagosome is expected to approximate the composition of the source membrane. Finally, to separate the autophagosomal membrane from contaminating cargo, styrene maleic acid (SMA) nanodiscs will be employed to rigorously assess the lipids that comprise the autophagosome. This proposed study will contribute new insight into the mechanism of autophagosome formation and answer a decades-long question in the field.