Membrane Fusion in Autophagy

Abstract

Macroautophagy is a pathway for the sequestration and degradation of cytosolic material when the cell is faced with stress conditions like starvation or infection. This cytosolic material is captured within a double-membraned vesicle (the autophagosome) which forms de novo and ultimately traffics to the lysosome for degradation. How membranes come together to form the autophagosome is unknown, but recent studies have suggested a possible role for the autophagosome-associated protein ATG8 in membrane tethering and fusion. Here we develop novel membrane platforms and protein-lipid coupling strategies to test fundamental aspects of ATG8 function, including the involvement of associated factors (ATG3 and ATG7) and the role of different lipid populations such as phosphatidylethanolamine. We compare the functionality of ATG8 to its mammalian homologue, MAP1LC3 and to a variety of mutants to establish which regions of the protein contribute to each activity. We utilize a range of different membrane dynamics assays including lipid-mixing, real-time membrane tethering, vesicle aggregation and cryo-electron microscopy. Our results suggest ATG8 may act as a “molecular glue”, bringing membranes and possibly protein cargo together, but cast doubt on the physiologic relevance of ATG8 as a membrane fusion protein.