In Vitro Insertion and Assembly of Outer Membrane Protein PhoE of Escherichia coli K-12 into the Outer Membrane

The assembly of the in vitro synthesized outer membrane protein PhoE into purified outer membranes was investigated. The assembly appeared to be strongly stimulated by the presence of low amounts of Triton X-100 (optimal 0.08%, w/v). The role of Triton X-100 in the in vitro system was further examined. Pretreating outer membranes with Triton X-100 did not make the membranes competent for correct assembly, indicating that the detergent did not act on the membrane but at the protein level. PhoE became assembly-incompetent with a half-life of approximately 12 min and 90 s at 37°C in the absence and presence, respectively, of 0.08% Triton X-100. Apparently, Triton X-100 induces an assembly-competent state in the PhoE protein with a very short half-life. Furthermore, the efficiency of correct assembly of PhoE was greatly reduced when outer membranes of deep rough lipopolysaccharide mutants were used, indicating an important role of lipopolysaccharides in the assembly of the porin.