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Investigation of the role of GABARAP family proteins in cargo recruitment to the autophagosome

Autophagy is a conserved cellular process critical for removing cellular waste and recycling nutrients. During autophagy cargo is engulfed by a crescent shaped membrane called the phagophore and subsequently degraded into reusable material. There are two types of autophagy: selective and bulk. Bulk autophagy is a non‐specific process, whereas in selective autophagy, only cargo tagged for degradation is enveloped by the phagophore. A defining characteristic of selective autophagy is the relationship between Atg8‐like membrane proteins and cargo adaptor proteins. A classically studied example of this relationship in mammalian systems is the interaction between cargo adaptor, p62 and Atg8‐like protein, LC3B. It has been shown that LC3B binds to the LC3‐Interacting Region (LIR) in p62. This interaction has been studied extensively, but mammals have two subfamilies of Atg8‐like proteins: LC3 and GABARAP. GABARAP family proteins have significant homology with LC3B and therefore should have binding affinity for proteins with LIR motifs. However, the interaction between GABARAP family proteins and cargo adaptors is not well characterized. The aim of this work is to investigate the ability of GABARAP family proteins to bind to synthetic lipid membranes, and recruit cargo adaptor proteins to these membranes. We used biochemical assays and fluorescence imaging to characterize the activity of GABARAP family proteins in vitro. Our preliminary results show via pulldown that GABARAPL2 has less affinity for cargo adaptor proteins, p62 and NDP52, than LC3B. We also demonstrated using fluorescence microscopy that GABARAPL2 is readily conjugated to lipid vesicles by Atg7, Atg3, and the Atg 5/12/16 complex. Taken together, these results show GABARAPL2 has a different role than LC3B in cargo recruitment to the autophagosome, although the specific role has yet to be elucidated.