In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components: Role of individual compnents and magnesium ions in reassembly

A method was developed for the reassembly of membranous vesicle from the sodium dcoxycholate-dissociated outer membrane components of Escherichia coli. The removal of the detergent by dialysis and the presence of Mg²⁺ were essential for the reassembly.Membrane protein alone did not form any membranous structure. Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialyzed. The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 Å, which is a characteristic of the native outer membrane. This characteristic trilaminar structure was shown to be due to the presence of lipopolysaccharide. Phospholipd was required for the vesicularization of membrane. Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture.The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic membrane proteins.