Autophagosome marker, LC3, is released extracellularly via several distinct pathways.

Autophagy-mediated secretion contributes to the maintenance of intracellular homeostasis by releasing cytoplasmic components into the extracellular space. However, several aspects of the process remain unclear. In this study, we developed an ultrasensitive detection system using HiBiT tag/NanoBiT technology to analyze the conditions that trigger the secretion of LC3, an autophagosome marker. In HiBiT-tagged knock-in cells, a detectable amount of HiBiT-dependent NanoLuc luciferase activity (HiBiT activity) from HiBiT-fused LC3 was observed in the culture supernatants. However, the levels were lower than those of CD63. HiBiT activity was detected only in the presence of detergent, indicating that LC3 was released from the lipid membranes. Treatment with bafilomycin A1 significantly increased the extracellular HiBiT activity, which was diminished in ATG5 or FIP200 knockout cells, suggesting that this release depends on autophagosome formation. However, some HiBiT-LC3 was detected in these knockout cells, indicating that LC3 may be released via an autophagy-independent mechanism. The introduction of a C-terminal truncation (ΔG) or the K51A/L53A mutation also reduced LC3 release, but did not completely inhibit it, suggesting that multiple pathways exist for LC3 release. This system is expected to elucidate the mechanisms underlying autophagy-mediated secretion.