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Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis

Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different pro...

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Autores principales: Uyeda, Atsuko, Watanabe, Takayoshi, Hohsaka, Takahiro, Matsuura, Tomoaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445883/
https://www.ncbi.nlm.nih.gov/pubmed/32995515
http://dx.doi.org/10.1093/synbio/ysy007
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author Uyeda, Atsuko
Watanabe, Takayoshi
Hohsaka, Takahiro
Matsuura, Tomoaki
author_facet Uyeda, Atsuko
Watanabe, Takayoshi
Hohsaka, Takahiro
Matsuura, Tomoaki
author_sort Uyeda, Atsuko
collection PubMed
description Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. While in vitro lipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using an in vitro transcription–translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes.
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spelling pubmed-74458832020-09-28 Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis Uyeda, Atsuko Watanabe, Takayoshi Hohsaka, Takahiro Matsuura, Tomoaki Synth Biol (Oxf) Research Article Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. While in vitro lipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using an in vitro transcription–translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes. Oxford University Press 2018-06-06 /pmc/articles/PMC7445883/ /pubmed/32995515 http://dx.doi.org/10.1093/synbio/ysy007 Text en © The Author(s) 2018. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Research Article
Uyeda, Atsuko
Watanabe, Takayoshi
Hohsaka, Takahiro
Matsuura, Tomoaki
Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title_full Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title_fullStr Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title_full_unstemmed Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title_short Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
title_sort different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445883/
https://www.ncbi.nlm.nih.gov/pubmed/32995515
http://dx.doi.org/10.1093/synbio/ysy007
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