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.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

“…Using normalized peak areas to estimate relative abundance, ATG8-PS represents approximately 10% (hLC3A) to 30% (hGABARAP) of the lipidated form, under these conditions. Similar results are also observed at endogenous expression levels (hGABARAPL2 knockin model, Figures 1I-1K) (Eck et al, 2020).…”

“…HeLa cells (female, human cervical adenicarcinoma epithelial) were cultured in DMEM (GIBCO, 41966-029) supplemented with 10% FBS (Sigma) and penicillin/streptomycin (100 U/ml, 100 mg/ml; GIBCO 15140-122) at 37 C, 5% CO 2 . Wild-type and endogenously HA-tagged GABARAPL2 HeLa cells were kindly provided by Dr Christian Behrends (Eck et al, 2020). Wild-type and ATG4BÀ/À HeLa cells expressing GFP-hLC3B.G120 were kindly provided by Dr Robin Ketteler (Agrotis et al, 2019).…”

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

“…Despite the discovery of UFMylation almost two decades ago, its structural basis, the full spectrum of UFMylated substrates, and its physiological role are still not fully resolved [17,40]. Studies in metazoans and our recent work have shown that UFMylation is involved in a wide range of homeostatic pathways, including ER stress tolerance, immunity, autophagy, lipid droplet biogenesis, and the DNA damage responses [14,15,23,[41][42][43][44][45][46]. In ER homeostasis, UFMylation is activated by stalling of ER-bound ribosomes and brings about the degradation of incomplete polypeptides, which can be toxic for the cell [14,23].…”

A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

“…NMR titration experiments with wild type R1-R2-R3 325–404 peptide revealed that A371 and adjacent residues are involved in GABARAPL2 binding at high GABARAPL2 concentrations. Again, taking into account that GABARAP and LC3 protein family members are proposed to recruit UBA5 to the ER membrane and play a critical role in the regulation of the ufmylation pathway [ 33 , 41 ], these results lead to the assumption that the A371T mutation plays a minor role in the ufmylation reaction itself, but might affect UBA5 localization and thus influences target ufmylation.…”