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

  • Fangfang Yang
  • Wenfeng Qu
  • Mengyan Du
  • Zihao Mai
  • Bin Wang
  • Yunyun Ma
  • Xiaoping WangEmail author
  • Tongsheng ChenEmail author
Original Article


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.


Affinity Bcl-2 proteins FRET Living cells Stoichiometry 



Fluorescence resonance energy transfer


Bcl-2 homology domains


Bcl-2-associated X protein


Bcl-2 antagonist killer


Mitochondrial outer membrane


B-cell lymphoma 2


B-cell lymphoma, long isoform


Bcl2L2, Bcl-2-like protein 2


Bcl-2-related protein A1


Myeloid cell leukemia sequence 1


Bcl-2 homology 3


BH3-interacting domain death agonist


Truncated Bid protein


Bcl-2-protein 11


p53 upregulated modulator of apoptosis


Bcl-2 antagonist of cell death


PMAIP1, Phorbol-12-myristate-13-acetate-induced protein 1


Fluorescence cross-correlation spectroscopy


Dulbecco’s modified Eagle’s medium


Fetal bovine serum




1,1′,3,3,3′,3′-Hexamethylin-dodicarbocyanine iodide


Partial acceptor photobleaching-based FRET


Regions of interest



We thank David W. Andrews for providing mCherry-Bad and mCherry-tBid plasmids and Andrew P. Gilmore for providing CFP-Bax plasmid. This work was supported by grants from the National Natural Science Foundation of China (NSFC), (Grant Nos. 61527825, 61875056 and 81572184).

Author contributions

FY designed and performed experiments, analyzed data, and wrote the manuscript. WQ, MD, ZM, BW, and YM performed experiments. XW and TC designed the study, planned experiments, and wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

18_2019_3286_MOESM1_ESM.docx (7 mb)
Supplementary material 1 (DOCX 7122 kb)


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Fangfang Yang
    • 1
  • Wenfeng Qu
    • 1
  • Mengyan Du
    • 1
  • Zihao Mai
    • 1
  • Bin Wang
    • 1
  • Yunyun Ma
    • 1
  • Xiaoping Wang
    • 2
    Email author
  • Tongsheng Chen
    • 1
    Email author
  1. 1.MOE Key Laboratory of Laser Life Science and College of BiophotonicsSouth China Normal UniversityGuangzhouChina
  2. 2.Department of Pain ManagementThe First Affiliated Hospital of Jinan UniversityGuangzhouChina

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