Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy

Mitochondria can be degraded by autophagy; this process is termed mitophagy. The Parkinson disease-associated ubiquitin ligase Parkin can trigger mitophagy of depolarized mitochondria. However, how the autophagy machinery is involved in this specific type of autophagy remains to be determined. It has been speculated that adaptor proteins such as p62 may mediate interaction between the autophagosomal LC3 family of proteins and ubiquitinated protein on mitochondria. Here, we describe our systematic analysis of the recruitment of Atg proteins in Parkin-dependent mitophagy. Structures containing upstream Atg proteins, including ULK1, Atg14, DFCP1, WIPI-1, and Atg16L1, can associate with depolarized mitochondria even in the absence of membrane-bound LC3. Atg9A structures are also recruited to these damaged mitochondria as well as the autophagosome formation site during starvation-induced canonical autophagy. At initial steps of Parkin-mediated mitophagy, the structures containing the ULK1 complex and Atg9A are independently recruited to depolarized mitochondria and both are required for further recruitment of downstream Atg proteins except LC3. Autophagosomal LC3 is important for efficient incorporation of damaged mitochondria into the autophagosome at a later stage. These findings suggest a process whereby the isolation membrane is generated de novo on damaged mitochondria as opposed to one where a preformed isolation membrane recognizes mitochondria.