Development of new tools to study lipidated mammalian ATG8

Abstract

Mammals conserve multiple mammalian ATG8 proteins (mATG8s) consisting of γ-aminobutyric acid receptor-associated protein (GABARAP) and microtubule-associated protein 1 light-chain 3 (LC3) subfamilies that tightly bind to the autophagic membranes in a lipidated form. They are crucial in selective autophagy and recruit proteins bearing LC3-interacting region (LIR) motifs. However, because limited research tools are available, information about the specific roles of each lipidated mATG8 in selective autophagy is scarce. Here, we identified LIR motifs specific to the lipidated form of each mATG8 and characterized the residues critical for their selective interaction using cell-based assays and structural analyses. Then, we used these selective LIR motifs to develop probes and irreversible deconjugases that targeted selective lipidated mATG8s in the autophagic membrane, revealing that lipidated GABARAP subfamily proteins regulate aggrephagy of amyotrophic lateral sclerosis-linked protein aggregates. Our tools will be useful in elucidating the functional significance of each mATG8 protein in autophagy research.