ABSTRACT
While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been 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 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. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.
Footnotes
Author contributions
Conceptualization: F.E., C.B.; Methodology: F.E., S.P., M.D.S., M.K., C.B.; Validation: F.E., S.P., M.D.S.; Formal analysis: F.E.; Investigation: F.E., S.P., M.D.S.; Resources: F.E.; Data curation: F.E., S.P., M.D.S.; Writing - original draft: F.E., C.B.; Writing - review & editing: F.E., M.K., C.B.; Visualization: F.E., S.P., M.D.S., C.B.; Supervision: S.W., H.F., C.B.; Project administration: C.B.; Funding acquisition: S.W., H.F., C.B.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation) within the framework of the Munich Cluster for Systems Neurology (EXC2145 - ID 390857198), the Collaborative Research Center 1177 (ID 259130777) and the project grant BE 4685/2-1. Hesso Farhan was supported by grants from the Norwegian Cancer Society (Kreftforeningen; 182815, 208015), from the Norwegian Research Council (Norges Forskningsråd; 262717) and from the Rakel go Otto Kr. Bruun legat. A Cancer Research UK Career Development Fellowship to S.W. (C20685/A12825) funded this work. M.D.S. was also funded by a Biotechnology and Biological Sciences Research Council (BBSRC) grant to S.W. (BB/N000315/1).
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD016734.
