Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane or...

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Main Authors: Akandé Rouchidane Eyitayo, Axel Boudier-Lemosquet, Stéphane Chaignepain, Muriel Priault, Stéphen Manon
Format: Article
Language:English
Published: MDPI AG 2023-05-01
Series:Biomolecules
Subjects:
Online Access:https://www.mdpi.com/2218-273X/13/6/876
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author Akandé Rouchidane Eyitayo
Axel Boudier-Lemosquet
Stéphane Chaignepain
Muriel Priault
Stéphen Manon
author_facet Akandé Rouchidane Eyitayo
Axel Boudier-Lemosquet
Stéphane Chaignepain
Muriel Priault
Stéphen Manon
author_sort Akandé Rouchidane Eyitayo
collection DOAJ
description The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in <i>E. coli</i>. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.
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spelling doaj.art-73cb29dd46384292aa08bbbb927f85322023-11-18T09:30:04ZengMDPI AGBiomolecules2218-273X2023-05-0113687610.3390/biom13060876Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis SystemAkandé Rouchidane Eyitayo0Axel Boudier-Lemosquet1Stéphane Chaignepain2Muriel Priault3Stéphen Manon4Institut de Biochimie et de Génétique Cellulaires, Université de Bordeaux, CNRS, UMR 5095, 33077 Bordeaux, FranceInstitut de Biochimie et de Génétique Cellulaires, Université de Bordeaux, CNRS, UMR 5095, 33077 Bordeaux, FranceInstitut de Biochimie et de Génétique Cellulaires, Université de Bordeaux, CNRS, UMR 5095, 33077 Bordeaux, FranceInstitut de Biochimie et de Génétique Cellulaires, Université de Bordeaux, CNRS, UMR 5095, 33077 Bordeaux, FranceInstitut de Biochimie et de Génétique Cellulaires, Université de Bordeaux, CNRS, UMR 5095, 33077 Bordeaux, FranceThe antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in <i>E. coli</i>. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.https://www.mdpi.com/2218-273X/13/6/876Bcl-xLBaxnanodiscscell-free synthesismembrane insertionMALDI
spellingShingle Akandé Rouchidane Eyitayo
Axel Boudier-Lemosquet
Stéphane Chaignepain
Muriel Priault
Stéphen Manon
Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
Biomolecules
Bcl-xL
Bax
nanodiscs
cell-free synthesis
membrane insertion
MALDI
title Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
title_full Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
title_fullStr Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
title_full_unstemmed Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
title_short Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System
title_sort bcl xl is spontaneously inserted into preassembled nanodiscs and stimulates bax insertion in a cell free protein synthesis system
topic Bcl-xL
Bax
nanodiscs
cell-free synthesis
membrane insertion
MALDI
url https://www.mdpi.com/2218-273X/13/6/876
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