title: In vitro membrane protein synthesis inside cell-sized vesicles reveals the dependence of membrane protein integration on vesicle volume.
abstract: Giant unilamellar vesicles (GUVs) are vesicles>1 μm in diameter that provide an environment in which the effect of a confined reaction volume on intravesicular reactions can be investigated. By synthesizing EmrE, a multidrug transporter from Escherichia coli, as a model membrane protein using a reconstituted in vitro transcription-translation system inside GUVs, we investigated the effect of a confined volume on the synthesis and membrane integration of EmrE. Flow cytometry was used to analyze multiple properties of the vesicles and to quantify EmrE synthesis inside GUVs composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. We found that EmrE was synthesized and integrated into the GUV membrane in its active form. We also found that the ratio of membrane-integrated EmrE to total synthesized EmrE increased with decreasing vesicle volume; this finding is explained by the effect of an increased surface-area-to-volume ratio in smaller vesicles. In vitro membrane synthesis inside GUVs is a useful approach to study quantitatively the properties of membrane proteins and their interaction with the membrane under cell-mimicking environments.
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka, Japan.
authors: Haruka
authors: H
authors: Soga
authors: Satoshi
authors: S
authors: Fujii
authors: Tetsuya
authors: T
authors: Yomo
authors: Yasuhiko
authors: Y
authors: Kato
authors: Hajime
authors: H
authors: Watanabe
authors: Tomoaki
authors: T
authors: Matsuura
mesh_terms: D000167:Acriflavine / Q000737:chemistry; D017920:Antiporters / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D004996:Ethidium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000138:chemical synthesis*; D010713:Phosphatidylcholines / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; C075436:EmrE protein, E coli; D000167:Acriflavine; D004247:DNA; D004996:Ethidium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine