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.

The PURE system for the cell-free synthesis of membrane proteins

“…One uses detergent in the preparation and another one does not, and they are selected according to the type of membrane protein and the subsequent activity measurement approach required. Although the membrane integration of the synthesized membrane protein is dependent on unregulated hydrophobic interactions between the protein and the lipid bilayer, some membrane proteins are able to maintain their functional state even after the spontaneous membrane integration in a co-translational manner [10][11][12][13][14][15][16] . Numerous examples of such membrane proteins have been reported so far [10][11][12][13][14][15][16] .…”

Communicating artificial cells

“…One solution would be to incorporate membrane protein sensors and transporters within the artificial cells. Advances in cellfree expression have led to the reconstitution of the secYEG translocon, ATP synthase [59], multidrug transporter [60], pheromone sensor [61], and ion channels [62] in phospholipid vesicles. The surface of vesicles can also be decorated with antibodies in order to sense specific target cells.…”

Construction de cellules synthétiques

“…L'objectif est que ces fonctions soient « codées » dans un génome minimal. L'expression de protéines membranaires in vitro est encore limitée malgré des avancées récentes [33,34]. Enfin, la division physique de la cellule minimale repose sur l'assemblage d'un cytosquelette actif à la membrane.…”