Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay

• Published in: Cellular and molecular life sciences : CMLS Vol. 77; no. 12; pp. 2387 - 2406
• Main Authors: Yang, Fangfang, Qu, Wenfeng, Du, Mengyan, Mai, Zihao, Wang, Bin, Ma, Yunyun, Wang, Xiaoping, Chen, Tongsheng
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.06.2020; Springer Nature B.V
• Subjects: Affinity; Apoptosis; Apoptosis - physiology; Apoptosis Regulatory Proteins - metabolism; BAX protein; Bcl-2 protein; bcl-2-Associated X Protein - metabolism; Bcl-x protein; bcl-X Protein - metabolism; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell Biology; Cell Line, Tumor; Cytosol; Dimers; Fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; HeLa Cells; Humans; Life Sciences; Mitochondria; Mitochondria - metabolism; Original; Original Article; Overexpression; Protein interaction; Protein Interaction Maps - physiology; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; Stoichiometry; Trimers; Stoichiometry; Affinity; Bcl-2 proteins; FRET; Living cells
• ISSN: 1420-682X; 1420-9071
• DOI: 10.1007/s00018-019-03286-z

The stoichiometry and affinity of Bcl-2 family complexes are essential information for understanding how their interactome network is orchestrated to regulate mitochondrial permeabilization and apoptosis. Based on over-expression model system, FRET analysis was used to quantify the protein–protein interactions among Bax, Bcl-xL, Bad and tBid in healthy and apoptotic cells. Our data indicate that the stoichiometry and affinity of Bcl-2 complexes are dependent on their membrane environment. Bcl-xL, Bad and tBid can form hetero-trimers in mitochondria. Bcl-xL binds preferentially to Bad, then to tBid and Bax in mitochondria, whilst Bcl-xL displays higher affinity to Bad or tBid than to itself. Strikingly, Bax can bind to Bcl-xL in cytosol. In cytosol of apoptotic cells, Bcl-xL associates with Bax to form hetero-trimer with 1:2 stoichiometry, while Bcl-xL associates with Bad to form hetero-trimer with 2:1 stoichiometry and Bcl-xL associates with tBid to form hetero-dimer. In mitochondria, Bcl-xL associates with Bax/Bad to form hetero-dimer in healthy cells, while Bcl-xL associates with Bad to form hetero-tetramer with 3:1 stoichiometry in apoptotic cells.