The B cell leukemia-2 (Bcl-2) protein family, including antiapoptotic members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1) and proapoptotic members (Bax, Bak, Bok) as well as BH3-only proteins (tBid, Bim, Puma, Bad, and Noxa), Bak and Bax play a key role in the regulation of apoptosis (García-Sáez, 2012, Chen et al., 2021, Wang et al., 2019a, Bleicken et al., 2017). It is well known that activated Bak or Bax can form homo-oligomers or hetero oligomers on mitochondria to cause mitochondrial outer membrane permeability (MOMP) (Garciaz et al., 2022, Dadsena et al., 2021). Sensitizer BH3-only proteins promote apoptosis either by disrupting the association of prosurvival proteins with Bax/Bak, while activators BH3-only proteins promote apoptosis by directly binding to and activating Bak or Bax (Kim et al., 2006, Leshchiner et al., 2013, Letai et al., 2002, Wei et al., 2000, Czabotar et al., 2013). Anti-apoptotic proteins inhibit apoptosis in two models (Dadsena et al., 2021, Leber et al., 2007). The ‘MODE 1’ is that prosurvival proteins sequester BH3-only proteins to block apoptosis upstream of Bak and Bax activat ion, and the ‘MODE 2’ is that prosurvival proteins prevent apoptosis by binding Bak/Bax to inhibit their oligomerization (Llambi et al., 2011, Ku et al., 2011, Ding et al., 2014, Akgul, 2009).

Bcl-2 family proteins form a network to regulate the critical balance between cellular life and death. For example, Puma not only directly bind to Bak to form hetero-oligomers to activating Bak and promoting apoptosis (Kim et al., 2006, Adams, 2003, Clohessy et al., 2006, Krajewski et al., 1995), but also can directly bind to Mcl-1 and thus prevent the inhibitory action of Mcl-1 on Bak activation (Clohessy et al., 2006, Han et al., 2004, Chen et al., 2005, Shimazu et al., 2007). Mcl-1 can inhibit apoptosis by not only binding directly to Bak to form hetero-dimers to inhibit their oligomerization but also sequestering Puma to block apoptosis upstream of Bak activation (Llambi et al., 2011). BimL has the same regulatory network as Puma (Kim et al., 2006, Czabotar et al., 2013). Recently, we used two-hybrid FRET assays to measure the stoichiometry and affinity between Bcl-xL and Bax/Bad/tBid in living cells, and for the first time revealed the possibility of tBid-Bcl-xL-Bad trimers (Yang et al., 2020).

This article aims to use quantitative fluorescence resonance energy transfer (FRET) imaging to study the binding priority among four Bcl-2 family proteins (Mcl-1, Bak, BimL, and Puma) and whether they can form heterotrimers in living cells. To this end, we constructed three plasmids, Bak-P2A-CFP-Mcl-1, BimL-P2A-CFP-Mcl-1 and Puma-P2A-CFP-Mcl-1, to achieve the co-expression of fluorescent protein untagged Bak/BimL/Puma and CFP-tagged Mcl-1. FRET analysis for the cells coexpressing Bak-P2A-CFP-Mcl-1 and YFP-Puma or YFP-BimL indicates that Bak makes the sites of Mcl-1 and Puma or BimL close by inserting into the Mcl-1-Puma or Mcl-1-BimL dimers to form Bak-Mcl-1-Puma or Bak-Mcl-1-BimL trimers. In addition, our data also suggest a notion that Mcl-1 binds preferentially to Puma/BimL in the presence of both Puma and Bak.