ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

While studies of ATG genes in knockout models led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3/GABARAP proteins are still poorly understood. A major drawback for their understanding is that the available interactome data was largely acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was exemplarily employed to map endogenous GABARAPL2 protein complexes in response to autophagic modulation using interaction proteomics. This approach identified the ER transmembrane protein and lipid droplet biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. Through this interaction, the GABARAPL2-interacting protein and UFM1-activating enzyme UBA5 becomes anchored at the ER membrane. Functional analysis unveiled ACSL3 and lipid droplet formation as novel regulators of the enigmatic UFM1 conjugation pathway.