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

Abstract

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 and 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 using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Further, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together, we describe ACSL3 as novel regulator of the enigmatic UFM1 conjugation pathway.