ATG12 Conjugation to ATG3 Modulates Mitochondrial Homeostasis and Cell Death

Autophagy is an intracellular response to starvation, which is conserved in eukaryotes from unicellular to metazoan organisms. While autophagy sustains an individual cell from death by a variety of cellular stresses, evidence suggests that it is also critically important on an organismal level for the prevention of cancer, neurodegenerative and autoimmune diseases. In light of the therapeutic implications of autophagy, much remains to be understood about the function of individual autophagy genes. Many of the autophagy genes participate in two ubiquitin-like conjugation systems. The first mediates conjugation of ATG12 to ATG5 and the second mediates lipidation of LC3/ATG8 by phosphatidylethanolamine. We hypothesized that ATG12 conjugates to other substrates in addition to ATG5, as is the case for most ubiquitin-like proteins (ubls). By increasing the cellular pool of ATG12, we were able to identify and characterize a novel covalent complex between ATG12 and ATG3. A small subset of the cellular pool of ATG3 is in complex with ATG12 at baseline conditions in mammalian cells. Formation of the ATG12-ATG3 complex requires activation by ATG7, the autophagy E1-like enzyme. ATG12 subsequently becomes auto-conjugated in cis by ATG3. Unlike ATG12-ATG5 complex formation, ATG12-ATG3 conjugation does not require the E2-like enzyme ATG10. The c-terminal glycine of ATG12 specifically binds to K243 of ATG3.The ATG12-ATG5 complex and ATG3 each modulate steps in early autophagosome expansion and closure. Therefore, we initially expected that the ATG12-ATG3 complex would affect autophagy. To our surprise, disruption of the ATG12-ATG3 complex had no effect on autophagy induced by starvation, rapamycin or chemical uncoupling of the mitochondria. Instead disruption of the ATG12-ATG3 complex leads to a highly fragmented mitochondrial network that is incapable of fusion, increased in mass and defective in mitochondrial autophagy. These phenotypes also correlate with resistance to mitochondrial cell death. In summary, we have demonstrated evidence for a previously unknown conjugate between ATG12 and ATG3. Conjugation between ATG12 and ATG3 endows each protein with an entirely separate function from either protein's known role in autophagosome biogenesis. Our work suggests that ATG12 may be a more broad-based protein modification than previously anticipated