Trans-Golgi network-associated noncanonical autophagy depends on the V-ATPase-ATG16L1 axis and mediates IL1B secretion.

Formation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3)-positive structures that does not require all of the core ATG (autophagy related) proteins is emerging in the process of noncanonical autophagy (NCA). While LC3 lipidation on endolysosomal membranes has been well characterized, the involvement of other membrane sources and the regulatory mechanisms governing LC3 lipidation in alternative forms of NCA remain poorly understood. Here, we demonstrate the occurrence of LC3 lipidation on the trans-Golgi network (TGN) platform. Different from canonical autophagosomes, these LC3-positive structures do not fuse with lysosomes, and fail to degrade long-lived proteins. In addition, the functional vacuolar-type H(+)-translocating ATPase (V-ATPase)-ATG16L1 axis is found to be essential for TGN-associated NCA. Notably, in this process, the cytosolic but not lysosomal V(1) complex of the V-ATPase assembles at the TGN and plays a pivotal role in further induction of NCA. Eventually, IL1B/IL-1β (interleukin 1 beta) secretion is found to be efficiently enhanced by such TGN-associated NCA, independently of GSDM (gasdermin)-mediated pore formation. Thus, besides the known endolysosome-related NCA, we identify a distinct form of TGN-associated NCA mediated by the V-ATPase-ATG16L1 axis. Such NCA might work as a protein-transport route for the extracellular secretion of IL1B, revealing a mechanism linking Golgi-derived NCA to inflammatory cytokines release.Abbreviation: ATG: autophagy related; Baf: bafilomycin A1; BFA: brefeldin A; CASM: conjugation of ATG8s to single membranes; CQ: chloroquine; ELISA: enzyme-linked immunosorbent assay; ER: endoplasmic reticulum; ERGIC: ER-Golgi intermediate compartment; GCA: golgicide A; GSDM: gasdermin; HCP: hexachlorophene; IL18/IL-18: interleukin 18; IL1B/IL-1β: interleukin 1 beta; LAP: LC3-associated phagocytosis; LPS: lipopolysaccharide; m-IL1B: mature IL1B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NCA: noncanonical autophagy; NIG: nigericin; NTZ: nitazoxanide; PBS: phosphate-buffered saline; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT: room temperature; SFG: salifungin; TGN: trans-Golgi network; UcPS: unconventional protein secretion; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-translocating ATPase; WDR: WD40 repeats; WT: wild-type.