ATG4B-dependent processing of tandem-domain BmLC3 exposes a Tyr136-regulated C-terminal fragment

Autophagy-related protein 8 (ATG8) family proteins are ubiquitin-like modifiers that undergo ATG4-mediated processing before lipid conjugation. However, the structural and functional diversification of insect ATG8 proteins remains poorly understood. Here, comparative sequence analysis identified a 248-amino-acid LC3C-related protein from the silkworm Bombyx mori (BmLC3) containing two tandem LC3-like regions separated by an internal glycine at position 135 (G135), in addition to the canonical C-terminal glycine at position 248 (G248). In HeLa cells, N-terminally GFP-tagged BmLC3 generated a lower-molecular-weight product, whereas substitution of G135 with alanine prevented its formation. BmLC3 processing was markedly reduced in ATG4B-knockout cells and restored by expression of B. mori ATG4B (BmATG4B), demonstrating ATG4B-dependent cleavage at the internal junction. An isolated GFP-tagged C-terminal BmLC3 region showed increased punctate localization following starvation and bafilomycin A1 treatment, while BmATG4B also processed membrane-tethered mammalian ATG8 reporters in a cell-based cleavage assay. In a tandem fluorescent BmLC3 reporter, cleavage generated a C-terminal product beginning with Tyr136 that showed little mRFP signal, whereas Y136M increased both mRFP fluorescence and the mRFP/GFP band-intensity ratio. Together, these findings reveal a previously unrecognized mode of ATG8 diversification and support a model in which internal proteolysis couples fragment generation to N-terminal residue-dependent control of C-terminal product stability.