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A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host
In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide–sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challen...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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American Society for Biochemistry and Molecular Biology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10011478/ https://www.ncbi.nlm.nih.gov/pubmed/36738791 http://dx.doi.org/10.1016/j.jbc.2023.102974 |
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author | Ferreras, Soledad Singh, Neha Pratap Le Borgne, Remi Bun, Philippe Binz, Thomas Parton, Robert G. Verbavatz, Jean-Marc Vannier, Christian Galli, Thierry |
author_facet | Ferreras, Soledad Singh, Neha Pratap Le Borgne, Remi Bun, Philippe Binz, Thomas Parton, Robert G. Verbavatz, Jean-Marc Vannier, Christian Galli, Thierry |
author_sort | Ferreras, Soledad |
collection | PubMed |
description | In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide–sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs. |
format | Online Article Text |
id | pubmed-10011478 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-100114782023-03-15 A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host Ferreras, Soledad Singh, Neha Pratap Le Borgne, Remi Bun, Philippe Binz, Thomas Parton, Robert G. Verbavatz, Jean-Marc Vannier, Christian Galli, Thierry J Biol Chem Research Article In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide–sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs. Syntaxin-1A/SNAP-25/VAMP-2 complexes were formed and regulated by SNARE partner protein Munc-18a in the presence of Caveolin. Additionally, Syntaxin-1A/SNAP-25/VAMP-2 synthesis provoked increased length of E. coli only in the presence of Caveolin. We found that cell elongation required SNAP-25 and was inhibited by tetanus neurotoxin. This elongation was not a result of cell division arrest. Furthermore, electron and super-resolution microscopies showed that synaptic SNAREs and Caveolin coexpression led to the partial loss of the cisternae, suggesting their fusion with the plasma membrane. In summary, we propose that this assay reconstitutes membrane fusion in a simple organism with an easy-to-observe phenotype and is amenable to structure-function studies of SNAREs. American Society for Biochemistry and Molecular Biology 2023-02-03 /pmc/articles/PMC10011478/ /pubmed/36738791 http://dx.doi.org/10.1016/j.jbc.2023.102974 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Ferreras, Soledad Singh, Neha Pratap Le Borgne, Remi Bun, Philippe Binz, Thomas Parton, Robert G. Verbavatz, Jean-Marc Vannier, Christian Galli, Thierry A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title | A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title_full | A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title_fullStr | A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title_full_unstemmed | A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title_short | A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host |
title_sort | synthetic organelle approach to probe snare-mediated membrane fusion in a bacterial host |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10011478/ https://www.ncbi.nlm.nih.gov/pubmed/36738791 http://dx.doi.org/10.1016/j.jbc.2023.102974 |
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