Development of new tools to study membrane-anchored mammalian Atg8 proteins

Mammals conserve multiple mammalian Atg8-family proteins (mATG8s) consisting of GABARAP (GABA type A receptor-associated protein) and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) subfamilies that tightly bind to autophagic membranes in a membrane-anchored form. These proteins are crucial for selective autophagy and recruit proteins bearing LC3-interacting region (LIR) motifs. However, because limited research tools are available, information on the specific roles of each membrane-anchored mATG8 in selective autophagy is scarce. In this study, we identified LIR motifs specific to the membrane-anchored form of each mATG8 and characterized the residues critical for their selective interaction using cell-based assays and structural analyses. We then used these selective LIR motifs to develop probes and irreversible deconjugases that targeted selective membrane-anchored mATG8s in the autophagic membrane, revealing that membrane-anchored GABARAP subfamily proteins regulate the aggrephagy of amyotrophic lateral sclerosis-linked protein aggregates. Our tools will be useful for elucidating the functional significance of each mATG8 protein on autophagic membranes in autophagy research.