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Evaluation of the Interaction between Bax and Hsp70 in Cells by Using a FRET System Consisting of a Fluorescent Amino Acid and YFP as a FRET Pair
Author(s) -
Park SeongHyun,
Ko Wooseok,
Park SangHyun,
Lee Hyun Soo,
Shin Injae
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900293
Subject(s) - yellow fluorescent protein , förster resonance energy transfer , brefeldin a , bafilomycin , endoplasmic reticulum , microbiology and biotechnology , chemistry , microtubule , immunoprecipitation , biophysics , biochemistry , biology , fluorescence , golgi apparatus , apoptosis , autophagy , physics , quantum mechanics , gene
To gain insight into factors that lead to dissociation of Bax from a complex with Hsp70 during apoptosis, we recently constructed a fluorescence resonance energy transfer (FRET) system composed of the Hsp70‐YFP (YFP=yellow fluorescent protein) fusion protein and fluorescent amino acid (ANAP=6‐acetyl(naphthalen‐2‐ylamino)‐2‐aminopropanoic acid)‐containing Bax (Bax‐ANAP), which was produced by using the genetic code expansion technique. In the current study, the FRET system was employed to elucidate how brefeldin A (an endoplasmic reticulum stress inducer), chlorpromazine and apoptozole (lysosomal membrane destabilizers), bafilomycin A1 (an inhibitor of lysosomal acidification) as well as raptinal and Az‐TPP‐O3 (mitochondria‐targeted apoptosis inducers) affect the interaction between Bax and Hsp70. Analyses of single live cell images together with results of co‐immunoprecipitation assays reveal that brefeldin A, chlorpromazine, and apoptozole promote dissociation of the Bax/Hsp70 complex through activation of the activator BH3‐only protein. However, the results show that bafilomycin A1, raptinal, and Az‐TPP‐O3 have no influence on the interaction of Bax with Hsp70. The combined observations made in the current and previous studies demonstrate that the FRET system consisting of Bax‐ANAP and Hsp70‐YFP is highly useful to understand apoptotic processes associated with the Bax–Hsp70 interaction.