Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation

Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GAB ARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GAB ARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosomelysosome fusion. We find that the GAB ARAP subfamily promotes PLE KHM1 recruitment and governs autophagosomelysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GAB ARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GAB ARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.