ATG8 Regulation and Function in Non-Canonical Autophagy

Canonical autophagy is a fundamental, catabolic process that targets cytoplasmic components for lysosomal degradation. In a defining step of this pathway, ATG8 subfamily proteins (LC3s and GABARAPs) are recruited to double membrane autophagosomes where they facilitate substrate capture, membrane biogenesis and maturation. LC3 proteins can also be recruited to single membrane, endolysosomal compartments through a distinct pathway known as “non-canonical autophagy”, associated with various macroendocytic engulfment events such as entosis and LC3-associated phagocytosis (LAP). This project explores the role of ATG8s in non-canonical autophagy, which is currently poorly understood. Findings demonstrate that in addition to LC3 recruitment, all three GABARAP family members (GABARAP, GABARAPL1 and GABARAPL2) are recruited to endolysosomal compartments targeted by non-canonical autophagy. Central to this membrane recruitment is an unusual lipidation process. In canonical autophagy, it is widely accepted that the lipid species conjugated to ATG8 proteins is phosphatidylethanolamine (PE). However, using mass spectrometry, this study shows for the first time in vivo that ATG8 proteins can become conjugated to both PE and phosphatidylserine (PS) upon stimulation of non-canonical autophagy. Furthermore, differential phosphorylation of serine 12 in LC3A is identified, as phosphorylation increases upon activation of non-canonical autophagy but not canonical autophagy. Excitingly, these data highlight different post-translational modifications of ATG8 proteins during non-canonical autophagy and indicate a complex level of regulation at distinct membrane structures. Such regulation may impact ATG8 functionality, known to be strongly shaped by its interactome. To date, few ATG8 binding partners have been described in the context of non-canonical autophagy. To tackle this gap in understanding, a candidate-based study and a mass spectrometry screen were implemented to identify ATG8 binding partners in this context. Whilst some ATG8 interactors appear specific to canonical autophagy, others such as the Rab7 GAP, TBC1D5, appear common to both pathways. Also presented within this study are potentially novel ATG8 interactors that may, upon further investigation, shed light on the unknown role of ATG8s during non-canonical autophagy.BBSRC DTP fundin