In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.
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.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Naoki
N
Ohta
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Yasuhiko
Y
Kato
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Hajime
H
Watanabe
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-tyou, Ikoma, Nara, Japan.
Hirotada
H
Mori
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Tomoaki
T
Matsuura
D017920:Antiporters / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D066298:In Vitro Techniques / Q000379:methods*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D010799:Phycoerythrin / Q000737:chemistry; D000069816:SEC Translocation Channels / Q000235:genetics / Q000378:metabolism
D017920:Antiporters; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D000069816:SEC Translocation Channels; C043426:inner membrane protein, E coli; D010799:Phycoerythrin; C075436:EmrE protein, E coli