Bak inserts into Mcl-1-puma and Mcl-1-BimL dimers to form hetero-trimers analyzed using quantitative live-cell FRET imaging

This report uses fluorescence resonance energy transfer (FRET) to explore protein-protein interactions and the relative priorities of Bcl-2 family proteins in living cells, focusing on four proteins: Mcl-1, Bak, BimL and Puma. FRET analysis of cells coexpressing CFP-Mcl-1 and YFP-Bak/Puma/BimL demonstrates the direct binding of Mcl-1 to Bak/Puma/BimL. We constructed Bak-P2A-CFP-Mcl-1, BimL-P2A-CFP-Mcl-1, and Puma-P2A-CFP-Mcl-1 plasmids to achieve the co-expression of Bak/Puma/BimL and Mcl-1. FRET analysis for the cells coexpressing Bak-P2A-CFP-Mcl-1 and YFP-Puma/BimL shows that co-expression of Bak and Mcl-1 significantly increased the EDmax values between CFP-Mcl-1 and YFP-Puma/BimL, indicating that Bak makes the sites of Mcl-1 and Puma/BimL close by inserting into the Mcl-1-Puma/BimL dimers to form Bak-Mcl-1-Puma/BimL trimers, which has also been further verified by Co-IP analysis. For the cells coexpressing Puma/BimL-P2A-CFP-Mcl-1 and YFP-Bak, the ED values between Mcl-1 and Bak were very small, demonstrating that Mcl-1 binds preferentially to Puma or BimL in the presence of both Puma or BimL and Bak. Collectively, Puma/BimL preferentially binds to Mcl-1 to form Mcl-1-Puma and Mcl-1-BimL dimers, and Bak can insert into the Mcl-1-Puma and Mcl-1-BimL hetero-dimers to form hetero-trimers (Bak-Mcl-1-Puma and Bak-Mcl-1-BimL).