An Investigation into the Roles of ATG8 Proteins in Autophagy Initiation

Abstract

Autophagy is an intracellular degradation process that occurs in all eukaryotes. Despite being critical for cellular and organismal homeostasis, the mechanism of autophagy remains poorly understood. To address this important question, we aimed to define protein-protein interactions of the Unc-51-like kinase 1 (ULK1) complex, a protein complex critical for transducing stress signals, such as nutrient starvation, to the autophagy machinery. In human cells, the ULK1 complex is composed of ULK1, ATG13, FIP200, and ATG101. Members ULK1, ATG13, and FIP200 were previously shown to bind to ATG8 proteins through their LC3-interacting regions (LIR), however the functional significance of the interaction remains unclear. Human cells express at least six proteins, referred to collectively as ATG8 proteins, which are homologous to the single yeast Atg8 protein. This study reveals that the binding of ATG8 proteins to the ULK1 complex plays an important role in activating ULK1 and initiating autophagy in response to starvation. ATG8 proteins are classified into two subfamilies, the LC3 and GABARAP subfamily. Using human cells depleted of each subfamily, we found that the GABARAP subfamily promotes ULK1 activity whereas the LC3 subfamily suppresses ULK1 activity. Furthermore, we determined that specific GABARAP subfamily members GABARAP and GABARAPL1 have redundant, compensatory functions in promoting ULK1 activity. We found that the LC3 members LC3B and LC3C suppress ULK1 activity and the expression of the GABARAP subfamily of proteins. By disrupting the endogenous ULK1-ATG8 interaction in human cells, we determined that ATG8 binding to ULK1 is important for ULK1 activity, the biogenesis of isolation membranes and autophagosomes, and autophagy activity. We also found that ATG8 binding to ATG13, but not FIP200, is important for ULK1 activity. Together, these results demonstrate that, by binding to ULK1 and ATG13, GABARAP and GABRAPL1 play a key role in ULK1 activation and autophagy initiation.