In Vitro Membrane Protein Synthesis Inside Cell-Sized Vesicles Reveals the Dependence of Membrane Protein Integration on Vesicle Volume

Giant unilamellar vesicles (GUVs) are vesicles >1 mu m 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-palmitoy1-2oleoyl-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 finding is explained by the effect of an increased surface-area-to-volume inside GUVs is a useful approach to study quantitatively the properties membrane under cell-mimicking environments.