In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.

Naoki Ohta,Yasuhiko Kato,Hajime Watanabe,Tomoaki Matsuura,Hirotada Mori

doi:10.1038/srep36466

Naoki Ohta, Yasuhiko Kato + Show 3 more

Open Access

https://doi.org/10.1038/srep36466

Copy DOI

Abstract

Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach.

Highlights

To properly fold and localize membrane proteins in the membrane
This work represents an essential step toward the construction of an artificial cell through a bottom-up approach[33,34,35]
We reconstituted the Sec translocon inside of cell-sized liposomes, which resulted in an increase in the production of active EmrE by approximately three-fold

Summary

Introduction

To properly fold and localize membrane proteins in the membrane. Previous methods, have relied on spontaneous insertion, without the help of translocons, to achieve membrane protein integration[21,22,23,24]. We previously encapsulated reconstituted IVTT, the protein synthesis using recombinant elements (PURE) system[31], inside cell-sized liposomes composed of defined components and succeeded in engineering[17,32] and characterizing[12,13] the mPOI. This technique, termed liposome display, lacked the translocon. By introducing the Sec translocon to the liposome display technique, the membrane integration efficiency of the mPOI is expected to increase This strategy should enable the display of membrane proteins that otherwise cannot be inserted. This work represents an essential step toward the construction of an artificial cell through a bottom-up approach[33,34,35]

Objectives

Methods

Results

Conclusion