Autophagy is a highly conserved pathway that degrades cytoplasmic components within the lysosome following sequestration in a double-membrane structure called the autophagosome. Autophagy serves as an essential pro-survival stress response pathway induced by a variety of stresses including starvation, hypoxia, and infection. In addition to its role during the stress response, autophagy plays an essential homeostatic role in the cell, promoting basal turnover of long-lived proteins and organelles and performing a "quality control" function by selectively degrading damaged cytoplasmic components.
Two ubiquitin-like conjugations systems are essential for autophagosome formation and elongation. The first attaches the ubiquitin-like molecule ATG12 to its substrate ATG5, and the second attaches LC3 to the lipid phosphatidylethanolamine. Our lab recently identified a novel conjugation between ATG12 and ATG3, the E2-like conjugating enzyme that mediates LC3 lipidation. Surprisingly, although both ATG12 and ATG3 mediate essential steps in the early autophagy pathway, cells lacking ATG12-ATG3 conjugation display normal LC3 lipidation and autophagosome formation following autophagy induction by starvation. However, in full nutrient conditions, cells lacking ATG12-ATG3 have increased numbers of autophagosomes, indicative of either increased autophagosome formation or decreased turnover.
In this study, we more carefully measured the effect of ATG12-ATG3 conjugation on basal autophagy and found that cells lacking ATG12-ATG3 exhibit decreased autolysosome formation under nutrient-rich conditions, perinuclear accumulation of late endosomes, and impaired late endosome function. We identify an interaction between the ATG12-ATG3 conjugate and the ESCRT accessory protein Alix and find that ATG12-ATG3 promotes Alix functions including exosome release and viral budding. Together, these data identify a molecular interaction between the core autophagy and ESCRT-associated machineries and reveal a role for the alternative ATG12-ATG3 conjugate in late endosome function that is independent of canonical autophagy.
