LC3 conjugation to lipid droplets

Abstract

Macroautophagy/autophagy and lipid droplet (LD) biology are intricately linked, with autophagosome-dependent degradation of LDs in response to different signals. LDs play crucial roles in forming autophagosomes possibly by providing essential lipids and serving as a supportive autophagosome assembly platform at the endoplasmic reticulum (ER)-LD interface. LDs and autophagosomes share common proteins, such as VPS13, ATG2, ZFYVE1/DFCP1, and ATG14, but their dual functions remain poorly understood. In our recent study, we found that prolonged starvation leads to ATG3 localizing to large LDs and lipidating LC3B, revealing a non-canonical autophagic role on LDs. In vitro, ATG3 associates with purified and artificial LDs, and conjugated Atg8-family proteins. In long-term starved cells, only LC3B is found on the specific large LDs, positioned near LC3B-positive membranes that undergo lysosome-mediated acidification. This implies that LD-lipidated LC3B acts as a tethering factor, connecting phagophores to LDs and promoting degradation. Our data also support the notion that certain LD surfaces may function as lipidation stations for LC3B, which may move to nearby sites of autophagosome formation. Overall, our study unveils an unknown non-canonical implication of LDs in autophagy processes.Abbreviation: ATG: autophagy-related enzyme, ATP: adenosine triphosphate, E2 enzyme: ubiquitin-conjugating enzyme, ER: endoplasmic reticulum, LD: lipid droplet, LIR motif: LC3-interacting region, MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta, PE: phosphatidylethanolamine, PLIN1: perilipin 1, PNPLA2/ATGL: patatin-like phospholipase domain containing 2, SQSTM1/p62: sequestosome 1, VSP13: vacuolar protein sorting 13, ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1.

Keywords: ATG3; LC3B; lipid droplets; membrane contact sites; noncanonical autophagy; prolonged starvation.

Figure 1.

LC3B lipidation to lipid droplets and implication for a noncanonical autophagy pathway. (A) during prolonged starvation, ATG3 binds alone to LDs and lipidates LC3B, delivered by ATG7. PLIN1 level on the LD is decreased on the LD that is lipidated. (B) Confocal imaging of EGFP-LC3B (green) and LDs (LipidTox, magenta) in differentiated adipocytes (3T3-L1) expressing EGFP-LC3B and incubated in EBSS for 48 h. The inset displays a 3D reconstructed image from z-stacks of the imaged LD. An LC3B-positive membrane, possibly autophagosomes, is in close apposition to the LC3B-lipidated LDs. (C) possible mechanisms. Lipidated LC3B on LD surfaces serves as a tethering factor for phagophore recruitment. The resulting autophagosomes fuse with lysosomes and undergo acidification, without the LD being fully engulfed. LDs may function as a platform for LC3B lipidation, with lipidated LC3B potentially being transferred to recruited/nascent autophagosomes close to the LD surface, directly or via the ER membrane.