Objective: To determine the interaction between nuclear receptor co-activator 7 (NCOA7) and microtubule-associated protein light chain 3 (LC3), and identify the LC3-binding sites in NCOA7. Methods: The mass spectrometry which was performed in gastric cancer AGS cells and GST-LC3 pulldown assay which was performed in HEK293 cells were used for identification of the LC3 binding proteins, the molecular cloning method for construction of the truncation and point mutants of NCOA7; Western blotting was used to detect the expression level of NCOA7 truncated ΔN462, ΔN650, ΔN650-I749/750A proteins, and the GST-LC3 pulldown assay to identify the LC3binding site in NCOA7. Results: The GST-LC3 pulldown assay combined with mass spectrometry identified NCOA7 as an LC3-binding protein. Western blotting results showed that different truncations and point mutants of NCOA7 were expressed. GST-LC3 pulldown NCOA7 mutants assay showed that the N-terminal WEDL motif and C-terminal WEII motif of NCOA7 were LC3 binding sites. However, binding to LC3 of the WEII motif was inhibited at the 651~720 amino acid sequence of NCOA7, which is an adjustable LC3 binding site. Conclusion: NCOA7 is an LC3 binding protein. Its N-terminal WEDL and C-terminal WEII motifs bind to LC3.

［Key words］nuclear receptor co-activator 7; autophagy; microtubule-associated protein light chain 3; LC3 interactive region; domain; motif; molecular cloning