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